diff --git a/.gitignore b/.gitignore
new file mode 100644
index 0000000..ebf2caa
--- /dev/null
+++ b/.gitignore
@@ -0,0 +1,3 @@
+.snakemake
+.ipynb_checkpoints
+__pycache__
\ No newline at end of file
diff --git a/README.md b/README.md
index 23d912f..8a08b66 100644
--- a/README.md
+++ b/README.md
@@ -32,26 +32,6 @@ For example, below are some commands for installing Miniconda and Snakemake.
`conda create -c conda-forge -c bioconda -n snakemake snakemake`
-Skipper requires several R packages. In order to install the precise versions used in the manuscript, we have scripts to install the used versions of R and corresponding packages from source.
-
-Use conda to create an environment for installing R:
-
-`conda env create -f documents/rskipper.yml`
-
-Use the get_R.sh script to complete installation of R. Expect the whole process to take around 4 hours. Provide your conda directory as the first argument and the directory you wish to install R as the second:
-
-`bash -l tools/get_R.sh /home/eboyle/miniconda3 /projects/ps-yeolab3/eboyle/encode/pipeline/gran`
-
-Alternatively, at least as of this writing, Skipper is compatible with the newest version of R and its packages. The required packages can be installed for an existing R installation as follows:
-
-`install.packages(c("tidyverse", "VGAM", "viridis", "ggrepel", "RColorBrewer", "Rtsne", "ggupset", "ggdendro", "cowplot"))`
-
-`if (!require("BiocManager", quietly = TRUE))`
- `install.packages("BiocManager")`
-`BiocManager::install(c("GenomicRanges","fgsea","rtracklayer"))`
-
-Paths to locally installed versions can be supplied in the config file, described below.
-
Preparing to run Skipper
Skipper uses a Snakemake workflow. The `Skipper.py` file contains the rules necessary to process CLIP data from fastqs. Skipper also supports running on BAMs - note that Skipper's analysis of repetitive elements will assume that non-uniquely mapping reads are contained within the BAM files.
@@ -66,14 +46,16 @@ Numerous resources must be entered in the `Skipper_config.py` file:
| MANIFEST | Information on samples to run |
| GENOME | Samtools- and STAR-indexed fasta of genome for the sample of interest |
| STAR_DIR | Path to STAR reference for aligning sequencing reads |
+| WORKDIR | Path to outputs |
+| protocol | ENCODE3 to run paired-end. ENCODE4 to run single-end |
Other paths to help Skipper run must be entered:
| Path | Description |
| ----------- | ----------- |
-| EXE_DIR | For convenience to point to stable locally installed software: it is added to PATH when Skipper runs |
| TOOL_DIR | Directory for the tools located in the GitHub |
+| RBPNET_PATH | Directory for Deep Learning code [RBPNet](https://github.com/algaebrown/RBPNet/)|
Information about the CLIP library to be analyzed is also required:
@@ -137,11 +119,42 @@ Remember to load the Snakemake environment before running
Use the dry run function to confirm that Snakemake can parse all the information:
-`snakemake -ns Skipper.py -j 1`
+```
+snakemake -kps Skipper.py \
+ --configfile $CONFIG \
+ --profile profiles/tscc2 -n
+```
Once Snakemake has confirmed DAG creation, submit the jobs using whatever high performance computing infrastructure options suit you:
-`snakemake -kps Skipper.py -w 15 -j 30 --cluster "qsub -e {params.error_file} -o {params.out_file} -l walltime={params.run_time} -l nodes=1:ppn={threads} -q home-yeo"`
+```
+snakemake -kps Skipper.py \
+ --configfile $CONFIG \
+ --profile profiles/tscc2 -n
+```
+
+Some deep learning rules will benefit from using GPU (temporary solution):
+
+```
+# Run on CPU util data preparation
+CONFIG=/tscc/nfs/home/hsher/projects/skipper/encode_configs/Skipper_pe_small_test.yaml
+
+snakemake -kps Skipper.py \
+ --configfile $CONFIG \
+ --profile profiles/tscc2 --until rbpnet_prepare_data
+
+# Perform training, validation and seqlet finding using GPU
+snakemake -kps Skipper.py \
+ --configfile $CONFIG \
+ --profile profiles/tscc2_gpu --until rbpnet_seqlet
+
+# Finish the remaining using CPU
+snakemake -kps Skipper.py \
+ --configfile $CONFIG \
+ --profile profiles/tscc2
+
+# Sorry this is not end-to-end yet. Snakemake 8 can do it end-to-end but with some refactoring.
+```
Did Skipper terminate? Sometimes jobs fail - inspect any error output and rerun the same command if there is no apparent explanation such as uninstalled dependencies or a misformatted input file. Snakemake will try to pick up where it left off.
@@ -164,3 +177,11 @@ Skipper produces a lot of output. The `output/figures` directory contains figure
Annotated reproducible enriched windows can be accessed at `output/reproducible_enriched_windows/` and Homer motif output is at `output/homer/`
Example CLIP fastqs and processed data are available at GEO and SRA: `https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213867`
+
+## Common problems:
+1. Pulling singularity and get "no space left on device"
+```
+export SINGULARITY_TMPDIR=/tscc/lustre/ddn/scratch/hsher/singularity_tmp
+export TMPDIR=/tscc/lustre/ddn/scratch/hsher/singularity_tmp
+export SINGULARITY_CACHEDIR=/tscc/lustre/ddn/scratch/hsher/singularity_cache
+```
\ No newline at end of file
diff --git a/Skipper.py b/Skipper.py
index 95f5759..5f85f4f 100644
--- a/Skipper.py
+++ b/Skipper.py
@@ -5,30 +5,38 @@
import sys
import glob
from time import sleep
+from pathlib import Path
+import warnings
-# example command
-# snakemake -kps Skipper.py -w 25 -j 30 --cluster "qsub -e {params.error_file} -o {params.out_file} -l walltime={params.run_time} -l nodes=1:ppn={threads} -q home-yeo"
-
-include: "Skipper_config.py"
+locals().update(config)
+workdir: config['WORKDIR']
if not os.path.exists("stderr"): os.makedirs("stderr")
if not os.path.exists("stdout"): os.makedirs("stdout")
-if EXE_DIR not in sys.path: os.environ["PATH"] = EXE_DIR + os.pathsep + os.environ["PATH"]
if OVERDISPERSION_MODE not in ["clip","input"]:
raise Exception("Overdispersion must be calculated using 'clip' or 'input' samples")
+# read and cleanup manifest
manifest = pd.read_csv(MANIFEST, comment = "#", index_col = False).dropna(subset=['Experiment','Sample'])
manifest["CLIP_replicate"] = pd.to_numeric(manifest.CLIP_replicate, downcast="integer")
manifest["Input_replicate"] = pd.to_numeric(manifest.Input_replicate, downcast="integer")
-manifest["Input_fastq"] = [name.strip() for name in manifest["Input_fastq"]]
-manifest["CLIP_fastq"] = [name.strip() for name in manifest["CLIP_fastq"]]
-manifest["Input_adapter"] = [name.strip() for name in manifest["Input_adapter"]]
-manifest["CLIP_adapter"] = [name.strip() for name in manifest["CLIP_adapter"]]
-if min(manifest.groupby("Experiment")["CLIP_fastq"].agg(lambda x: len(set(x)))) < 2:
- sys.stderr.write("WARNING: NONZERO EXPERIMENTS HAVE ONLY ONE CLIP REPLICATE.\nPIPELINE MUST HALT AFTER GENERATING RAW COUNTS\nThis usually means your manifest is incorrectly formatted\n")
- sleep(5)
+
+for col in manifest.columns[manifest.columns.str.contains('_fastq') | manifest.columns.str.contains('_adapter')]:
+ manifest[col] = manifest[col].str.strip()
+
+# check numbers are correct
+try:
+ if min(manifest.groupby("Experiment")["CLIP_fastq"].agg(lambda x: len(set(x)))) < 2:
+ sys.stderr.write("WARNING: NONZERO EXPERIMENTS HAVE ONLY ONE CLIP REPLICATE.\nPIPELINE MUST HALT AFTER GENERATING RAW COUNTS\nThis usually means your manifest is incorrectly formatted\n")
+ print(manifest.groupby("Experiment")["CLIP_fastq"].agg(lambda x: len(set(x))))
+ sleep(5)
+except:
+ if min(manifest.groupby("Experiment")["CLIP_fastq_1"].agg(lambda x: len(set(x)))) < 2:
+ sys.stderr.write("WARNING: NONZERO EXPERIMENTS HAVE ONLY ONE CLIP REPLICATE.\nPIPELINE MUST HALT AFTER GENERATING RAW COUNTS\nThis usually means your manifest is incorrectly formatted\n")
+ print(manifest.groupby("Experiment")["CLIP_fastq_1"].agg(lambda x: len(set(x))))
+ sleep(5)
if max(manifest.groupby("Sample")["Input_replicate"].agg(lambda x: min(x))) > 1:
raise Exception("Input replicates for samples in manifest do not increment from 1 as expected")
@@ -36,11 +44,15 @@
if max(manifest.groupby("Sample")["CLIP_replicate"].agg(lambda x: min(x))) > 1:
raise Exception("CLIP replicates for samples in manifest do not increment from 1 as expected")
+# create label for IN and CLIP:
+# Sample = DEK_HepG2_4020, replicate_label: DEK_HepG2_4020_IN_1 and DEK_HepG2_4020_IP_1
manifest["Input_replicate_label"] = [(str(sample) + "_IN_" + str(replicate)).replace(" ","") for replicate, sample in zip(manifest.Input_replicate.tolist(),manifest.Sample.tolist())]
manifest["CLIP_replicate_label"] = [(str(sample) + "_IP_" + str(replicate)).replace(" ","") for replicate, sample in zip(manifest.CLIP_replicate.tolist(),manifest.Sample.tolist())]
-input_replicates = manifest.loc[:,manifest.columns.isin(["Input_replicate_label","Input_fastq","Input_bam","Input_adapter"])].drop_duplicates()
-clip_replicates = manifest.loc[:,manifest.columns.isin(["CLIP_replicate_label","CLIP_fastq","CLIP_bam","CLIP_adapter"])].drop_duplicates()
+input_replicates = manifest.loc[:,manifest.columns.isin(["Input_replicate_label","Input_fastq","Input_fastq_1", "Input_fastq_2","Input_bam","Input_adapter","Input_adapter_1","Input_adapter_2"])].drop_duplicates()
+clip_replicates = manifest.loc[:,manifest.columns.isin(["CLIP_replicate_label","CLIP_fastq","CLIP_fastq_1","CLIP_fastq_2","CLIP_bam","CLIP_adapter","CLIP_adapter_1","CLIP_adapter_2"])].drop_duplicates()
+
+
if len(input_replicates) != len(input_replicates[["Input_replicate_label"]].drop_duplicates()) or \
len(clip_replicates) != len(clip_replicates[["CLIP_replicate_label"]].drop_duplicates()):
@@ -49,23 +61,42 @@
input_replicate_labels = input_replicates.Input_replicate_label.tolist()
clip_replicate_labels = clip_replicates.CLIP_replicate_label.tolist()
replicate_labels = pd.Series(input_replicate_labels + clip_replicate_labels)
+config['replicate_labels']= replicate_labels
+
+# if all(bam in manifest.columns.tolist() for bam in ["Input_bam", "CLIP_bam"]):
+# replicate_label_to_bams = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_bam.tolist() + clip_replicates.CLIP_bam.tolist()))
+# else:
+
+# FASTQ and ADAPTOR
+if "Input_fastq" in manifest.columns and config['protocol']=='ENCODE4':
+ config['replicate_label_to_fastqs'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_fastq.tolist() + clip_replicates.CLIP_fastq.tolist()))
+ config['replicate_label_to_adapter'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_adapter.tolist() + clip_replicates.CLIP_adapter.tolist()))
+elif config['protocol']=='ENCODE3':
+ config['replicate_label_to_fastq_1'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_fastq_1.tolist() + clip_replicates.CLIP_fastq_1.tolist()))
+ config['replicate_label_to_fastq_2'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_fastq_2.tolist() + clip_replicates.CLIP_fastq_2.tolist()))
+ config['replicate_label_to_adapter_1'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_adapter_1.tolist() + clip_replicates.CLIP_adapter_1.tolist()))
+ config['replicate_label_to_adapter_2'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_adapter_2.tolist() + clip_replicates.CLIP_adapter_2.tolist()))
+else:
+ raise Exception("protocol does not fit in ENCODE3 or ENCODE4")
-if all(bam in manifest.columns.tolist() for bam in ["Input_bam", "CLIP_bam"]):
- replicate_label_to_bams = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_bam.tolist() + clip_replicates.CLIP_bam.tolist()))
+# BAMs
+if config['protocol']=='ENCODE4':
+ config['replicate_label_to_bams'] = dict(zip(input_replicate_labels + clip_replicate_labels, ["output/bams/dedup/genome/" + replicate_label + ".genome.Aligned.sort.dedup.bam" for replicate_label in input_replicate_labels + clip_replicate_labels] ))
+elif config['protocol']=='ENCODE3':
+ config['replicate_label_to_bams'] = dict(zip(input_replicate_labels + clip_replicate_labels, [f"output/bams/dedup/genome_R{INFORMATIVE_READ}/" + replicate_label + f".genome.Aligned.sort.dedup.R{INFORMATIVE_READ}.bam" for replicate_label in input_replicate_labels + clip_replicate_labels] ))
else:
- replicate_label_to_bams = dict(zip(input_replicate_labels + clip_replicate_labels, ["output/bams/dedup/genome/" + replicate_label + ".genome.Aligned.sort.dedup.bam" for replicate_label in input_replicate_labels + clip_replicate_labels] ))
+ raise Exception("protocol does not fit in ENCODE3 or ENCODE4")
-experiment_labels = pd.Series(manifest.Experiment.drop_duplicates().tolist())
+# EXPERIMENT LABELS
+config['experiment_labels'] = pd.Series(manifest.Experiment.drop_duplicates().tolist())
experiment_data = manifest.groupby("Experiment").agg({"CLIP_replicate_label": list, "Input_replicate_label" : list})
-if "Input_fastq" in manifest.columns:
- replicate_label_to_fastqs = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_fastq.tolist() + clip_replicates.CLIP_fastq.tolist()))
- replicate_label_to_adapter = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_adapter.tolist() + clip_replicates.CLIP_adapter.tolist()))
-overdispersion_replicate_lookup = dict(zip(manifest.CLIP_replicate_label.tolist(), manifest.Input_replicate_label.tolist() if OVERDISPERSION_MODE == "input" else manifest.CLIP_replicate_label.tolist()))
-clip_to_input_replicate_label = dict(zip(manifest.CLIP_replicate_label.tolist(), manifest.Input_replicate_label.tolist()))
-experiment_to_replicate_labels = dict(zip(experiment_data.index.tolist(), [reduce(lambda agg, x: agg if x in agg else agg + [x], inputs, []) + clips for inputs, clips in zip(experiment_data.Input_replicate_label, experiment_data.CLIP_replicate_label)]))
-experiment_to_clip_replicate_labels = dict(zip(experiment_data.index.tolist(), experiment_data.CLIP_replicate_label))
+# OVERDISPERSION and BACKGROUND PAIRING
+config['overdispersion_replicate_lookup'] = dict(zip(manifest.CLIP_replicate_label.tolist(), manifest.Input_replicate_label.tolist() if OVERDISPERSION_MODE == "input" else manifest.CLIP_replicate_label.tolist()))
+config['clip_to_input_replicate_label'] = dict(zip(manifest.CLIP_replicate_label.tolist(), manifest.Input_replicate_label.tolist()))
+config['experiment_to_replicate_labels'] = dict(zip(experiment_data.index.tolist(), [reduce(lambda agg, x: agg if x in agg else agg + [x], inputs, []) + clips for inputs, clips in zip(experiment_data.Input_replicate_label, experiment_data.CLIP_replicate_label)]))
+config['experiment_to_clip_replicate_labels'] = dict(zip(experiment_data.index.tolist(), experiment_data.CLIP_replicate_label))
experiment_to_input_replicate_labels = {}
for experiment_label, label_list in zip(experiment_data.index, experiment_data.Input_replicate_label):
@@ -76,710 +107,162 @@
if other_entry != entry:
replicates.add(other_entry)
experiment_to_input_replicate_labels[experiment_label].update({entry : list(replicates)})
+config['experiment_to_input_replicate_labels']=experiment_to_input_replicate_labels
+
+# Fool-proof Detect misalignment for GFF and PARTITION
+if Path(GFF).name.replace('.gff3.gz', '') != Path(FEATURE_ANNOTATIONS).name.replace('.tiled_partition.features.tsv.gz', ''):
+ warnings.warn(f'''Detected Name Mismatch in GFF and FEATURE ANNOTATIONS:
+ FEATURE_ANNOTATIONS={FEATURE_ANNOTATIONS}
+ GFF={GFF}
+ Check if they are the same cell line
+ ''')
+
+config['manifest'] = manifest
+
+# access config file path
+if '--configfile' in sys.argv:
+ i = sys.argv.index('--configfile')
+elif '--configfiles' in sys.argv:
+ i = sys.argv.index('--configfiles')
+config['CONFIG_PATH']=sys.argv[i+1]
+print(config['CONFIG_PATH'])
+locals().update(config)
rule all:
input:
- expand("output/fastqc/initial/{replicate_label}_fastqc.html", replicate_label = replicate_labels),
- expand("output/fastqc/processed/{replicate_label}.trimmed.umi_fastqc.html", replicate_label = replicate_labels),
+ # expand("output/fastqc/initial/{replicate_label}_fastqc.html", replicate_label = replicate_labels
+ # ) if config['protocol']=='ENCODE4' else ,
+ # expand("output/fastqc/processed/{replicate_label}.trimmed.umi_fastqc.html", replicate_label = replicate_labels),
expand("output/bams/dedup/genome/{replicate_label}.genome.Aligned.sort.dedup.bam", replicate_label = replicate_labels),
expand("output/bams/dedup/genome/{replicate_label}.genome.Aligned.sort.dedup.bam.bai", replicate_label = replicate_labels),
- # expand("output/bigwigs/unscaled/plus/{replicate_label}.unscaled.plus.bw", replicate_label = replicate_labels),
- # expand("output/bigwigs/scaled/plus/{replicate_label}.scaled.plus.bw", replicate_label = replicate_labels),
- expand("output/counts/repeats/vectors/{replicate_label}.counts", replicate_label = replicate_labels),
+ expand("output/bigwigs/unscaled/plus/{replicate_label}.unscaled.plus.bw", replicate_label = replicate_labels),
+ expand("output/bigwigs/scaled/plus/{replicate_label}.scaled.plus.bw", replicate_label = replicate_labels),
+ expand("output/bigwigs/scaled/plus/{replicate_label}.scaled.cov.plus.bw", replicate_label = replicate_labels),
+ # expand("output/counts/repeats/vectors/{replicate_label}.counts", replicate_label = replicate_labels),
expand("output/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_windows.tsv.gz", zip, experiment_label = manifest.Experiment, clip_replicate_label = manifest.CLIP_replicate_label),
expand("output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz", experiment_label = manifest.Experiment),
- expand("output/figures/enrichment_reproducibility/{experiment_label}.enrichment_reproducibility.pdf", experiment_label = manifest.Experiment),
- expand("output/counts/repeats/tables/family/{experiment_label}.tsv.gz", experiment_label = manifest.Experiment),
+ # expand("output/figures/enrichment_reproducibility/{experiment_label}.enrichment_reproducibility.pdf", experiment_label = manifest.Experiment),
+ # expand("output/counts/repeats/tables/family/{experiment_label}.tsv.gz", experiment_label = manifest.Experiment),
expand("output/reproducible_enriched_re/{experiment_label}.reproducible_enriched_re.tsv.gz", experiment_label = manifest.Experiment),
expand("output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz", experiment_label = manifest.Experiment),
+ expand("output/finemapping/both_tested_sites/{experiment_label}.both_tested_windows.bed",experiment_label = manifest.Experiment),
expand("output/homer/finemapped_results/{experiment_label}/homerResults.html", experiment_label = manifest.Experiment),
expand("output/gene_sets/{experiment_label}.enriched_terms.tsv.gz", experiment_label = manifest.Experiment),
"output/figures/tsne/skipper.tsne_query.pdf",
- output:
- "land_ho.txt"
- threads: 1
- params:
- error_file = "stderr/all.err",
- out_file = "stdout/all.out",
- run_time = "00:04:00",
- memory = "20",
- job_name = "all"
- shell:
- "echo $(date) > {output};"
- "echo created by Evan Boyle and the Yeo lab >> {output}"
-
-rule parse_gff:
- input:
- gff = ancient(GFF),
- rankings = ancient(ACCESSION_RANKINGS),
- output:
- partition = PARTITION,
- feature_annotations = FEATURE_ANNOTATIONS,
- threads: 4
- params:
- error_file = "stderr/parse_gff.err",
- out_file = "stdout/parse_gff.out",
- run_time = "3:00:00",
- job_name = "parse_gff"
- benchmark: "benchmarks/parse_gff.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/parse_gff.R {input.gff} {input.rankings} {output.partition} {output.feature_annotations}"
-
-rule run_initial_fastqc:
- input:
- fq = lambda wildcards: replicate_label_to_fastqs[wildcards.replicate_label].split(" "),
- output:
- report = "output/fastqc/initial/{replicate_label}_fastqc.html",
- zip_file = "output/fastqc/initial/{replicate_label}_fastqc.zip",
- directory = directory("output/fastqc/initial/{replicate_label}_fastqc")
- threads: 1
- params:
- error_file = "stderr/{replicate_label}.fastqc_initial.err",
- out_file = "stdout/{replicate_label}.fastqc_initial.out",
- run_time = "3:00:00",
- job_name = "run_initial_fastqc"
- benchmark: "benchmarks/fastqc/unassigned_experiment.{replicate_label}.initial_fastqc.txt"
- shell:
- "less {input.fq} | fastqc stdin:{wildcards.replicate_label} --extract --outdir output/fastqc/initial -t {threads}"
-
-rule trim_fastq:
- input:
- fq = lambda wildcards: replicate_label_to_fastqs[wildcards.replicate_label].split(" "),
- adapter = lambda wildcards: replicate_label_to_adapter[wildcards.replicate_label],
- output:
- fq_trimmed = temp("output/fastqs/trimmed/{replicate_label}-trimmed.fastq.gz"),
- metrics = "output/fastqs/trimmed/{replicate_label}-trimmed.log"
- threads: 8
- params:
- run_time = "3:30:00",
- memory = "15000",
- error_file = "stderr/{replicate_label}.trim.err",
- out_file = "stdout/{replicate_label}.trim.out",
- job_name = "trim_fastq"
- benchmark: "benchmarks/trim/unassigned_experiment.{replicate_label}.trim.txt"
- shell:
- "less {input.fq} | skewer "
- "-t {threads} "
- "-x {input.adapter} "
- "-o output/fastqs/trimmed/{wildcards.replicate_label} "
- "-z -r 0.2 -d 0.2 -q 13 -l 20 -"
-
-rule extract_umi:
- input:
- fq = "output/fastqs/trimmed/{replicate_label}-trimmed.fastq.gz",
- output:
- fq_umi = "output/fastqs/umi/{replicate_label}.trimmed.umi.fq.gz",
- json = "output/fastp/{replicate_label}.fastp.json",
- html = "output/fastp/{replicate_label}.fastp.html",
- threads: 8
- params:
- error_file = "stderr/{replicate_label}.extract_umi.err",
- out_file = "stdout/{replicate_label}.extract_umi.out",
- run_time = "45:00",
- memory = "10000",
- job_name = "extract_umi",
- umi_length = UMI_SIZE,
- benchmark: "benchmarks/umi/unassigned_experiment.{replicate_label}.extract_umi.txt"
- shell:
- "fastp "
- "-i {input.fq} "
- "-o {output.fq_umi} "
- "-A "
- "-U "
- "--umi_len={params.umi_length} "
- "--umi_loc=read1 "
- "-j output/fastp/{wildcards.replicate_label}.fastp.json "
- "-h output/fastp/{wildcards.replicate_label}.fastp.html "
- "-w {threads}"
-
-
-rule run_trimmed_fastqc:
- input:
- "output/fastqs/umi/{replicate_label}.trimmed.umi.fq.gz",
- output:
- report = "output/fastqc/processed/{replicate_label}.trimmed.umi_fastqc.html",
- zip_file = "output/fastqc/processed/{replicate_label}.trimmed.umi_fastqc.zip",
- threads: 1
- params:
- outdir="output/fastqc/processed/",
- run_time = "03:00:00",
- memory = "15000",
- error_file = "stderr/{replicate_label}.run_trimmed_fastqc.err",
- out_file = "stdout/{replicate_label}.run_trimmed_fastqc.out",
- job_name = "run_trimmed_fastqc"
- benchmark: "benchmarks/fastqc/unassigned_experiment.{replicate_label}.trimmed_fastqc.txt"
- shell:
- "fastqc {input} --extract --outdir output/fastqc/processed -t {threads}"
-
-rule align_reads:
- input:
- fq= "output/fastqs/umi/{replicate_label}.trimmed.umi.fq.gz",
- output:
- ubam = temp("output/bams/raw/genome/{replicate_label}.genome.Aligned.out.bam"),
- # unmapped= "output/bams/raw/genome/{replicate_label}.genome.Unmapped.out.mate1",
- log= "output/bams/raw/genome/{replicate_label}.genome.Log.final.out",
- threads: 8
- params:
- error_file = "stderr/{replicate_label}.align_reads_genome.err",
- out_file = "stdout/{replicate_label}.align_reads_genome.out",
- run_time = "02:00:00",
- memory = "40000",
- job_name = "align_reads",
- star_sjdb = STAR_DIR,
- outprefix = "output/bams/raw/genome/{replicate_label}.genome.",
- rg = "{replicate_label}"
- benchmark: "benchmarks/align/unassigned_experiment.{replicate_label}.align_reads_genome.txt"
- shell:
- "STAR "
- "--alignEndsType EndToEnd "
- "--genomeDir {params.star_sjdb} "
- "--genomeLoad NoSharedMemory "
- "--outBAMcompression 10 "
- "--outFileNamePrefix {params.outprefix} "
- "--winAnchorMultimapNmax 100 "
- "--outFilterMultimapNmax 100 "
- "--outFilterMultimapScoreRange 1 "
- "--outSAMmultNmax 1 "
- "--outMultimapperOrder Random "
- "--outFilterScoreMin 10 "
- "--outFilterType BySJout "
- "--limitOutSJcollapsed 5000000 "
- "--outReadsUnmapped None "
- "--outSAMattrRGline ID:{wildcards.replicate_label} "
- "--outSAMattributes All "
- "--outSAMmode Full "
- "--outSAMtype BAM Unsorted "
- "--outSAMunmapped Within "
- "--readFilesCommand zcat "
- "--outStd Log "
- "--readFilesIn {input.fq} "
- "--runMode alignReads "
- "--runThreadN {threads}"
-
-rule sort_bam:
- input:
- bam="output/bams/raw/{ref}/{replicate_label}.{ref}.Aligned.out.bam",
- output:
- sort = "output/bams/raw/{ref}/{replicate_label}.{ref}.Aligned.sort.bam",
- threads: 2
- params:
- error_file = "stderr/{ref}_{replicate_label}.sort_bam.err",
- out_file = "stdout/{ref}_{replicate_label}.sort_bam.out",
- run_time = "00:30:00",
- memory = "10000",
- job_name = "sortbam",
- benchmark: "benchmarks/sort/{ref}/unassigned_experiment.{replicate_label}.sort_bam.txt"
- shell:
- "samtools sort -T {wildcards.replicate_label} -@ {threads} -o {output.sort} {input.bam};"
-
-
-rule index_bams:
- input:
- bam = "output/bams/{round}/{ref}/{replicate_label}.Aligned.{mid}.bam"
- output:
- ibam = "output/bams/{round}/{ref}/{replicate_label}.Aligned.{mid}.bam.bai"
- threads: 2
- params:
- error_file = "stderr/{round}_{ref}_{mid}_{replicate_label}.index_bams.err",
- out_file = "stdout/{round}_{ref}_{mid}_{replicate_label}.index_bams.out",
- run_time = "10:00",
- memory = "1000",
- job_name = "index_bam"
- benchmark: "benchmarks/index_bam/{round}/{ref}/{mid}/unassigned_experiment.{replicate_label}.index_bam.txt"
- shell:
- "samtools index -@ {threads} {input.bam};"
-
-
-rule dedup_umi:
- input:
- bam="output/bams/raw/genome/{replicate_label}.genome.Aligned.sort.bam",
- ibam = "output/bams/raw/genome/{replicate_label}.genome.Aligned.sort.bam.bai"
- output:
- bam_dedup="output/bams/dedup/genome/{replicate_label}.genome.Aligned.sort.dedup.bam"
- params:
- error_file = "stderr/{replicate_label}.dedup_umi.err",
- out_file = "stdout/{replicate_label}.dedup_umi.out",
- run_time = "1:00:00",
- memory = "10000",
- job_name = "dedup_bam",
- prefix='output/bams/dedup/genome/{replicate_label}.genome.sort'
- benchmark: "benchmarks/dedup/genome/unassigned_experiment.{replicate_label}.dedup_umi.txt"
- shell:
- "{JAVA_EXE} -server -Xms8G -Xmx8G -Xss20M -jar {UMICOLLAPSE_DIR}/umicollapse.jar bam "
- "-i {input.bam} -o {output.bam_dedup} --umi-sep : --two-pass"
-
-rule make_unscaled_bigwig:
- input:
- CHROM_SIZES,
- bam = lambda wildcards: replicate_label_to_bams[wildcards.replicate_label],
- output:
- bg_plus = temp("output/bedgraphs/unscaled/plus/{replicate_label}.unscaled.plus.bg"),
- bg_minus = temp("output/bedgraphs/unscaled/minus/{replicate_label}.unscaled.minus.bg"),
- bw_plus = "output/bigwigs/unscaled/plus/{replicate_label}.unscaled.plus.bw",
- bw_minus = "output/bigwigs/unscaled/minus/{replicate_label}.unscaled.minus.bw",
- params:
- error_file = "stderr/{replicate_label}.make_bigwig.err",
- out_file = "stdout/{replicate_label}.make_bigwig.out",
- run_time = "40:00",
- memory = "1000",
- job_name = "make_bigwig"
- benchmark: "benchmarks/bigwigs/unassigned_experiment.{replicate_label}.make_bigwig.txt"
- shell:
- "bedtools genomecov -5 -strand + -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_plus};"
- "bedtools genomecov -5 -strand - -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_minus};"
- "{TOOL_DIR}/bedGraphToBigWig {output.bg_plus} {CHROM_SIZES} {output.bw_plus};"
- "{TOOL_DIR}/bedGraphToBigWig {output.bg_minus} {CHROM_SIZES} {output.bw_minus};"
-
-rule make_scaled_bigwig:
- input:
- CHROM_SIZES,
- bam = lambda wildcards: replicate_label_to_bams[wildcards.replicate_label],
- output:
- bg_plus = temp("output/bedgraphs/scaled/plus/{replicate_label}.scaled.plus.bg"),
- bg_minus = temp("output/bedgraphs/scaled/minus/{replicate_label}.scaled.minus.bg"),
- bw_plus = "output/bigwigs/scaled/plus/{replicate_label}.scaled.plus.bw",
- bw_minus = "output/bigwigs/scaled/minus/{replicate_label}.scaled.minus.bw",
- params:
- error_file = "stderr/{replicate_label}.make_bigwig.err",
- out_file = "stdout/{replicate_label}.make_bigwig.out",
- run_time = "40:00",
- memory = "1000",
- job_name = "make_bigwig"
- benchmark: "benchmarks/bigwigs/unassigned_experiment.{replicate_label}.make_bigwig.txt"
- shell:
- "factor=$(samtools idxstats {input.bam} | awk '{{sum += $3}} END {{print 10**6 / sum}}');"
- "bedtools genomecov -scale $factor -5 -strand + -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_plus};"
- "bedtools genomecov -scale $factor -5 -strand - -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_minus};"
- "{TOOL_DIR}/bedGraphToBigWig {output.bg_plus} {CHROM_SIZES} {output.bw_plus};"
- "{TOOL_DIR}/bedGraphToBigWig {output.bg_minus} {CHROM_SIZES} {output.bw_minus};"
-
-rule uniq_repeats:
- input:
- repeatmasker = ancient(REPEAT_TABLE),
- genome = ancient(GENOME)
- output:
- sorted_bed = temp("repeats.sort.temp.bed.gz"),
- unique_repeats = REPEAT_BED
- params:
- error_file = "stderr/calc_partition_nuc.err",
- out_file = "stdout/calc_partition_nuc.out",
- run_time = "40:00",
- memory = "1000",
- job_name = "uniq_repeats_nuc"
- benchmark: "benchmarks/uniq_repeats.txt"
- shell:
- "zcat {REPEAT_TABLE} | awk -v OFS=\"\\t\" '{{print $6,$7,$8,$11 \":\" name_count[$11]++, $2, $10,$11,$12,$13}} "
- "$13 == \"L1\" || $13 == \"Alu\" {{$11 = $11 \"_AS\"; $12 = $12 \"_AS\"; $13 = $13 \"_AS\"; "
- "if($10 == \"+\") {{$10 = \"-\"}} else {{$10 = \"+\"}}; print $6,$7,$8,$11 \":\" name_count[$11]++, $2, $10,$11,$12,$13}}' | "
- "tail -n +2 | bedtools sort -i - | gzip > {output.sorted_bed}; "
- "bedtools coverage -s -d -a {output.sorted_bed} -b {output.sorted_bed} | awk -v OFS=\"\\t\" "
- "'$NF >1 {{print $1,$2+$(NF-1)-1,$2+$(NF-1),$4,$5,$6}}' | "
- "bedtools sort -i - | "
- "bedtools merge -c 4,5,6 -o distinct -s -i - | "
- "bedtools subtract -s -a {output.sorted_bed} -b - | "
- "bedtools nuc -s -fi {input.genome} -bed - | awk -v OFS=\"\\t\" 'NR > 1 {{print $1,$2,$3,$4,$5,$6,$7,$8,$9,$11}}' | "
- "gzip -c > {output.unique_repeats}"
-
-
-rule quantify_repeats:
- input:
- CHROM_SIZES,
- bam = lambda wildcards: replicate_label_to_bams[wildcards.replicate_label],
- repeats = REPEAT_BED
- output:
- counts = "output/counts/repeats/vectors/{replicate_label}.counts"
- params:
- error_file = "stderr/{replicate_label}.quantify_repeats.err",
- out_file = "stdout/{replicate_label}.quantify_repeats.out",
- run_time = "15:00",
- memory = "20000",
- job_name = "dedup_bam",
- prefix='output/bams/dedup/genome/{replicate_label}.genome.sort'
- benchmark: "benchmarks/repeats/unassigned_experiment.{replicate_label}.quantify_repeats.txt"
- shell:
- "bedtools bamtobed -i {input.bam} | awk '($1 != \"chrEBV\") && ($4 !~ \"/{UNINFORMATIVE_READ}$\")' | "
- "bedtools flank -s -l 1 -r 0 -g {CHROM_SIZES} -i - | "
- "bedtools shift -p 1 -m -1 -g {CHROM_SIZES} -i - | "
- "bedtools sort -i - | "
- "bedtools coverage -s -counts -a {input.repeats} -b - | "
- "awk 'BEGIN {{print \"{wildcards.replicate_label}\"}} {{print $NF}}' > {output.counts}"
-
-rule make_repeat_count_tables:
- input:
- unique_repeats = REPEAT_BED,
- replicate_counts = lambda wildcards: expand("output/counts/repeats/vectors/{replicate_label}.counts", replicate_label = experiment_to_replicate_labels[wildcards.experiment_label]),
- output:
- name_table = "output/counts/repeats/tables/name/{experiment_label}.tsv.gz",
- class_table = "output/counts/repeats/tables/class/{experiment_label}.tsv.gz",
- family_table = "output/counts/repeats/tables/family/{experiment_label}.tsv.gz",
- params:
- error_file = "stderr/{experiment_label}.make_repeat_count_tables.err",
- out_file = "stdout/{experiment_label}.make_repeat_count_tables.out",
- run_time = "00:15:00",
- cores = "1",
- memory = "200",
- job_name = "make_repeat_count_tables"
- benchmark: "benchmarks/counts/{experiment_label}.all_replicates.make_repeat_count_table.txt"
- shell:
- "echo \"repeat_name\" | paste - {input.replicate_counts} | sed -n '1p' | gzip > {output.name_table};"
- "echo \"repeat_class\" | paste - {input.replicate_counts} | sed -n '1p' | gzip > {output.class_table};"
- "echo \"repeat_family\" | paste - {input.replicate_counts} | sed -n '1p' | gzip > {output.family_table};"
- "paste <(zcat {input.unique_repeats} | awk -v OFS=\"\\t\" 'BEGIN {{print \"repeat_name\";}} {{print $7}}') {input.replicate_counts} | "
- "awk -v OFS=\"\\t\" 'NR > 1 {{for(i = 2; i <= NF; i++) {{tabulation[$1][i] += $i}} }} END {{for(name in tabulation) {{ printf name; for(i = 2; i <= NF; i++) {{printf \"\\t\" tabulation[name][i]}} print \"\";}} }}' | sort -k 1,1 | gzip >> {output.name_table};"
- "paste <(zcat {input.unique_repeats} | awk -v OFS=\"\\t\" 'BEGIN {{print \"repeat_class\";}} {{print $8}}') {input.replicate_counts} | "
- "awk -v OFS=\"\\t\" 'NR > 1 {{for(i = 2; i <= NF; i++) {{tabulation[$1][i] += $i}} }} END {{for(name in tabulation) {{ printf name; for(i = 2; i <= NF; i++) {{printf \"\\t\" tabulation[name][i]}} print \"\";}} }}' | sort -k 1,1 | gzip >> {output.class_table};"
- "paste <(zcat {input.unique_repeats} | awk -v OFS=\"\\t\" 'BEGIN {{print \"repeat_family\";}} {{print $9}}') {input.replicate_counts} | "
- "awk -v OFS=\"\\t\" 'NR > 1 {{for(i = 2; i <= NF; i++) {{tabulation[$1][i] += $i}} }} END {{for(name in tabulation) {{ printf name; for(i = 2; i <= NF; i++) {{printf \"\\t\" tabulation[name][i]}} print \"\";}} }}' | sort -k 1,1 | gzip >> {output.family_table};"
-
-rule fit_clip_betabinomial_re_model:
- input:
- table = "output/counts/repeats/tables/name/{experiment_label}.tsv.gz",
- output:
- coef = "output/clip_model_coef_re/{experiment_label}.{clip_replicate_label}.tsv",
- # plot = lambda wildcards: expand("output/figures/clip_distributions/{{experiment_label}}.{{clip_replicate_label}}.{other_label}.clip_distribution.pdf", other_label = experiment_to_input_replicate_labels[wildcards.experiment_label][wildcards.Input_replicate_label])
- params:
- error_file = "stderr/{experiment_label}.{clip_replicate_label}.fit_clip_betabinomial_re_model.err",
- out_file = "stdout/{experiment_label}.{clip_replicate_label}.fit_clip_betabinomial_re_model.out",
- run_time = "10:00",
- memory = "2000",
- job_name = "fit_clip_betabinomial_re_model"
- benchmark: "benchmarks/fit_clip_betabinomial_re_model/{experiment_label}.{clip_replicate_label}.fit_clip.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/fit_clip_betabinom_re.R {input.table} {wildcards.experiment_label} {wildcards.clip_replicate_label}"
-
-rule fit_input_betabinomial_re_model:
- input:
- table = "output/counts/repeats/tables/name/{experiment_label}.tsv.gz",
- output:
- coef = "output/input_model_coef_re/{experiment_label}.{input_replicate_label}.tsv",
- # plot = lambda wildcards: expand("output/figures/input_distributions/{{experiment_label}}.{{input_replicate_label}}.{other_label}.input_distribution.pdf", other_label = experiment_to_input_replicate_labels[wildcards.experiment_label][wildcards.Input_replicate_label])
- params:
- error_file = "stderr/{experiment_label}.{input_replicate_label}.fit_input_betabinomial_re_model.err",
- out_file = "stdout/{experiment_label}.{input_replicate_label}.fit_input_betabinomial_re_model.out",
- run_time = "10:00",
- memory = "2000",
- job_name = "fit_input_betabinomial_re_model"
- benchmark: "benchmarks/fit_input_betabinomial_re_model/{experiment_label}.{input_replicate_label}.fit_input.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/fit_input_betabinom_re.R {input.table} {wildcards.experiment_label} {wildcards.input_replicate_label}"
-
-rule call_enriched_re:
- input:
- table = "output/counts/repeats/tables/name/{experiment_label}.tsv.gz",
- replicate = lambda wildcards: "output/counts/repeats/vectors/" + re.sub("IP_\d$","IP_2",wildcards.clip_replicate_label) + ".counts",
- repeats = REPEAT_BED,
- parameters = lambda wildcards: "output/" + OVERDISPERSION_MODE + "_model_coef_re/{experiment_label}." + overdispersion_replicate_lookup[wildcards.clip_replicate_label] + ".tsv",
- output:
- "output/figures/clip_scatter_re/{experiment_label}.{clip_replicate_label}.clip_test_distribution.pdf",
- "output/enriched_re/{experiment_label}.{clip_replicate_label}.enriched_re.tsv.gz"
- params:
- input_replicate_label = lambda wildcards: clip_to_input_replicate_label[wildcards.clip_replicate_label],
- error_file = "stderr/{experiment_label}.{clip_replicate_label}.call_enriched_re.err",
- out_file = "stdout/{experiment_label}.{clip_replicate_label}.call_enriched_re.out",
- run_time = "00:25:00",
- memory = "3000",
- job_name = "call_enriched_re"
- benchmark: "benchmarks/call_enriched_re/{experiment_label}.{clip_replicate_label}.call_enriched_re.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/call_enriched_re.R {input.table} {input.repeats} {input.parameters} {params.input_replicate_label} {wildcards.clip_replicate_label} {wildcards.experiment_label}.{wildcards.clip_replicate_label}"
-
-rule find_reproducible_enriched_re:
- input:
- windows = lambda wildcards: expand("output/enriched_re/{{experiment_label}}.{clip_replicate_label}.enriched_re.tsv.gz", clip_replicate_label = experiment_to_clip_replicate_labels[wildcards.experiment_label])
- output:
- reproducible_windows = "output/reproducible_enriched_re/{experiment_label}.reproducible_enriched_re.tsv.gz",
- params:
- error_file = "stderr/{experiment_label}.find_reproducible_enriched_re.err",
- out_file = "stdout/{experiment_label}.find_reproducible_enriched_re.out",
- run_time = "5:00",
- memory = "1000",
- job_name = "find_reproducible_enriched_re"
- benchmark: "benchmarks/find_reproducible_enriched_re/{experiment_label}.all_replicates.reproducible.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/identify_reproducible_re.R output/enriched_re/ {wildcards.experiment_label}"
-
-rule partition_bam_reads:
- input:
- CHROM_SIZES,
- bam = lambda wildcards: replicate_label_to_bams[wildcards.replicate_label],
- region_partition = PARTITION,
- output:
- counts = "output/counts/genome/vectors/{replicate_label}.counts",
- params:
- error_file = "stderr/{replicate_label}.partition_bam_reads.err",
- out_file = "stdout/{replicate_label}.partition_bam_reads.out",
- run_time = "20:00",
- cores = "1",
- memory = "10000",
- job_name = "partition_bam_reads"
- benchmark: "benchmarks/counts/unassigned_experiment.{replicate_label}.partition_bam_reads.txt"
- shell:
- "bedtools bamtobed -i {input.bam} | awk '($1 != \"chrEBV\") && ($4 !~ \"/{UNINFORMATIVE_READ}$\")' | "
- "bedtools flank -s -l 1 -r 0 -g {CHROM_SIZES} -i - | "
- "bedtools shift -p 1 -m -1 -g {CHROM_SIZES} -i - | "
- "bedtools sort -i - | "
- "bedtools coverage -counts -s -a {input.region_partition} -b - | cut -f 7 | "
- "awk 'BEGIN {{print \"{wildcards.replicate_label}\"}} {{print}}' > {output.counts};"
-
-rule calc_partition_nuc:
- input:
- partition = PARTITION,
- genome = GENOME
- output:
- nuc = PARTITION.replace(".bed", ".nuc")
- params:
- error_file = "stderr/calc_partition_nuc.err",
- out_file = "stdout/calc_partition_nuc.out",
- run_time = "00:10:00",
- memory = "1000",
- job_name = "calc_partition_nuc"
- benchmark: "benchmarks/partition_nuc.txt"
- shell:
- "bedtools nuc -s -fi {input.genome} -bed {input.partition} | gzip -c > {output.nuc}"
-
-rule make_genome_count_table:
- input:
- partition = rules.calc_partition_nuc.output.nuc,
- replicate_counts = lambda wildcards: expand("output/counts/genome/vectors/{replicate_label}.counts", replicate_label = experiment_to_replicate_labels[wildcards.experiment_label]),
- output:
- count_table = "output/counts/genome/tables/{experiment_label}.tsv.gz",
- params:
- error_file = "stderr/{experiment_label}.make_count_table.err",
- out_file = "stdout/{experiment_label}.make_count_table.out",
- run_time = "00:05:00",
- cores = "1",
- memory = "200",
- job_name = "make_genome_count_table"
- benchmark: "benchmarks/counts/{experiment_label}.all_replicates.make_genome_count_table.txt"
- shell:
- "paste <(zcat {input.partition} | awk -v OFS=\"\\t\" 'BEGIN {{print \"chr\\tstart\\tend\\tname\\tscore\\tstrand\\tgc\"}} NR > 1 {{print $1,$2,$3,$4,$5,$6,$8}}' ) {input.replicate_counts} | gzip -c > {output.count_table};"
-
-rule fit_input_betabinomial_model:
- input:
- table = "output/counts/genome/tables/{experiment_label}.tsv.gz"
- output:
- coef = "output/input_model_coef/{experiment_label}.{input_replicate_label}.tsv",
- # plot = lambda wildcards: expand("output/figures/input_distributions/{{experiment_label}}.{{input_replicate_label}}.{other_label}.input_distribution.pdf", other_label = experiment_to_input_replicate_labels[wildcards.experiment_label][wildcards.Input_replicate_label])
- params:
- error_file = "stderr/{experiment_label}.{input_replicate_label}.fit_input_betabinom.err",
- out_file = "stdout/{experiment_label}.{input_replicate_label}.fit_input_betabinom.out",
- run_time = "1:00:00",
- memory = "10000",
- job_name = "fit_input_betabinomial_model"
- benchmark: "benchmarks/betabinomial/{experiment_label}.{input_replicate_label}.fit_input.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/fit_input_betabinom.R {input.table} {wildcards.experiment_label} {wildcards.input_replicate_label}"
-
-rule fit_clip_betabinomial_model:
- input:
- table = "output/counts/genome/tables/{experiment_label}.tsv.gz"
- output:
- coef = "output/clip_model_coef/{experiment_label}.{clip_replicate_label}.tsv",
- # plot = lambda wildcards: expand("output/figures/clip_distributions/{{experiment_label}}.{{clip_replicate_label}}.{other_label}.clip_distribution.pdf", other_label = experiment_to_input_replicate_labels[wildcards.experiment_label][wildcards.Input_replicate_label])
- params:
- error_file = "stderr/{experiment_label}.{clip_replicate_label}.fit_clip_betabinomial_model.err",
- out_file = "stdout/{experiment_label}.{clip_replicate_label}.fit_clip_betabinomial_model.out",
- run_time = "1:00:00",
- memory = "1000",
- job_name = "fit_clip_betabinomial_model"
- benchmark: "benchmarks/fit_clip_betabinomial_model/{experiment_label}.{clip_replicate_label}.fit_clip.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/fit_clip_betabinom.R {input.table} {wildcards.experiment_label} {wildcards.clip_replicate_label}"
-
-rule call_enriched_windows:
- input:
- feature_annotations = ancient(FEATURE_ANNOTATIONS),
- accession_rankings = ancient(ACCESSION_RANKINGS),
- replicate = lambda wildcards: "output/counts/genome/vectors/" + re.sub("IP_\d$","IP_2",wildcards.clip_replicate_label) + ".counts",
- table = "output/counts/genome/tables/{experiment_label}.tsv.gz",
- parameters = lambda wildcards: "output/" + OVERDISPERSION_MODE + "_model_coef/{experiment_label}." + overdispersion_replicate_lookup[wildcards.clip_replicate_label] + ".tsv",
- # parameters = lambda wildcards: "output/clip_model_coef/{experiment_label}.{wildcards.clip_replicate_label}.tsv",
- output:
- "output/threshold_scan/{experiment_label}.{clip_replicate_label}.threshold_data.tsv",
- "output/tested_windows/{experiment_label}.{clip_replicate_label}.tested_windows.tsv.gz",
- "output/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_windows.tsv.gz",
- "output/enrichment_summaries/{experiment_label}.{clip_replicate_label}.enriched_window_feature_summary.tsv",
- "output/enrichment_summaries/{experiment_label}.{clip_replicate_label}.enriched_window_transcript_summary.tsv",
- "output/enrichment_summaries/{experiment_label}.{clip_replicate_label}.enriched_window_gene_summary.tsv",
- "output/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_fractions_feature_data.tsv",
- "output/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds_feature_data.tsv",
- "output/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds_transcript_data.tsv",
- "output/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds_feature_gc_data.tsv",
- "output/figures/threshold_scan/{experiment_label}.{clip_replicate_label}.threshold_scan.pdf",
- "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_coverage.pdf",
- "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_rates.pdf",
- "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_counts.linear.pdf",
- "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_counts.log10.pdf",
- "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_odds.feature.pdf",
- "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_odds.all_transcript_types.pdf",
- "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_odds.select_transcript_types.pdf",
- "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_counts.per_gene_feature.pdf",
- "output/figures/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_fractions.feature.pdf",
- "output/figures/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds.feature.pdf",
- "output/figures/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds.all_transcript_types.pdf",
- "output/figures/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds.feature_gc.pdf"
- params:
- input_replicate_label = lambda wildcards: clip_to_input_replicate_label[wildcards.clip_replicate_label],
- error_file = "stderr/{experiment_label}.{clip_replicate_label}.call_enriched_windows.err",
- out_file = "stdout/{experiment_label}.{clip_replicate_label}.call_enriched_windows.out",
- run_time = "00:45:00",
- memory = "6000",
- job_name = "call_enriched_windows"
- benchmark: "benchmarks/call_enriched_windows/{experiment_label}.{clip_replicate_label}.call_enriched_windows.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/call_enriched_windows.R {input.table} {input.accession_rankings} {input.feature_annotations} {input.parameters} {params.input_replicate_label} {wildcards.clip_replicate_label} {wildcards.experiment_label}.{wildcards.clip_replicate_label}"
-
-rule check_window_concordance:
- input:
- windows = lambda wildcards: expand("output/tested_windows/{{experiment_label}}.{clip_replicate_label}.tested_windows.tsv.gz", clip_replicate_label = experiment_to_clip_replicate_labels[wildcards.experiment_label])
- output:
- "output/figures/enrichment_reproducibility/{experiment_label}.enrichment_reproducibility.pdf",
- "output/enrichment_reproducibility/{experiment_label}.enrichment_reproducibility.tsv"
- params:
- error_file = "stderr/{experiment_label}.check_window_concordance.err",
- out_file = "stdout/{experiment_label}.check_window_concordance.out",
- run_time = "0:15:00",
- memory = "1000",
- job_name = "check_window_concordance"
- benchmark: "benchmarks/check_window_concordance/{experiment_label}.all_replicates.concordance.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/check_window_concordance.R output/tested_windows {wildcards.experiment_label} " + (BLACKLIST if BLACKLIST is not None else "")
-
-rule find_reproducible_enriched_windows:
- input:
- windows = lambda wildcards: expand("output/enriched_windows/{{experiment_label}}.{clip_replicate_label}.enriched_windows.tsv.gz", clip_replicate_label = experiment_to_clip_replicate_labels[wildcards.experiment_label])
- output:
- reproducible_windows = "output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz",
- linear_bar = "output/figures/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_window_counts.linear.pdf",
- log_bar = "output/figures/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_window_counts.log10.pdf"
- params:
- error_file = "stderr/{experiment_label}.find_reproducible_enriched_windows.err",
- out_file = "stdout/{experiment_label}.find_reproducible_enriched_windows.out",
- run_time = "5:00",
- memory = "2000",
- job_name = "find_reproducible_enriched_windows"
- benchmark: "benchmarks/find_reproducible_enriched_windows/{experiment_label}.all_replicates.reproducible.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/identify_reproducible_windows.R output/enriched_windows/ {wildcards.experiment_label} " + (BLACKLIST if BLACKLIST is not None else "")
-
-rule sample_background_windows_by_region:
- input:
- enriched_windows = "output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz",
- all_windows = ancient(FEATURE_ANNOTATIONS),
- output:
- variable_windows = "output/homer/region_matched_background/variable/{experiment_label}.sampled_variable_windows.bed.gz",
- fixed_windows = "output/homer/region_matched_background/fixed/{experiment_label}.sampled_fixed_windows.bed.gz"
- params:
- error_file = "stderr/{experiment_label}.sample_background_windows_by_region.err",
- out_file = "stdout/{experiment_label}.sample_background_windows_by_region.out",
- run_time = "10:00",
- memory = "3000",
- job_name = "sample_background_windows"
- benchmark: "benchmarks/sample_background_windows_by_region/{experiment_label}.sample_background_windows_by_region.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/sample_matched_background_by_region.R {input.enriched_windows} {input.all_windows} 75 output/homer/region_matched_background {wildcards.experiment_label};"
-
-rule get_nt_coverage:
- input:
- windows = "output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz",
- clip_bams = lambda wildcards: [replicate_label_to_bams[clip_replicate_label] for clip_replicate_label in experiment_to_clip_replicate_labels[wildcards.experiment_label]],
- input_bams = lambda wildcards: [replicate_label_to_bams[input_replicate_label] for input_replicate_label in experiment_to_input_replicate_labels[wildcards.experiment_label]],
- output:
- nt_census = temp("output/finemapping/nt_coverage/{experiment_label}.nt_census.bed"),
- nt_input_counts = temp("output/finemapping/nt_coverage/{experiment_label}.nt_coverage.input.counts"),
- nt_clip_counts = temp("output/finemapping/nt_coverage/{experiment_label}.nt_coverage.clip.counts"),
- nt_coverage = "output/finemapping/nt_coverage/{experiment_label}.nt_coverage.bed"
- params:
- error_file = "stderr/{experiment_label}.get_nt_coverage.err",
- out_file = "stdout/{experiment_label}.get_nt_coverage.out",
- run_time = "1:00:00",
- memory = "15000",
- job_name = "get_nt_coverage"
- benchmark: "benchmarks/get_nt_coverage/{experiment_label}.all_replicates.reproducible.txt"
- shell:
- "zcat {input.windows} | tail -n +2 | sort -k1,1 -k2,2n | awk -v OFS=\"\t\" '{{print $1, $2 -37, $3+37,$4,$5,$6}}' | "
- "bedtools merge -i - -s -c 6 -o distinct | awk -v OFS=\"\t\" '{{for(i=$2;i< $3;i++) {{print $1,i,i+1,\"MW:\" NR \":\" i - $2,0,$4, NR}} }}' > {output.nt_census}; "
- "samtools cat {input.input_bams} | bedtools intersect -s -wa -a - -b {output.nt_census} | "
- "bedtools bamtobed -i - | awk '($1 != \"chrEBV\") && ($4 !~ \"/{UNINFORMATIVE_READ}$\")' | "
- "bedtools flank -s -l 1 -r 0 -g {CHROM_SIZES} -i - | "
- "bedtools shift -p 1 -m -1 -g {CHROM_SIZES} -i - | "
- "bedtools sort -i - | "
- "bedtools coverage -counts -s -a {output.nt_census} -b - | awk '{{print $NF}}' > {output.nt_input_counts};"
- "samtools cat {input.clip_bams} | bedtools intersect -s -wa -a - -b {output.nt_census} | "
- "bedtools bamtobed -i - | awk '($1 != \"chrEBV\") && ($4 !~ \"/{UNINFORMATIVE_READ}$\")' | "
- "bedtools flank -s -l 1 -r 0 -g {CHROM_SIZES} -i - | "
- "bedtools shift -p 1 -m -1 -g {CHROM_SIZES} -i - | "
- "bedtools sort -i - | "
- "bedtools coverage -counts -s -a {output.nt_census} -b - | awk '{{print $NF}}' > {output.nt_clip_counts};"
- "paste {output.nt_census} {output.nt_input_counts} {output.nt_clip_counts} > {output.nt_coverage}"
-
-rule finemap_windows:
- input:
- nt_coverage = "output/finemapping/nt_coverage/{experiment_label}.nt_coverage.bed",
- output:
- finemapped_windows = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz"
- params:
- error_file = "stderr/{experiment_label}.finemap_windows.err",
- out_file = "stdout/{experiment_label}.finemap_windows.out",
- run_time = "1:00:00",
- memory = "10000",
- job_name = "finemap_windows"
- benchmark: "benchmarks/finemap_windows/{experiment_label}.all_replicates.reproducible.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/finemap_enriched_windows.R {input.nt_coverage} output/finemapping/mapped_sites/ {wildcards.experiment_label}"
-
-rule run_homer:
- input:
- finemapped_windows = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz",
- background = "output/homer/region_matched_background/fixed/{experiment_label}.sampled_fixed_windows.bed.gz",
- genome = GENOME
- output:
- report = "output/homer/finemapped_results/{experiment_label}/homerResults.html"
- params:
- error_file = "stderr/{experiment_label}.run_homer.err",
- out_file = "stdout/{experiment_label}.run_homer.out",
- run_time = "40:00",
- memory = "2000",
- job_name = "run_homer"
- benchmark: "benchmarks/run_homer/{experiment_label}.all_replicates.reproducible.txt"
- shell:
- "findMotifsGenome.pl <(less {input.finemapped_windows} | awk -v OFS=\"\t\" '{{print $4 \":\"$9,$1,$2+1,$3,$6}}') "
- "{input.genome} output/homer/finemapped_results/{wildcards.experiment_label} -preparsedDir output/homer/preparsed -size given -rna -nofacts -S 20 -len 5,6,7,8,9 -nlen 1 "
- "-bg <(zcat {input.background} | awk -v OFS=\"\t\" '{{print $4,$1,$2+1,$3,$6}}') "
-
-rule consult_encode_reference:
- input:
- enriched_windows = lambda wildcards: expand("output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz", experiment_label = experiment_labels),
- enriched_re = lambda wildcards: expand("output/reproducible_enriched_re/{experiment_label}.reproducible_enriched_re.tsv.gz", experiment_label = experiment_labels),
- encode_references = lambda wildcards: expand(TOOL_DIR + "/{reference}.reference.tsv", reference = ["encode3_feature_summary", "encode3_eclip_enrichment", "encode3_class_assignment"])
- output:
- tsne_coordinates = "output/tsne/skipper.tsne_query.tsv",
- tsne_plot = "output/figures/tsne/skipper.tsne_query.pdf"
- params:
- error_file = "stderr/skipper.consult_encode_reference.err",
- out_file = "stdout/skipper.consult_encode_reference.out",
- run_time = "10:00",
- memory = "1000",
- job_name = "consult_encode_reference"
- benchmark: "benchmarks/consult_encode_reference/skipper.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/consult_encode_reference.R output/reproducible_enriched_windows output/reproducible_enriched_re {TOOL_DIR} skipper "
-
-rule consult_term_reference:
- input:
- enriched_windows = "output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz",
- gene_sets = GENE_SETS,
- gene_set_reference = GENE_SET_REFERENCE,
- gene_set_distance = GENE_SET_DISTANCE
- output:
- enrichment_results = "output/gene_sets/{experiment_label}.enriched_terms.tsv.gz",
- enrichment_plot = "output/figures/gene_sets/{experiment_label}.clustered_top_terms.pdf"
- params:
- error_file = "stderr/{experiment_label}.consult_term_reference.err",
- out_file = "stdout/{experiment_label}.consult_term_reference.out",
- run_time = "15:00",
- memory = "1000",
- job_name = "consult_term_reference"
- benchmark: "benchmarks/consult_term_reference/{experiment_label}.all_replicates.reproducible.txt"
- shell:
- "{R_EXE} --vanilla {TOOL_DIR}/consult_term_reference.R {input.enriched_windows} {input.gene_sets} {input.gene_set_reference} {input.gene_set_distance} {wildcards.experiment_label} "
+ # Quality control
+ # expand("output/multiqc/{experiment_label}/multiqc_data", experiment_label = manifest.Experiment),
+ # expand("output/multiqc/{experiment_label}/multiqc_plots", experiment_label = manifest.Experiment),
+ # expand("output/multiqc/{experiment_label}/multiqc_report.html", experiment_label = manifest.Experiment),
+ expand("output/counts/genome/megatables/{genome_type}.tsv.gz", genome_type = ["feature_type_top","transcript_type_top"]),
+ expand("output/counts/repeats/megatables/{repeat_type}.tsv.gz", repeat_type = ['name', 'class', 'family']),
+ "output/QC/unique_fragments.csv",
+ # expand("output/ml/gkmsvm/{experiment_label}.cvpred.txt", experiment_label = manifest.Experiment),
+ # "output/ml/gkmsvm/AUPRC.txt",
+ # "variants_done.txt",
+ # expand("output/joined_reproducible_windows/l2or.{type}.csv",
+ # type =['EXON_SMALL','INTRON', 'SS5_ADJ,SS5_PROX,SS3_ADJ,SS3_PROX,SSB_ADJ,SSB_PROX',
+ # 'UTR5,CDS_START,CDS,CDS_STOP,CDS_SOLITARY,UTR3','EXON_PSEUDO','EXON_LNCRNA',
+ # 'EXON_MRNA']
+ # ),
+ # "output/joined_reproducible_re/l2or.csv",
+ # "output/joined_reproducible_windows/all.csv",
+ # expand("output/conservation/UKBB_DR/{experiment_label}.csv", experiment_label = manifest.Experiment)
+ expand("output/ml/rbpnet_model/{experiment_label}/valid/test_data_metric.csv",
+ experiment_label = manifest.Experiment),
+ expand("output/ml/rbpnet_model/{experiment_label}/motif_done",
+ experiment_label = manifest.Experiment),
+ expand("output/variants/gnomAD_roulette/{experiment_label}.total.csv",
+ experiment_label = manifest.Experiment),
+ expand("output/variants/clinvar/{experiment_label}.vep.tsv",
+ experiment_label = manifest.Experiment),
+ expand("output/variant_analysis/{experiment_label}.clinvar_variants.csv",
+ experiment_label = manifest.Experiment)
+
+module se_preprocess:
+ snakefile:
+ "rules/se_preprocess.smk"
+ config:
+ config
+
+module pe_preprocess:
+ snakefile:
+ "rules/pe_preprocess.smk"
+ config:
+ config
+
+module qc:
+ snakefile:
+ "rules/qc.smk"
+ config: config
+
+module genome:
+ snakefile:
+ "rules/genome_windows.smk"
+ config: config
+
+module repeat:
+ snakefile:
+ "rules/repeat.smk"
+ config: config
+
+module finemap:
+ snakefile:
+ "rules/finemap.smk"
+ config: config
+
+module analysis:
+ snakefile:
+ "rules/analysis.smk"
+ config: config
+
+module meta_analysis:
+ snakefile:
+ "rules/meta_analysis.smk"
+ config:
+ config
+module bigwig:
+ snakefile:
+ "rules/bigwig.smk"
+ config:
+ config
+module prep_ml:
+ snakefile:
+ "rules/prep_ml.smk"
+ config:
+ config
+
+module rbpnet:
+ snakefile:
+ "rules/train_rbpnet.smk"
+ config:
+ config
+
+# module variants:
+# snakefile:
+# "rules/variants.smk"
+# config:
+# config
+
+module variants_rbpnet:
+ snakefile:
+ "rules/variants_rbpnet.smk"
+ config:
+ config
+
+
+if config['protocol']=='ENCODE4':
+ use rule * from se_preprocess as se_*
+else:
+ use rule * from pe_preprocess as pe_*
+
+use rule * from bigwig
+use rule * from qc
+use rule * from genome
+use rule * from repeat
+use rule * from finemap
+use rule * from analysis
+use rule * from meta_analysis
+use rule * from prep_ml as ml_*
+# use rule * from variants as variants_*
+use rule * from rbpnet as rbpnet_*
+use rule * from variants_rbpnet as rbpnet_variants_*
diff --git a/Skipper_ark.py b/Skipper_ark.py
new file mode 100644
index 0000000..a8a8cdf
--- /dev/null
+++ b/Skipper_ark.py
@@ -0,0 +1,272 @@
+import pandas as pd
+from functools import reduce
+import re
+import os
+import sys
+import glob
+from time import sleep
+from pathlib import Path
+import warnings
+
+# example command
+"""
+# ENCODE 4
+snakemake -kps Skipper.py \
+ -j 30 \
+ --cluster "qsub -e {params.error_file} -o {params.out_file} -l walltime={params.run_time} -l nodes=1:ppn={threads} -q home-yeo" \
+ --configfile /home/hsher/projects/skipper/encore_configs/Skipper_config_small_test.yaml \
+ --conda-prefix /home/hsher/snakeconda \
+ --use-conda \
+ --use-singularity \
+ --singularity-prefix /home/hsher/scratch/singularity \
+ --singularity-args "--bind /oasis --bind /projects --bind /scratch" \
+ -n
+
+# ENCODE 3
+snakemake -kps Skipper.py \
+ -j 30 \
+ --cluster "qsub -e {params.error_file} -o {params.out_file} -l walltime={params.run_time} -l nodes=1:ppn={threads} -q home-yeo" \
+ --configfile /home/hsher/projects/skipper/encode_configs/Skipper_pe_small_test.yaml \
+ --conda-prefix /home/hsher/snakeconda \
+ --use-conda \
+ --use-singularity \
+ --singularity-prefix /home/hsher/scratch/singularity \
+ --singularity-args "--bind /oasis --bind /projects --bind /scratch" \
+ output/ml/gkmsvm/RBFOX2_HepG2_ENCSR987FTF.cvpred.txt
+
+
+"""
+
+locals().update(config)
+
+workdir: config['WORKDIR']
+
+if not os.path.exists("stderr"): os.makedirs("stderr")
+if not os.path.exists("stdout"): os.makedirs("stdout")
+
+
+if OVERDISPERSION_MODE not in ["clip","input"]:
+ raise Exception("Overdispersion must be calculated using 'clip' or 'input' samples")
+
+# read and cleanup manifest
+manifest = pd.read_csv(MANIFEST, comment = "#", index_col = False).dropna(subset=['Experiment','Sample'])
+manifest["CLIP_replicate"] = pd.to_numeric(manifest.CLIP_replicate, downcast="integer")
+manifest["Input_replicate"] = pd.to_numeric(manifest.Input_replicate, downcast="integer")
+
+for col in manifest.columns[manifest.columns.str.contains('_fastq') | manifest.columns.str.contains('_adapter')]:
+ manifest[col] = manifest[col].str.strip()
+
+# check numbers are correct
+try:
+ if min(manifest.groupby("Experiment")["CLIP_fastq"].agg(lambda x: len(set(x)))) < 2:
+ sys.stderr.write("WARNING: NONZERO EXPERIMENTS HAVE ONLY ONE CLIP REPLICATE.\nPIPELINE MUST HALT AFTER GENERATING RAW COUNTS\nThis usually means your manifest is incorrectly formatted\n")
+ sleep(5)
+except:
+ if min(manifest.groupby("Experiment")["CLIP_fastq_1"].agg(lambda x: len(set(x)))) < 2:
+ sys.stderr.write("WARNING: NONZERO EXPERIMENTS HAVE ONLY ONE CLIP REPLICATE.\nPIPELINE MUST HALT AFTER GENERATING RAW COUNTS\nThis usually means your manifest is incorrectly formatted\n")
+ sleep(5)
+
+if max(manifest.groupby("Sample")["Input_replicate"].agg(lambda x: min(x))) > 1:
+ raise Exception("Input replicates for samples in manifest do not increment from 1 as expected")
+
+if max(manifest.groupby("Sample")["CLIP_replicate"].agg(lambda x: min(x))) > 1:
+ raise Exception("CLIP replicates for samples in manifest do not increment from 1 as expected")
+
+# create label for IN and CLIP:
+# Sample = DEK_HepG2_4020, replicate_label: DEK_HepG2_4020_IN_1 and DEK_HepG2_4020_IP_1
+manifest["Input_replicate_label"] = [(str(sample) + "_IN_" + str(replicate)).replace(" ","") for replicate, sample in zip(manifest.Input_replicate.tolist(),manifest.Sample.tolist())]
+manifest["CLIP_replicate_label"] = [(str(sample) + "_IP_" + str(replicate)).replace(" ","") for replicate, sample in zip(manifest.CLIP_replicate.tolist(),manifest.Sample.tolist())]
+
+input_replicates = manifest.loc[:,manifest.columns.isin(["Input_replicate_label","Input_fastq","Input_fastq_1", "Input_fastq_2","Input_bam","Input_adapter","Input_adapter_1","Input_adapter_2"])].drop_duplicates()
+clip_replicates = manifest.loc[:,manifest.columns.isin(["CLIP_replicate_label","CLIP_fastq","CLIP_fastq_1","CLIP_fastq_2","CLIP_bam","CLIP_adapter","CLIP_adapter_1","CLIP_adapter_2"])].drop_duplicates()
+
+
+
+if len(input_replicates) != len(input_replicates[["Input_replicate_label"]].drop_duplicates()) or \
+ len(clip_replicates) != len(clip_replicates[["CLIP_replicate_label"]].drop_duplicates()):
+ raise Exception("Manifest files are not consistent across replicates")
+
+input_replicate_labels = input_replicates.Input_replicate_label.tolist()
+clip_replicate_labels = clip_replicates.CLIP_replicate_label.tolist()
+replicate_labels = pd.Series(input_replicate_labels + clip_replicate_labels)
+config['replicate_labels']= replicate_labels
+
+# if all(bam in manifest.columns.tolist() for bam in ["Input_bam", "CLIP_bam"]):
+# replicate_label_to_bams = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_bam.tolist() + clip_replicates.CLIP_bam.tolist()))
+# else:
+
+# FASTQ and ADAPTOR
+if "Input_fastq" in manifest.columns and config['protocol']=='ENCODE4':
+ config['replicate_label_to_fastqs'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_fastq.tolist() + clip_replicates.CLIP_fastq.tolist()))
+ config['replicate_label_to_adapter'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_adapter.tolist() + clip_replicates.CLIP_adapter.tolist()))
+elif config['protocol']=='ENCODE3':
+ config['replicate_label_to_fastq_1'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_fastq_1.tolist() + clip_replicates.CLIP_fastq_1.tolist()))
+ config['replicate_label_to_fastq_2'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_fastq_2.tolist() + clip_replicates.CLIP_fastq_2.tolist()))
+ config['replicate_label_to_adapter_1'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_adapter_1.tolist() + clip_replicates.CLIP_adapter_1.tolist()))
+ config['replicate_label_to_adapter_2'] = dict(zip(input_replicate_labels + clip_replicate_labels, input_replicates.Input_adapter_2.tolist() + clip_replicates.CLIP_adapter_2.tolist()))
+else:
+ raise Exception("protocol does not fit in ENCODE3 or ENCODE4")
+
+# BAMs
+if config['protocol']=='ENCODE4':
+ config['replicate_label_to_bams'] = dict(zip(input_replicate_labels + clip_replicate_labels, ["output/bams/dedup/genome/" + replicate_label + ".genome.Aligned.sort.dedup.bam" for replicate_label in input_replicate_labels + clip_replicate_labels] ))
+elif config['protocol']=='ENCODE3':
+ config['replicate_label_to_bams'] = dict(zip(input_replicate_labels + clip_replicate_labels, [f"output/bams/dedup/genome_R{INFORMATIVE_READ}/" + replicate_label + f".genome.Aligned.sort.dedup.R{INFORMATIVE_READ}.bam" for replicate_label in input_replicate_labels + clip_replicate_labels] ))
+else:
+ raise Exception("protocol does not fit in ENCODE3 or ENCODE4")
+
+# EXPERIMENT LABELS
+config['experiment_labels'] = pd.Series(manifest.Experiment.drop_duplicates().tolist())
+experiment_data = manifest.groupby("Experiment").agg({"CLIP_replicate_label": list, "Input_replicate_label" : list})
+
+
+# OVERDISPERSION and BACKGROUND PAIRING
+config['overdispersion_replicate_lookup'] = dict(zip(manifest.CLIP_replicate_label.tolist(), manifest.Input_replicate_label.tolist() if OVERDISPERSION_MODE == "input" else manifest.CLIP_replicate_label.tolist()))
+config['clip_to_input_replicate_label'] = dict(zip(manifest.CLIP_replicate_label.tolist(), manifest.Input_replicate_label.tolist()))
+config['experiment_to_replicate_labels'] = dict(zip(experiment_data.index.tolist(), [reduce(lambda agg, x: agg if x in agg else agg + [x], inputs, []) + clips for inputs, clips in zip(experiment_data.Input_replicate_label, experiment_data.CLIP_replicate_label)]))
+config['experiment_to_clip_replicate_labels'] = dict(zip(experiment_data.index.tolist(), experiment_data.CLIP_replicate_label))
+
+experiment_to_input_replicate_labels = {}
+for experiment_label, label_list in zip(experiment_data.index, experiment_data.Input_replicate_label):
+ experiment_to_input_replicate_labels[experiment_label] = {}
+ for entry in label_list:
+ replicates = set()
+ for other_entry in label_list:
+ if other_entry != entry:
+ replicates.add(other_entry)
+ experiment_to_input_replicate_labels[experiment_label].update({entry : list(replicates)})
+config['experiment_to_input_replicate_labels']=experiment_to_input_replicate_labels
+
+# Fool-proof Detect misalignment for GFF and PARTITION
+if Path(GFF).name.replace('.gff3.gz', '') != Path(FEATURE_ANNOTATIONS).name.replace('.tiled_partition.features.tsv.gz', ''):
+ warnings.warn(f'''Detected Name Mismatch in GFF and FEATURE ANNOTATIONS:
+ FEATURE_ANNOTATIONS={FEATURE_ANNOTATIONS}
+ GFF={GFF}
+ Check if they are the same cell line
+ ''')
+
+config['manifest'] = manifest
+
+
+rule all:
+ input:
+ # expand("output/fastqc/initial/{replicate_label}_fastqc.html", replicate_label = replicate_labels
+ # ) if config['protocol']=='ENCODE4' else ,
+ # expand("output/fastqc/processed/{replicate_label}.trimmed.umi_fastqc.html", replicate_label = replicate_labels),
+ expand("output/bams/dedup/genome/{replicate_label}.genome.Aligned.sort.dedup.bam", replicate_label = replicate_labels),
+ expand("output/bams/dedup/genome/{replicate_label}.genome.Aligned.sort.dedup.bam.bai", replicate_label = replicate_labels),
+ expand("output/bigwigs/unscaled/plus/{replicate_label}.unscaled.plus.bw", replicate_label = replicate_labels),
+ expand("output/bigwigs/scaled/plus/{replicate_label}.scaled.plus.bw", replicate_label = replicate_labels),
+ expand("output/counts/repeats/vectors/{replicate_label}.counts", replicate_label = replicate_labels),
+ expand("output/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_windows.tsv.gz", zip, experiment_label = manifest.Experiment, clip_replicate_label = manifest.CLIP_replicate_label),
+ expand("output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz", experiment_label = manifest.Experiment),
+ expand("output/figures/enrichment_reproducibility/{experiment_label}.enrichment_reproducibility.pdf", experiment_label = manifest.Experiment),
+ expand("output/counts/repeats/tables/family/{experiment_label}.tsv.gz", experiment_label = manifest.Experiment),
+ expand("output/reproducible_enriched_re/{experiment_label}.reproducible_enriched_re.tsv.gz", experiment_label = manifest.Experiment),
+ expand("output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz", experiment_label = manifest.Experiment),
+ expand("output/homer/finemapped_results/{experiment_label}/homerResults.html", experiment_label = manifest.Experiment),
+ expand("output/gene_sets/{experiment_label}.enriched_terms.tsv.gz", experiment_label = manifest.Experiment),
+ "output/figures/tsne/skipper.tsne_query.pdf",
+ expand("output/multiqc/{experiment_label}/multiqc_data", experiment_label = manifest.Experiment),
+ expand("output/multiqc/{experiment_label}/multiqc_plots", experiment_label = manifest.Experiment),
+ expand("output/multiqc/{experiment_label}/multiqc_report.html", experiment_label = manifest.Experiment),
+ expand("output/counts/genome/megatables/{genome_type}.tsv.gz", genome_type = ["feature_type_top","transcript_type_top"]),
+ expand("output/counts/repeats/megatables/{repeat_type}.tsv.gz", repeat_type = ['name', 'class', 'family']),
+ "output/QC/unique_fragments.csv",
+ # expand("output/variants/gnomAD/{experiment_label}.{chr}.vcf", experiment_label = manifest.Experiment, chr=[f'chr{i}' for i in list(range(1,23))+['X','Y']]),
+ # expand("output/ml/sequence/{experiment_label}.foreground.fa", experiment_label = manifest.Experiment),
+ # expand("output/ml/gkmsvm/{experiment_label}.cvpred.txt", experiment_label = manifest.Experiment),
+ # expand("output/ml/gkmsvm/{experiment_label}.model.txt", experiment_label = manifest.Experiment),
+ # "output/ml/gkmsvm/AUPRC.txt",
+ # "variants_done.txt"
+ output:
+ "land_ho.txt"
+ threads: 1
+ params:
+ error_file = "stderr/all.err",
+ out_file = "stdout/all.out",
+ run_time = "00:04:00",
+ memory = "200",
+ job_name = "all"
+ shell:
+ "echo $(date) > {output};"
+ "echo Version: 1.99.0 >> {output};"
+ "echo RBP-ARK >> {output};"
+ "echo Created by Evan Boyle and the Yeo lab >> {output}"
+
+module se_preprocess:
+ snakefile:
+ "rules/se_preprocess.smk"
+ config:
+ config
+
+module pe_preprocess:
+ snakefile:
+ "rules/pe_preprocess.smk"
+ config:
+ config
+
+module qc:
+ snakefile:
+ "rules/qc.smk"
+ config: config
+
+module genome:
+ snakefile:
+ "rules/genome_windows.smk"
+ config: config
+
+module repeat:
+ snakefile:
+ "rules/repeat.smk"
+ config: config
+
+module finemap:
+ snakefile:
+ "rules/finemap.smk"
+ config: config
+
+module analysis:
+ snakefile:
+ "rules/analysis.smk"
+ config: config
+
+module meta_analysis:
+ snakefile:
+ "rules/meta_analysis.smk"
+ config:
+ config
+module bigwig:
+ snakefile:
+ "rules/bigwig.smk"
+ config:
+ config
+module prep_ml:
+ snakefile:
+ "rules/prep_ml.smk"
+ config:
+ config
+
+module variants:
+ snakefile:
+ "rules/variants.smk"
+ config:
+ config
+
+
+
+if config['protocol']=='ENCODE4':
+ use rule * from se_preprocess as se_*
+else:
+ use rule * from pe_preprocess as pe_*
+
+use rule * from bigwig
+use rule * from qc
+use rule * from genome
+use rule * from repeat
+use rule * from finemap
+use rule * from analysis
+use rule * from meta_analysis
+use rule * from prep_ml as ml_*
+use rule * from variants as variants_*
+
diff --git a/Skipper_config.py b/Skipper_config.py
deleted file mode 100644
index add4573..0000000
--- a/Skipper_config.py
+++ /dev/null
@@ -1,56 +0,0 @@
-# Configuration file
-
-import os
-import sys
-import glob
-
-REPO_PATH = "/projects/ps-yeolab3/eboyle/encode/pipeline/github/skipper"
-# Input data
-# Use a GFF filtered for genes expressed in the cell type of interest
-# Skipper will partition the transcriptome and create feature annotations from the GFF
-MANIFEST = REPO_PATH + "/example/translation_manifest.csv"
-GFF = REPO_PATH + "/annotations/gencode.v38.annotation.k562_totalrna.gt1.gff3.gz"
-PARTITION = REPO_PATH + "/annotations/gencode.v38.annotation.k562_totalrna.gt1.tiled_partition.bed.gz"
-FEATURE_ANNOTATIONS = REPO_PATH + "/annotations/gencode.v38.annotation.k562_totalrna.gt1.tiled_partition.features.tsv.gz"
-
-# Information about CLIP library
-UMI_SIZE = 10
-# Single-end: enter 1. Paired-end: enter read (1 or 2) corresponding to crosslink site
-INFORMATIVE_READ = 1
-# Use multiple input replicates to estimate overdispersion (preferred), or use multiple CLIP replicates
-# Skipper requires replicates to model the variance in read counts.
-OVERDISPERSION_MODE = "input" # input or clip
-
-# Paths to custom tools
-# Path to tools packaged with Skipper
-TOOL_DIR = REPO_PATH + "/tools"
-# Path to additional installed tools (enter "." for the current directory if all tools are globally installed)
-EXE_DIR = "/projects/ps-yeolab3/eboyle/software/bin/"
-
-# General resources
-# STAR reference
-STAR_DIR = "/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/star_2_7_gencode29_sjdb"
-# Path to R with installed dependencies: VGAM, viridis, fgsea, GenomicRanges, ggrepel, RColorBrewer, Rtsne, ggupset, cowplot, ggdendro
-R_EXE = "/projects/ps-yeolab4/software/R-4.1.2/bin/Rscript"
-# Downloaded from https://github.com/Daniel-Liu-c0deb0t/UMICollapse
-UMICOLLAPSE_DIR = "/projects/ps-yeolab3/eboyle/software/UMICollapse"
-# Java executable for UMICollapse: enter "java" for globally installed version
-JAVA_EXE = "/projects/ps-yeolab3/eboyle/software/jdk-18.0.1.1/bin/java"
-# indexed genome fasta
-GENOME = "/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta"
-# Downloaded from UCSC genome browser
-CHROM_SIZES = REPO_PATH + "/annotations/hg38.chrom.sizes"
-# https://genome.ucsc.edu/cgi-bin/hgTables?hgsid=1435901349_MASaYmzfsYNwQY34JLgNir7HOyVh&clade=mammal&org=Human&db=hg38
-REPEAT_TABLE = "/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.tsv.gz"
-
-# Customizable, with defaults
-BLACKLIST = REPO_PATH + "/annotations/encode3_eclip_blacklist.bed" # set to None for no blacklisting
-GENE_SETS = REPO_PATH + "/annotations/c5.go.v7.5.1.symbols.gmt"
-GENE_SET_REFERENCE = REPO_PATH + "/annotations/encode3_go_terms.reference.tsv.gz"
-GENE_SET_DISTANCE = REPO_PATH + "/annotations/encode3_go_terms.jaccard_index.rds"
-REPEAT_BED = "/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz"
-# Ranked list of gene and transcript types found in GFF annotations
-ACCESSION_RANKINGS = REPO_PATH + "/annotations/accession_type_ranking.txt"
-
-# Internal use
-UNINFORMATIVE_READ = 3 - INFORMATIVE_READ
diff --git a/encode_configs/Skipper_config_HepG2_failed_20230620.yaml b/encode_configs/Skipper_config_HepG2_failed_20230620.yaml
new file mode 100644
index 0000000..9d45b2c
--- /dev/null
+++ b/encode_configs/Skipper_config_HepG2_failed_20230620.yaml
@@ -0,0 +1,22 @@
+WORKDIR : /tscc/nfs/home/hsher/scratch/ENCODE3_HepG2_failed
+protocol: ENCODE3
+MANIFEST : /tscc/projects/ps-yeolab5/encode/processing/rerun_failed/Skipper_manifest_HepG2_20230620_bam.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 2
+OVERDISPERSION_MODE : clip
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 1
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+RBPNET_PATH: /tscc/nfs/home/hsher/projects/RBPNet/
diff --git a/encode_configs/Skipper_config_K562_failed_20230620.yaml b/encode_configs/Skipper_config_K562_failed_20230620.yaml
new file mode 100644
index 0000000..9744014
--- /dev/null
+++ b/encode_configs/Skipper_config_K562_failed_20230620.yaml
@@ -0,0 +1,21 @@
+MANIFEST: /projects/ps-yeolab5/encode/processing/rerun_failed/Skipper_manifest_K562_20230620_bam.csv
+GFF: /projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.gff3.gz
+PARTITION: /projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS: /projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS: /projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE: 10
+INFORMATIVE_READ: 2
+OVERDISPERSION_MODE: clip
+TOOL_DIR : /projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR: /projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+
+GENOME: /projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES: /projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE: /projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST: /projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS: /projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE: /projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE: /projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ: 1
+REPEAT_BED: /projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+RBPNET_PATH: /tscc/nfs/home/hsher/projects/RBPNet/
diff --git a/encode_configs/Skipper_pe_manifest_small.csv b/encode_configs/Skipper_pe_manifest_small.csv
new file mode 100644
index 0000000..21f72c8
--- /dev/null
+++ b/encode_configs/Skipper_pe_manifest_small.csv
@@ -0,0 +1,5 @@
+Experiment,Sample,Cells,Input_replicate,Input_adapter_1,Input_adapter_2,Input_fastq_1,Input_fastq_2,CLIP_replicate,CLIP_adapter_1,CLIP_adapter_2,CLIP_fastq_1,CLIP_fastq_2,Accession,Barcode 1,Barcode 2,RBP
+PUM1_K562_ENCSR308YNT,PUM1_K562_ENCSR308YNT,K562,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF475RBN.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF294VPI.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/A03_G07.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF046QBX.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF353ZJC.fastq.gz,ENCSR308YNT,A03,G07,PUM1
+PUM2_K562_ENCSR661ICQ,PUM2_K562_ENCSR661ICQ,K562,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF616FCF.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF495ZPY.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/A03_G07.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF956TOZ.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF133DNM.fastq.gz,ENCSR661ICQ,A03,G07,PUM2
+PUM1_K562_ENCSR308YNT,PUM1_K562_ENCSR308YNT,K562,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF475RBN.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF294VPI.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/A04_F05.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF116YKN.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF019LLG.fastq.gz,ENCSR308YNT,A04,F05,PUM1
+PUM2_K562_ENCSR661ICQ,PUM2_K562_ENCSR661ICQ,K562,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF616FCF.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF495ZPY.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/A04_F05.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF041KJT.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF462SCV.fastq.gz,ENCSR661ICQ,A04,F05,PUM2
diff --git a/encode_configs/Skipper_pe_small_test.yaml b/encode_configs/Skipper_pe_small_test.yaml
new file mode 100644
index 0000000..9b69492
--- /dev/null
+++ b/encode_configs/Skipper_pe_small_test.yaml
@@ -0,0 +1,23 @@
+WORKDIR: /tscc/nfs/home/hsher/scratch/testing_branch_ENCODE3
+protocol: ENCODE3
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/encode_configs/Skipper_pe_manifest_small.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 2
+OVERDISPERSION_MODE : clip
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 1
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+RBPNET_PATH: /tscc/nfs/home/hsher/projects/RBPNet/
diff --git a/encode_configs/encode3_manifest_H_fastq.csv b/encode_configs/encode3_manifest_H_fastq.csv
new file mode 100644
index 0000000..4253a69
--- /dev/null
+++ b/encode_configs/encode3_manifest_H_fastq.csv
@@ -0,0 +1,207 @@
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diff --git a/encode_configs/encode3_manifest_K_fastq.csv b/encode_configs/encode3_manifest_K_fastq.csv
new file mode 100644
index 0000000..41efc54
--- /dev/null
+++ b/encode_configs/encode3_manifest_K_fastq.csv
@@ -0,0 +1,241 @@
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+DHX30_K562_ENCSR529GSJ,DHX30_K562_ENCSR529GSJ,K562,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF418SOC.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF245WDH.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF238BOS.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF510RNY.fastq.gz,ENCSR529GSJ,X2A,X2B,DHX30
+GEMIN5_K562_ENCSR238CLX,GEMIN5_K562_ENCSR238CLX,K562,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF064TEN.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF381NWX.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF384BDA.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF175TMY.fastq.gz,ENCSR238CLX,X2A,X2B,GEMIN5
+PHF6_K562_ENCSR001KKZ,PHF6_K562_ENCSR001KKZ,K562,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF928TNJ.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/sm_input/ENCFF804TGO.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/fwd_encode3.fasta,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/RiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF461CTN.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/eclip_fastq/clip/ENCFF165KAR.fastq.gz,ENCSR001KKZ,X2A,X2B,PHF6
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\ No newline at end of file
diff --git a/encode_configs/encode_pe_rules_config_HepG2_20230620.yaml b/encode_configs/encode_pe_rules_config_HepG2_20230620.yaml
new file mode 100644
index 0000000..adcdae8
--- /dev/null
+++ b/encode_configs/encode_pe_rules_config_HepG2_20230620.yaml
@@ -0,0 +1,22 @@
+WORKDIR: /tscc/nfs/home/hsher/scratch/ENCODE3_HepG2
+protocol: ENCODE3
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/encode_configs/encode3_manifest_H_fastq.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 2
+OVERDISPERSION_MODE : clip
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab3/bay001/annotations/hg38/star_2_7_gencode40_sjdb
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab3/bay001/annotations/hg38/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 1
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+RBPNET_PATH: /tscc/nfs/home/hsher/projects/RBPNet/
diff --git a/encode_configs/encode_pe_rules_config_K562_20230929.yaml b/encode_configs/encode_pe_rules_config_K562_20230929.yaml
new file mode 100644
index 0000000..ef182bd
--- /dev/null
+++ b/encode_configs/encode_pe_rules_config_K562_20230929.yaml
@@ -0,0 +1,22 @@
+WORKDIR: /tscc/nfs/home/hsher/scratch/ENCODE3_K562
+protocol: ENCODE3
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/encode_configs/encode3_manifest_K_fastq.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 2
+OVERDISPERSION_MODE : clip
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 1
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+RBPNET_PATH: /tscc/nfs/home/hsher/projects/RBPNet/
diff --git a/encore_configs/HepG2.csv b/encore_configs/HepG2.csv
new file mode 100644
index 0000000..aca4957
--- /dev/null
+++ b/encore_configs/HepG2.csv
@@ -0,0 +1,87 @@
+,Experiment,Sample,Cells,Input_replicate,Input_adapter,Input_fastq,CLIP_replicate,CLIP_adapter,CLIP_fastq,RBP,Accession,phase
+0,PABPN1_HepG2_5062,PABPN1_HepG2_5062,HepG2,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5062_INPUT1_S31_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5062_IP1_S32_L001_R1_001.fastq.gz,PABPN1,5062,ENCORE2
+1,PABPN1_HepG2_5062,PABPN1_HepG2_5062,HepG2,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5062_INPUT2_S33_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5062_IP2_S34_L001_R1_001.fastq.gz,PABPN1,5062,ENCORE2
+2,CDK9_HepG2_5063,CDK9_HepG2_5063,HepG2,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5063_INPUT1_S35_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5063_IP1_S36_L001_R1_001.fastq.gz,CDK9,5063,ENCORE2
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+4,RACK1_HepG2_5067,RACK1_HepG2_5067,HepG2,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5067_INPUT1_S51_L002_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5067_IP1_S52_L002_R1_001.fastq.gz,RACK1,5067,ENCORE2
+5,RACK1_HepG2_5067,RACK1_HepG2_5067,HepG2,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5067_INPUT2_S53_L002_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20230507_maya_zfp_cutrun_pe50/5067_IP2_S54_L002_R1_001.fastq.gz,RACK1,5067,ENCORE2
+0,DEK_HepG2_4020,DEK_HepG2_4020,HepG2,1,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA5.fasta,/tscc/projects/ps-yeolab4/seqdata/20190314_Encode_Stefan_Chaim_eCLIP/igm-storage1.ucsd.edu/190312_K00180_0768_AHYGGTBBXX_SR75_Combo/4020_INPUT1_S17_L007_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20190314_Encode_Stefan_Chaim_eCLIP/igm-storage1.ucsd.edu/190312_K00180_0768_AHYGGTBBXX_SR75_Combo/4020_CLIP1_S18_L007_R1_001.fastq.gz,DEK,4020,ENCORE
+1,DEK_HepG2_4020,DEK_HepG2_4020,HepG2,2,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA5.fasta,/tscc/projects/ps-yeolab4/seqdata/20190314_Encode_Stefan_Chaim_eCLIP/igm-storage1.ucsd.edu/190312_K00180_0768_AHYGGTBBXX_SR75_Combo/4020_INPUT2_S19_L007_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20190314_Encode_Stefan_Chaim_eCLIP/igm-storage1.ucsd.edu/190312_K00180_0768_AHYGGTBBXX_SR75_Combo/4020_CLIP2_S20_L007_R1_001.fastq.gz,DEK,4020,ENCORE
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diff --git a/encore_configs/K562.csv b/encore_configs/K562.csv
new file mode 100644
index 0000000..14cbdb4
--- /dev/null
+++ b/encore_configs/K562.csv
@@ -0,0 +1,291 @@
+,Experiment,Sample,Cells,Input_replicate,Input_adapter,Input_fastq,CLIP_replicate,CLIP_adapter,CLIP_fastq,RBP,Accession,phase
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+149,RPL29_K562_4243,RPL29_K562_4243,K562,2,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20211008_encode_ribo_evan/RPL29_IN2_S22_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20211008_encode_ribo_evan/RPL29_IP2_S24_L001_R1_001.fastq.gz,RPL29,4243,ENCORE
+150,RPS21_K562_9990,RPS21_K562_9990,K562,1,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20211103_maya_jas_steven/RPS21_IN1_S7_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20211103_maya_jas_steven/RPS21_IP1_S15_L001_R1_001.fastq.gz,RPS21,9990,ENCORE
+151,RPS21_K562_9990,RPS21_K562_9990,K562,2,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20211103_maya_jas_steven/RPS21_IN2_S8_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20211103_maya_jas_steven/RPS21_IP2_S16_L001_R1_001.fastq.gz,RPS21,9990,ENCORE
+152,RPS19_K562_9991,RPS19_K562_9991,K562,1,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20211103_maya_jas_steven/RPS19_IN1_S5_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20211103_maya_jas_steven/RPS19_IP1_S13_L001_R1_001.fastq.gz,RPS19,9991,ENCORE
+153,RPS19_K562_9991,RPS19_K562_9991,K562,2,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/20211103_maya_jas_steven/RPS19_IN2_S6_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/20211103_maya_jas_steven/RPS19_IP2_S14_L001_R1_001.fastq.gz,RPS19,9991,ENCORE
diff --git a/encore_configs/Skipper_config_HepG2_20230620.yaml b/encore_configs/Skipper_config_HepG2_20230620.yaml
new file mode 100644
index 0000000..f25bfed
--- /dev/null
+++ b/encore_configs/Skipper_config_HepG2_20230620.yaml
@@ -0,0 +1,23 @@
+WORKDIR: /tscc/nfs/home/hsher/scratch/ENCORE_HepG2
+protocol: ENCODE4
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/encore_configs/HepG2.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+TMPDIR: /tscc/nfs/home/hsher/scratch/
+RBPNET_PATH: /tscc/nfs/home/hsher/projects/RBPNet/
\ No newline at end of file
diff --git a/encore_configs/Skipper_config_K562_20230620.yaml b/encore_configs/Skipper_config_K562_20230620.yaml
new file mode 100644
index 0000000..6ef06ad
--- /dev/null
+++ b/encore_configs/Skipper_config_K562_20230620.yaml
@@ -0,0 +1,23 @@
+WORKDIR: /tscc/nfs/home/hsher/scratch/ENCORE_K562
+protocol: ENCODE4
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/encore_configs/K562.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.k562_totalrna.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+TMPDIR: /tscc/nfs/home/hsher/scratch/
+RBPNET_PATH: /tscc/nfs/home/hsher/projects/RBPNet/
\ No newline at end of file
diff --git a/encore_configs/Skipper_config_small_test.yaml b/encore_configs/Skipper_config_small_test.yaml
new file mode 100644
index 0000000..f8c6b79
--- /dev/null
+++ b/encore_configs/Skipper_config_small_test.yaml
@@ -0,0 +1,23 @@
+WORKDIR: /tscc/nfs/home/hsher/scratch/ENCORE_test_2
+protocol: ENCODE4
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/encore_configs/Skipper_manifest_small.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/gencode.v40.annotation.hepg2_totalrna.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+RBPNET_PATH: /tscc/nfs/home/hsher/projects/RBPNet/
diff --git a/other_configs/AlexT_Puf60.yaml b/other_configs/AlexT_Puf60.yaml
new file mode 100644
index 0000000..84aabf0
--- /dev/null
+++ b/other_configs/AlexT_Puf60.yaml
@@ -0,0 +1,22 @@
+WORKDIR: /tscc/projects/ps-yeolab5/hsher/Alex_PUF60
+protocol: ENCODE4
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/other_configs/puf60_skipper_manifest_wdiff.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/85d899b/bin/skipper/annotations/GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_median_tpm.Breast_Mammary_Tissue.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/85d899b/bin/skipper/annotations/GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_median_tpm.Breast_Mammary_Tissue.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/85d899b/bin/skipper/annotations/GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_median_tpm.Breast_Mammary_Tissue.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+TMPDIR: /tscc/projects/ps-yeolab3/scratch_tmp
diff --git a/other_configs/Alicia_RBM18.yaml b/other_configs/Alicia_RBM18.yaml
new file mode 100644
index 0000000..211d1bf
--- /dev/null
+++ b/other_configs/Alicia_RBM18.yaml
@@ -0,0 +1,22 @@
+WORKDIR: /tscc/projects/ps-yeolab5/hsher/alicia_RBM18_new
+protocol: ENCODE4
+MANIFEST : /tscc/projects/ps-yeolab5/hsher/alicia_RBM18/aliciaenoos_clips-csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+TMPDIR: /tscc/projects/ps-yeolab3/scratch_tmp
diff --git a/other_configs/Alicia_RBM18_ipsc.csv b/other_configs/Alicia_RBM18_ipsc.csv
new file mode 100644
index 0000000..01ed3c7
--- /dev/null
+++ b/other_configs/Alicia_RBM18_ipsc.csv
@@ -0,0 +1,5 @@
+,Experiment,Input_replicate,CLIP_fastq,Input_fastq,Sample,CLIP_replicate,Cells,RBP,CLIP_adapter,Input_adapter,Accession,Author,Email,Name,Org
+0,RBM18,1,/tscc/nfs/home/mgosztyl/seqdata/20240418_seq_results/igm-storage.ucsd.edu/240415_LH00444_0092_A227CY2LT4/FASTQ/RBM18_IN1_S16_L004_R1_001.fastq.gz,/tscc/nfs/home/mgosztyl/seqdata/20240418_seq_results/igm-storage.ucsd.edu/240415_LH00444_0092_A227CY2LT4/FASTQ/RBM18_IP1_S24_L004_R1_001.fastq.gz,RBM18,1,iPSC,RBM18,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta,,,,,
+1,RBM18,2,/tscc/nfs/home/mgosztyl/seqdata/20240418_seq_results/igm-storage.ucsd.edu/240415_LH00444_0092_A227CY2LT4/FASTQ/RBM18_IN2_S17_L004_R1_001.fastq.gz,/tscc/nfs/home/mgosztyl/seqdata/20240418_seq_results/igm-storage.ucsd.edu/240415_LH00444_0092_A227CY2LT4/FASTQ/RBM18_IP2_S25_L004_R1_001.fastq.gz,RBM18,2,iPSC,RBM18,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta,,,,,
+0,RBM18_HEK293T,1,/tscc/projects/ps-yeolab4/seqdata/20231220_Phuong_Elliot_Jack/igm-storage.ucsd.edu/231213_LH00444_0028_B22F25CLT3/RBM18_IP1_S61_L005_R1_001.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/20231220_Phuong_Elliot_Jack/igm-storage.ucsd.edu/231213_LH00444_0028_B22F25CLT3/RBM18_IN1_S47_L005_R1_001.fastq.gz,RBM18_HEK293T,1,293T,RBM18,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRiL19.fasta,RBM18_1,krothamel@gmail.com,krothamel@gmail.com,Katie Rothamel,UCSD
+1,RBM18_HEK293T,2,/tscc/projects/ps-yeolab4/seqdata/20231220_Phuong_Elliot_Jack/igm-storage.ucsd.edu/231213_LH00444_0028_B22F25CLT3/RBM18_IP2_S62_L005_R1_001.fastq.gz,/tscc/projects/ps-yeolab4/seqdata/20231220_Phuong_Elliot_Jack/igm-storage.ucsd.edu/231213_LH00444_0028_B22F25CLT3/RBM18_IN2_S48_L005_R1_001.fastq.gz,RBM18_HEK293T,2,293T,RBM18,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRNA1.fasta,/tscc/projects/ps-yeolab4/software/skipper/bb63a25/examples/inputs/InvRiL19.fasta,RBM18_2,krothamel@gmail.com,krothamel@gmail.com,Katie Rothamel,UCSD
diff --git a/other_configs/Alicia_RBM18_ipsc.yaml b/other_configs/Alicia_RBM18_ipsc.yaml
new file mode 100644
index 0000000..4e93883
--- /dev/null
+++ b/other_configs/Alicia_RBM18_ipsc.yaml
@@ -0,0 +1,22 @@
+WORKDIR: /tscc/projects/ps-yeolab5/hsher/alicia_RBM18_ipsc_allref
+protocol: ENCODE4
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/other_configs/Alicia_RBM18_ipsc.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+TMPDIR: /tscc/projects/ps-yeolab3/scratch_tmp
diff --git a/other_configs/Alicia_RBM18_ipsc_hek_ref.yaml b/other_configs/Alicia_RBM18_ipsc_hek_ref.yaml
new file mode 100644
index 0000000..bb152d3
--- /dev/null
+++ b/other_configs/Alicia_RBM18_ipsc_hek_ref.yaml
@@ -0,0 +1,22 @@
+WORKDIR: /tscc/projects/ps-yeolab5/hsher/alicia_RBM18_ipsc
+protocol: ENCODE4
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/other_configs/Alicia_RBM18_ipsc.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+TMPDIR: /tscc/projects/ps-yeolab3/scratch_tmp
diff --git a/other_configs/NONO_R_S_DMSO_charl_fixed.csv b/other_configs/NONO_R_S_DMSO_charl_fixed.csv
new file mode 100644
index 0000000..fcba567
--- /dev/null
+++ b/other_configs/NONO_R_S_DMSO_charl_fixed.csv
@@ -0,0 +1,7 @@
+,Experiment,Input_replicate,CLIP_fastq,Input_fastq,Sample,CLIP_replicate,Cells,RBP,CLIP_adapter,Input_adapter
+0,10R,1,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/10R_CLIP_rep1.fastq.gz,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/10R_INPUT_rep1.fastq.gz,10R,1,22Rv1 human prostate cancer,NONO,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA3_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta
+1,10R,2,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/10R_CLIP_rep2.fastq.gz,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/10R_INPUT_rep2.fastq.gz,10R,2,22Rv1 human prostate cancer,NONO,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA4_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta
+2,10S,1,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/10S_CLIP_rep1.fastq.gz,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/10S_INPUT_rep1.fastq.gz,10S,1,22Rv1 human prostate cancer,NONO,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA3_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta
+3,10S,2,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/10S_CLIP_rep2.fastq.gz,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/10S_INPUT_rep2.fastq.gz,10S,2,22Rv1 human prostate cancer,NONO,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA4_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta
+4,DMSO,1,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/DMSO_CLIP_rep1.fastq.gz,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/DMSO_INPUT_rep1.fastq.gz,DMSO,1,22Rv1 human prostate cancer,NONO,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA3_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta
+5,DMSO,2,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/DMSO_CLIP_rep2.fastq.gz,/tscc/nfs/home/hsher/projects/stefan_nono/geo_upload/clip_data/DMSO_INPUT_rep2.fastq.gz,DMSO,2,22Rv1 human prostate cancer,NONO,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA4_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta
diff --git a/other_configs/Nadav_IGF2BP3.yaml b/other_configs/Nadav_IGF2BP3.yaml
new file mode 100644
index 0000000..cc6cb99
--- /dev/null
+++ b/other_configs/Nadav_IGF2BP3.yaml
@@ -0,0 +1,22 @@
+WORKDIR: /tscc/nfs/home/hsher/scratch/Nadav_IGF2BP3
+protocol: ENCODE4
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/other_configs/hek293_IGF2BP3_MY10A_B_DMSO.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+TMPDIR: /tscc/projects/ps-yeolab3/scratch_tmp
diff --git a/other_configs/Stefan_NONO.yaml b/other_configs/Stefan_NONO.yaml
new file mode 100644
index 0000000..d646554
--- /dev/null
+++ b/other_configs/Stefan_NONO.yaml
@@ -0,0 +1,22 @@
+WORKDIR: /tscc/nfs/home/hsher/scratch/Stefan_NONO
+protocol: ENCODE4
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/other_configs/NONO_R_S_DMSO_charl_fixed.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/85d899b/bin/skipper/annotations/GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_median_tpm.Prostate.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/85d899b/bin/skipper/annotations/GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_median_tpm.Prostate.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/85d899b/bin/skipper/annotations/GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_median_tpm.Prostate.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+TMPDIR: /tscc/projects/ps-yeolab3/scratch_tmp
diff --git a/other_configs/Tao_RBM15.csv b/other_configs/Tao_RBM15.csv
new file mode 100644
index 0000000..de82ec1
--- /dev/null
+++ b/other_configs/Tao_RBM15.csv
@@ -0,0 +1,3 @@
+,Experiment,Input_replicate,CLIP_fastq,Input_fastq,Sample,CLIP_replicate,Cells,RBP,CLIP_adapter,Input_adapter
+0,RBM15,1,/tscc/nfs/home/mgosztyl/seqdata/20240418_seq_results/igm-storage.ucsd.edu/240415_LH00444_0092_A227CY2LT4/FASTQ/RBM15_IN1_S14_L004_R1_001.fastq.gz,/tscc/nfs/home/mgosztyl/seqdata/20240418_seq_results/igm-storage.ucsd.edu/240415_LH00444_0092_A227CY2LT4/FASTQ/RBM15_IP1_S22_L004_R1_001.fastq.gz,RBM15,1,HEK,RBM15,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta
+1,RBM15,2,/tscc/nfs/home/mgosztyl/seqdata/20240418_seq_results/igm-storage.ucsd.edu/240415_LH00444_0092_A227CY2LT4/FASTQ/RBM15_IN2_S15_L004_R1_001.fastq.gz,/tscc/nfs/home/mgosztyl/seqdata/20240418_seq_results/igm-storage.ucsd.edu/240415_LH00444_0092_A227CY2LT4/FASTQ/RBM15_IP2_S23_L004_R1_001.fastq.gz,RBM15,2,HEK,RBM15,/tscc/projects/ps-yeolab4/software/eclip/0.7.0/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta
diff --git a/other_configs/Tao_RBM15.yaml b/other_configs/Tao_RBM15.yaml
new file mode 100644
index 0000000..81205b5
--- /dev/null
+++ b/other_configs/Tao_RBM15.yaml
@@ -0,0 +1,22 @@
+WORKDIR: /tscc/projects/ps-yeolab5/hsher/alicia_RBM15
+protocol: ENCODE4
+MANIFEST : /tscc/nfs/home/hsher/projects/skipper/other_configs/Tao_RBM15.csv
+GFF : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.gff3.gz
+PARTITION : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.tiled_partition.bed.gz
+FEATURE_ANNOTATIONS : /tscc/projects/ps-yeolab4/software/skipper/d0055ff/bin/skipper/annotations/gencode.v38.annotation.hek293t.gt1.tiled_partition.features.tsv.gz
+ACCESSION_RANKINGS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/accession_type_ranking.txt
+UMI_SIZE : 10
+INFORMATIVE_READ : 1
+OVERDISPERSION_MODE : input
+TOOL_DIR : /tscc/projects/ps-yeolab3/hsher/skipper/tools
+STAR_DIR : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/
+GENOME : /tscc/projects/ps-yeolab4/genomes/GRCh38/chromosomes/GRCh38_no_alt_analysis_set_GCA_000001405.15.fasta
+CHROM_SIZES : /tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/star_2_7_gencode40_sjdb/chrNameLength.txt
+REPEAT_TABLE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/repeatmasker.grch38.tsv.gz
+BLACKLIST : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_eclip_blacklist.bed
+GENE_SETS : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/c5.go.v7.5.1.symbols.gmt
+GENE_SET_REFERENCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.reference.tsv.gz
+GENE_SET_DISTANCE : /tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/encode3_go_terms.jaccard_index.rds
+UNINFORMATIVE_READ : 2
+REPEAT_BED : /tscc/projects/ps-yeolab3/eboyle/encode/pipeline/05_20220720/repeatmasker.grch38.sort.unique.bed.gz
+TMPDIR: /tscc/projects/ps-yeolab3/scratch_tmp
diff --git a/other_configs/hek293_IGF2BP3_MY10A_B_DMSO.csv b/other_configs/hek293_IGF2BP3_MY10A_B_DMSO.csv
new file mode 100644
index 0000000..5f91b56
--- /dev/null
+++ b/other_configs/hek293_IGF2BP3_MY10A_B_DMSO.csv
@@ -0,0 +1,7 @@
+Experiment,Sample,Cells,Input_replicate,Input_adapter,Input_fastq,CLIP_replicate,CLIP_adapter,CLIP_fastq,RBP,Accession
+MY10A,MY10A,Hek_293T,1,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_A1_IN_S81_L003_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_A1_IP_S78_L003_R1_001.fastq.gz,IGF2BP3,INV1
+MY10B,MY10B,Hek_293T,1,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_B1_IN_S82_L003_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_B2_IP_S79_L003_R1_001.fastq.gz,IGF2BP3,INV2
+DMSO,DMSO,Hek_293T,1,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_DMSO_2_IP_S80_L003_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_DMSO2_IP_S83_L003_R1_001.fastq.gz,IGF2BP3,INV3
+MY10A,MY10A,Hek_293T,2,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_IN_A_S72_L003_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_IP_A_S75_L003_R1_001.fastq.gz,IGF2BP3,INV4
+MY10B,MY10B,Hek_293T,2,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_IN_B_S73_L003_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_IP_B_S76_L003_R1_001.fastq.gz,IGF2BP3,INV5
+DMSO,DMSO,Hek_293T,2,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRiL19_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_IN_DMSO_S74_L003_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/eclip/0.7.1/examples/inputs/InvRNA1_adapters.fasta,/tscc/projects/ps-yeolab4/seqdata/20240318_SB_PL_OM_NA_NW_MG_KM_TY_EC/igm-storage2.ucsd.edu/240311_LH00444_0071_A2257TNLT4/NW_IP_DMSO_S77_L003_R1_001.fastq.gz,IGF2BP3,INV6
\ No newline at end of file
diff --git a/other_configs/puf60_skipper_manifest.csv b/other_configs/puf60_skipper_manifest.csv
new file mode 100644
index 0000000..63dfde0
--- /dev/null
+++ b/other_configs/puf60_skipper_manifest.csv
@@ -0,0 +1,17 @@
+# Filter gene annotation files to genes expressed in the cell type to improve sensitivity,,,,,,,,,
+# Replicates of the same sample must have identical experiment and sample fields. Replicates are numbered per experiment.,,,,,,,,,
+Experiment,Sample,Cells,Input_replicate,Input_adapter,Input_fastq,CLIP_replicate,CLIP_adapter,CLIP_fastq,Notes
+PUF60-MDAMB231,PUF60-MDAMB231,MDAMB231,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/231-IN1_S17_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/231-IP1_S18_R1_001.fastq.gz,
+PUF60-MDAMB231,PUF60-MDAMB231,MDAMB231,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/231-IN2_S19_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/231-IP2_S20_R1_001.fastq.gz,
+PUF60-MDAMB436,PUF60-MDAMB436,MDAMB436,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/436_PUF60_IN1_S53_L005_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/436_PUF60_IP1_S67_L005_R1_001.fastq.gz,
+PUF60-MDAMB436,PUF60-MDAMB436,MDAMB436,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/436_PUF60_IN2_S54_L005_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/436_PUF60_IP2_S68_L005_R1_001.fastq.gz,
+PUF60-MCF10A,PUF60-MCF10A,MCF10A,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/10A_rep1N_S20_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA3.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/10A_rep1P_S23_R1_001.fastq.gz,
+PUF60-MCF10A,PUF60-MCF10A,MCF10A,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/10A_rep2N_S30_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA3.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/10A_rep2P_S37_R1_001.fastq.gz,
+PUF60-SUM149,PUF60-SUM149,SUM149,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/149_rep1N_S5_L003_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/149_rep1P_S6_L003_R1_001.fastq.gz,
+PUF60-SUM149,PUF60-SUM149,SUM149,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/149_rep2N_S7_L003_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/149_rep2P_S8_L003_R1_001.fastq.gz,
+L140P-PUF60,L140P-PUF60,MDAMB231,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IN1_S36_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IP1_S44_L001_R1_001.fastq.gz
+L140P-PUF60,L140P-PUF60,MDAMB231,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IN2_S37_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IP2_S45_L001_R1_001.fastq.gz
+WT-PUF60,WT-PUF60,MDAMB436,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IN1_S38_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IP1_S46_L001_R1_001.fastq.gz
+WT-PUF60,WT-PUF60,MDAMB436,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IN2_S39_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IP2_S47_L001_R1_001.fastq.gz
+V5,V5,MCF10A,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/V5_IN1_S27_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/V5_IP1_S29_L001_R1_001.fastq.gz
+V5,V5,MCF10A,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/V5_IN2_S28_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/V5_IP2_S30_L001_R1_001.fastq.gz
\ No newline at end of file
diff --git a/other_configs/puf60_skipper_manifest_wdiff.csv b/other_configs/puf60_skipper_manifest_wdiff.csv
new file mode 100644
index 0000000..407a5d1
--- /dev/null
+++ b/other_configs/puf60_skipper_manifest_wdiff.csv
@@ -0,0 +1,19 @@
+,Experiment,Sample,Cells,Input_replicate,Input_adapter,Input_fastq,CLIP_replicate,CLIP_adapter,CLIP_fastq,Notes
+0,PUF60-MDAMB231,PUF60-MDAMB231,MDAMB231,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/231-IN1_S17_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/231-IP1_S18_R1_001.fastq.gz,
+1,PUF60-MDAMB231,PUF60-MDAMB231,MDAMB231,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/231-IN2_S19_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/231-IP2_S20_R1_001.fastq.gz,
+2,PUF60-MDAMB436,PUF60-MDAMB436,MDAMB436,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/436_PUF60_IN1_S53_L005_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/436_PUF60_IP1_S67_L005_R1_001.fastq.gz,
+3,PUF60-MDAMB436,PUF60-MDAMB436,MDAMB436,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/436_PUF60_IN2_S54_L005_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/436_PUF60_IP2_S68_L005_R1_001.fastq.gz,
+4,PUF60-MCF10A,PUF60-MCF10A,MCF10A,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/10A_rep1N_S20_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA3.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/10A_rep1P_S23_R1_001.fastq.gz,
+5,PUF60-MCF10A,PUF60-MCF10A,MCF10A,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/10A_rep2N_S30_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA3.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/10A_rep2P_S37_R1_001.fastq.gz,
+6,PUF60-SUM149,PUF60-SUM149,SUM149,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/149_rep1N_S5_L003_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/149_rep1P_S6_L003_R1_001.fastq.gz,
+7,PUF60-SUM149,PUF60-SUM149,SUM149,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/149_rep2N_S7_L003_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRNA1.fasta,/tscc/projects/ps-yeolab4/seqdata/tankka_alex/final_puf60_clips/149_rep2P_S8_L003_R1_001.fastq.gz,
+8,L140P-PUF60,L140P-PUF60,MDAMB231,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IN1_S36_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IP1_S44_L001_R1_001.fastq.gz,
+9,L140P-PUF60,L140P-PUF60,MDAMB231,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IN2_S37_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IP2_S45_L001_R1_001.fastq.gz,
+10,WT-PUF60,WT-PUF60,MDAMB436,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IN1_S38_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IP1_S46_L001_R1_001.fastq.gz,
+11,WT-PUF60,WT-PUF60,MDAMB436,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IN2_S39_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IP2_S47_L001_R1_001.fastq.gz,
+12,V5,V5,MCF10A,1,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/V5_IN1_S27_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/V5_IP1_S29_L001_R1_001.fastq.gz,
+13,V5,V5,MCF10A,2,/tscc/projects/ps-yeolab3/eboyle/encode/pipeline/adapters/InvRiL19.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/V5_IN2_S28_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/V5_IP2_S30_L001_R1_001.fastq.gz,
+0,L140P-vs-WT,L140P-vs-WT,MDAMB231,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IP1_S46_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IP1_S44_L001_R1_001.fastq.gz,
+1,L140P-vs-WT,L140P-vs-WT,MDAMB231,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IP2_S47_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IP2_S45_L001_R1_001.fastq.gz,
+0,WT-vs-L140P,WT-vs-L140P,MDAMB231,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IP1_S44_L001_R1_001.fastq.gz,1,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IP1_S46_L001_R1_001.fastq.gz,
+1,WT-vs-L140P,WT-vs-L140P,MDAMB231,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/L14OP_IP2_S45_L001_R1_001.fastq.gz,2,/tscc/projects/ps-yeolab4/software/skipper/1.99.0/examples/inputs/InvRNA1.fasta,/tscc/nfs/home/atankka/seqdata/tankka_alex/20240221_L140P/WT_IP2_S47_L001_R1_001.fastq.gz,
diff --git a/profiles/ark-yeo/config.yaml b/profiles/ark-yeo/config.yaml
new file mode 100644
index 0000000..fb9acaf
--- /dev/null
+++ b/profiles/ark-yeo/config.yaml
@@ -0,0 +1,15 @@
+verbose: true
+notemp: false
+latency: 60
+printshellcmds: true
+cores: 1
+skip-script-cleanup: true
+nolock: true
+keep-going: true
+--singularity-prefix: /tscc/projects/ps-yeolab4/software/skipper/1.99.0/bin/.singularity
+use-singularity: true
+singularity-args: "--bind /tscc"
+retries: 3
+resources: mem_mb=16000
+cluster: "sbatch --partition condo --qos=condo --account csd792 --nodes=1 --tasks-per-node={threads} --error={params.error_file} --output={params.out_file} --time={params.run_time} --mem={params.memory}M"
+jobs: 16
diff --git a/profiles/ark/config.yaml b/profiles/ark/config.yaml
new file mode 100644
index 0000000..1b32237
--- /dev/null
+++ b/profiles/ark/config.yaml
@@ -0,0 +1,13 @@
+verbose: true
+notemp: false
+latency: 60
+printshellcmds: true
+cores: 8
+skip-script-cleanup: true
+nolock: true
+keep-going: true
+--singularity-prefix: /tscc/projects/ps-yeolab4/software/skipper/1.99.0/bin/.singularity/
+use-singularity: true
+singularity-args: "--bind /tscc"
+retries: 3
+resources: mem_mb=16000
diff --git a/profiles/tscc2/config.yaml b/profiles/tscc2/config.yaml
new file mode 100644
index 0000000..9aa5a8d
--- /dev/null
+++ b/profiles/tscc2/config.yaml
@@ -0,0 +1,16 @@
+verbose: true
+notemp: true
+latency: 60
+printshellcmds: true
+skip-script-cleanup: true
+nolock: true
+keep-going: true
+use-singularity: true
+singularity-args: "--bind /tscc"
+singularity-prefix: /tscc/nfs/home/hsher/scratch/singularity
+use-conda: true
+conda-prefix: "/tscc/nfs/home/hsher/snakeconda"
+conda-frontend: conda
+jobs: 30
+cluster: "sbatch -t {params.run_time} -e {params.error_file} -o {params.out_file} -p gold -q hcg-csd792 -A csd792 --mem {params.memory} --tasks-per-node {threads} -J {rule}"
+cluster-cancel: "scancel"
diff --git a/profiles/tscc2_gpu/config.yaml b/profiles/tscc2_gpu/config.yaml
new file mode 100644
index 0000000..42d60f2
--- /dev/null
+++ b/profiles/tscc2_gpu/config.yaml
@@ -0,0 +1,16 @@
+verbose: true
+notemp: true
+latency: 60
+printshellcmds: true
+skip-script-cleanup: true
+nolock: true
+keep-going: true
+use-singularity: true
+singularity-args: "--bind /tscc --nv"
+singularity-prefix: /tscc/nfs/home/hsher/scratch/singularity
+use-conda: true
+conda-prefix: "/tscc/nfs/home/hsher/snakeconda"
+conda-frontend: conda
+jobs: 30
+cluster: "sbatch -t {params.run_time} -e {params.error_file} -o {params.out_file} -p rtx3090 -q condo-gpu -A csd792 --gpus 1 -J {rule}"
+cluster-cancel: "scancel"
\ No newline at end of file
diff --git a/profiles/tscc2_single/config.yaml b/profiles/tscc2_single/config.yaml
new file mode 100644
index 0000000..1d24bf2
--- /dev/null
+++ b/profiles/tscc2_single/config.yaml
@@ -0,0 +1,14 @@
+verbose: false
+notemp: true
+latency: 60
+printshellcmds: true
+skip-script-cleanup: true
+nolock: true
+keep-going: true
+use-singularity: true
+singularity-args: "--bind /tscc"
+singularity-prefix: /tscc/nfs/home/hsher/scratch/singularity
+use-conda: true
+conda-prefix: "/tscc/nfs/home/hsher/snakeconda"
+conda-frontend: conda
+jobs: 8
\ No newline at end of file
diff --git a/profiles/tscc2_snakemake8/config.yaml b/profiles/tscc2_snakemake8/config.yaml
new file mode 100644
index 0000000..6f058ae
--- /dev/null
+++ b/profiles/tscc2_snakemake8/config.yaml
@@ -0,0 +1,29 @@
+verbose: true
+notemp: true
+latency: 60
+printshellcmds: true
+skip-script-cleanup: true
+nolock: true
+keep-going: true
+retries: 3
+use-singularity: "true"
+singularity-args: "--bind /tscc"
+singularity-prefix: /tscc/nfs/home/hsher/scratch/singularity
+use-conda: true
+conda-prefix: "/tscc/nfs/home/hsher/snakeconda"
+conda-not-block-search-path-envvars: true
+conda-frontend: conda
+jobs: 30
+executor: slurm
+default-resources:
+ slurm_partition: "gold"
+ slurm_account: "csd792"
+ slurm_extra: "--qos=hcg-csd792"
+ runtime: 30
+ mem_mb: 32000
+set-resources:
+ train_model:
+ slurm_partition: "rtx3090"
+ slurm_account: "csd792"
+ slurm_extra: "--qos=condo-gpu --gpus=1 --exclude tscc-gpu-9-1,tscc-gpu-5-16"
+ runtime: "2h"
diff --git a/rules/analysis.smk b/rules/analysis.smk
new file mode 100644
index 0000000..002e372
--- /dev/null
+++ b/rules/analysis.smk
@@ -0,0 +1,53 @@
+locals().update(config)
+rule run_homer:
+ input:
+ finemapped_windows = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz",
+ background = "output/homer/region_matched_background/fixed/{experiment_label}.sampled_fixed_windows.bed.gz",
+ genome = GENOME
+ output:
+ report = "output/homer/finemapped_results/{experiment_label}/homerResults.html"
+ resources:
+ mem_mb=8000,
+ runtime=50
+ benchmark: "benchmarks/run_homer/{experiment_label}.all_replicates.reproducible.txt"
+ container:
+ "docker://howardxu520/skipper:Homer_4.11"
+ shell:
+ "findMotifsGenome.pl <(zcat {input.finemapped_windows} | awk -v OFS=\"\t\" '{{print $4 \":\"$9,$1,$2+1,$3,$6}}') "
+ "{input.genome} output/homer/finemapped_results/{wildcards.experiment_label} -preparsedDir output/homer/preparsed -size given -rna -nofacts -S 20 -len 5,6,7,8,9 -nlen 1 "
+ "-bg <(zcat {input.background} | awk -v OFS=\"\t\" '{{print $4,$1,$2+1,$3,$6}}') "
+
+rule consult_encode_reference:
+ input:
+ enriched_windows = lambda wildcards: expand("output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz", experiment_label = experiment_labels),
+ enriched_re = lambda wildcards: expand("output/reproducible_enriched_re/{experiment_label}.reproducible_enriched_re.tsv.gz", experiment_label = experiment_labels),
+ encode_references = lambda wildcards: expand(TOOL_DIR + "/{reference}.reference.tsv", reference = ["encode3_feature_summary", "encode3_eclip_enrichment", "encode3_class_assignment"])
+ output:
+ tsne_coordinates = "output/tsne/skipper.tsne_query.tsv",
+ tsne_plot = "output/figures/tsne/skipper.tsne_query.pdf"
+ resources:
+ mem_mb=lambda wildcards, attempt: 4000 * (2 ** (attempt - 1)),
+ runtime=lambda wildcards, attempt: 60 * (2 ** (attempt - 1)),
+ benchmark: "benchmarks/consult_encode_reference/skipper.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/consult_encode_reference.R output/reproducible_enriched_windows output/reproducible_enriched_re {TOOL_DIR} skipper "
+
+rule consult_term_reference:
+ input:
+ enriched_windows = "output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz",
+ gene_sets = GENE_SETS,
+ gene_set_reference = GENE_SET_REFERENCE,
+ gene_set_distance = GENE_SET_DISTANCE
+ output:
+ enrichment_results = "output/gene_sets/{experiment_label}.enriched_terms.tsv.gz",
+ enrichment_plot = "output/figures/gene_sets/{experiment_label}.clustered_top_terms.pdf"
+ resources:
+ mem_mb=1000,
+ runtime=15
+ benchmark: "benchmarks/consult_term_reference/{experiment_label}.all_replicates.reproducible.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/consult_term_reference.R {input.enriched_windows} {input.gene_sets} {input.gene_set_reference} {input.gene_set_distance} {wildcards.experiment_label} "
diff --git a/rules/bigwig.smk b/rules/bigwig.smk
new file mode 100644
index 0000000..7dfa970
--- /dev/null
+++ b/rules/bigwig.smk
@@ -0,0 +1,70 @@
+locals().update(config)
+rule make_unscaled_bigwig:
+ input:
+ CHROM_SIZES,
+ bam = lambda wildcards: config['replicate_label_to_bams'][wildcards.replicate_label],
+ output:
+ bg_plus = temp("output/bedgraphs/unscaled/plus/{replicate_label}.unscaled.plus.bg"),
+ bg_minus = temp("output/bedgraphs/unscaled/minus/{replicate_label}.unscaled.minus.bg"),
+ bw_plus = "output/bigwigs/unscaled/plus/{replicate_label}.unscaled.plus.bw",
+ bw_minus = "output/bigwigs/unscaled/minus/{replicate_label}.unscaled.minus.bw",
+ benchmark: "benchmarks/bigwigs/unassigned_experiment.{replicate_label}.make_bigwig.txt"
+ container:
+ "docker://howardxu520/skipper:bigwig_1.0"
+ resources:
+ mem_mb=10000,
+ tmpdir=TMPDIR,
+ runtime=120
+ shell:
+ "samtools index {input.bam};"
+ "bedtools genomecov -split -strand + -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_plus};"
+ "bedtools genomecov -split -strand - -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_minus};"
+ "bedGraphToBigWig {output.bg_plus} {CHROM_SIZES} {output.bw_plus};"
+ "bedGraphToBigWig {output.bg_minus} {CHROM_SIZES} {output.bw_minus};"
+
+rule make_scaled_bigwig:
+ input:
+ CHROM_SIZES,
+ bam = lambda wildcards: config['replicate_label_to_bams'][wildcards.replicate_label],
+ output:
+ bg_plus = temp("output/bedgraphs/scaled/plus/{replicate_label}.scaled.plus.bg"),
+ bg_minus = temp("output/bedgraphs/scaled/minus/{replicate_label}.scaled.minus.bg"),
+ bw_plus = "output/bigwigs/scaled/plus/{replicate_label}.scaled.plus.bw",
+ bw_minus = "output/bigwigs/scaled/minus/{replicate_label}.scaled.minus.bw",
+ resources:
+ mem_mb=10000,
+ tmpdir=TMPDIR,
+ runtime=120
+ benchmark: "benchmarks/bigwigs/unassigned_experiment.{replicate_label}.make_bigwig.txt"
+ container:
+ "docker://howardxu520/skipper:bigwig_1.0"
+ shell:
+ "samtools index {input.bam};"
+ "FACTOR=$(samtools idxstats {input.bam} | cut -f 3 | paste -sd+ | bc | xargs -I {{}} echo 'scale=6; 10^6 / {{}}' | bc);"
+ "bedtools genomecov -scale $FACTOR -split -strand + -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_plus};"
+ "bedtools genomecov -scale $FACTOR -split -strand - -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_minus};"
+ "bedGraphToBigWig {output.bg_plus} {CHROM_SIZES} {output.bw_plus};"
+ "bedGraphToBigWig {output.bg_minus} {CHROM_SIZES} {output.bw_minus};"
+
+rule make_scaled_bigwig_coverage:
+ input:
+ CHROM_SIZES,
+ bam = lambda wildcards: config['replicate_label_to_bams'][wildcards.replicate_label],
+ output:
+ bg_plus = temp("output/bedgraphs/scaled/plus/{replicate_label}.scaled.cov.plus.bg"),
+ bg_minus = temp("output/bedgraphs/scaled/minus/{replicate_label}.scaled.cov.minus.bg"),
+ bw_plus = "output/bigwigs/scaled/plus/{replicate_label}.scaled.cov.plus.bw",
+ bw_minus = "output/bigwigs/scaled/minus/{replicate_label}.scaled.cov.minus.bw",
+ benchmark: "benchmarks/bigwigs/unassigned_experiment.{replicate_label}.make_bigwig.txt"
+ resources:
+ mem_mb=10000,
+ tmpdir=TMPDIR,
+ runtime=120
+ container:
+ "docker://howardxu520/skipper:bigwig_1.0"
+ shell:
+ "factor=$(samtools idxstats {input.bam} | cut -f 3 | paste -sd+ | bc | xargs -I {{}} echo 'scale=6; 10^6 / {{}}' | bc);"
+ "bedtools genomecov -scale $factor -strand + -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_plus};"
+ "bedtools genomecov -scale $factor -strand - -bg -ibam {input.bam} | sort -k1,1 -k2,2n | grep -v EBV > {output.bg_minus};"
+ "bedGraphToBigWig {output.bg_plus} {CHROM_SIZES} {output.bw_plus};"
+ "bedGraphToBigWig {output.bg_minus} {CHROM_SIZES} {output.bw_minus};"
diff --git a/rules/ctk_mcross.smk b/rules/ctk_mcross.smk
new file mode 100644
index 0000000..a68620f
--- /dev/null
+++ b/rules/ctk_mcross.smk
@@ -0,0 +1,69 @@
+rule ctk:
+ input:
+ bam_ip_umap = "output/bams/genome/{replicate_label}.genome.Aligned.sort.dedup.umap.bam"
+ output:
+ mdtag = temp("output/bams/genome/{replicate_label}.genome.Aligned.sort.dedup.umap.md.sam.gz"),
+ mutation_file = "output/ctk/{replicate_label}.mutation.txt",
+ tagbed = "output/ctk/{replicate_label}.tag.bed",
+ peak = "output/ctk/{replicate_label}.uniq.peak.sig.bed",
+ peak_bd = "output/ctk/{replicate_label}.uniq.peak.sig.boundary.bed",
+ peak_PH = "output/ctk/{replicate_label}.uniq.peak.sig.halfPH.bed",
+ params:
+ error_out_file = "error_files/ctk.{replicate_label}.err",
+ out_file = "stdout/ctk.{replicate_label}.out",
+ run_time = "2:10:00",
+ memory = 10000,
+ cores = 1,
+ conda:
+ "envs/ctk.yaml"
+ shell:
+ """
+ samtools fillmd {input.bam_ip_umap} {GENOMEFA} | gzip -c > {output.mdtag}
+ parseAlignment.pl \
+ -v --map-qual 1 \
+ --min-len 18 \
+ --mutation-file {output.mutation_file} \
+ {output.mdtag} - > {output.tagbed}
+
+ tag2peak.pl -big -ss \
+ -v --valley-seeking -p 0.05 --valley-depth 0.9 \
+ --multi-test --dbkey hg38 \
+ {output.tagbed} \
+ {output.peak} \
+ --out-boundary {output.peak_bd} \
+ --out-half-PH {output.peak_PH}
+ """
+
+rule mcross_get_kmer_seed:
+ input:
+ foreground = rules.fetch_sequence.output.finemapped_fa,
+ background = rules.fetch_sequence.output.background_fa,
+ output:
+ kmer_enrichment = "output/ctk/mcross/{experiment_label}.kmer.txt",
+ config = "output/ctk/mcross/{experiment_label}.config.txt",
+ topn_kmer_matrix = "output/ctk/mcross/{experiment_label}.w7.zcore.mat.txt",
+ top_peak = "output/ctk/mcross/top7mer/top.{experiment_label}.txt",
+ params:
+ error_file = "error_files/mcross_kmer.{experiment_label}.err",
+ out_file = "stdout/mcross_kmer.{experiment_label}.out",
+ run_time = "2:00:00",
+ memory = 10000,
+ cores = 1,
+ conda:
+ "envs/ctk.yaml"
+ shell:
+ """
+ word_enrich.pl -w 7 \
+ -test binom -v \
+ {input.foreground} \
+ {input.background} \
+ {output.kmer_enrichment}
+
+ # generate config
+ echo '{output.kmer_enrichment}"\t\"{wildcards.experiment_label}' > {output.config}
+
+ gen_word_enrich_matrix.pl \
+ {output.config} {output.topn_kmer_matrix}
+
+ topword.R {output.topn_kmer_matrix} {wildcards.experiment_label}_top7mer
+ """
\ No newline at end of file
diff --git a/rules/envs/bcftools.yaml b/rules/envs/bcftools.yaml
new file mode 100644
index 0000000..b348d25
--- /dev/null
+++ b/rules/envs/bcftools.yaml
@@ -0,0 +1,8 @@
+name: ls-gkm
+channels:
+ - conda-forge
+ - bioconda
+ - defaults
+dependencies:
+ - bcftools
+prefix: /home/hsher/miniconda3/envs/ls-gkm
diff --git a/rules/envs/eugene.yaml b/rules/envs/eugene.yaml
new file mode 100644
index 0000000..2e2608b
--- /dev/null
+++ b/rules/envs/eugene.yaml
@@ -0,0 +1,118 @@
+name: eugene2
+channels:
+ - defaults
+dependencies:
+ - _libgcc_mutex=0.1=main
+ - _openmp_mutex=5.1=1_gnu
+ - ca-certificates=2024.7.2=h06a4308_0
+ - ld_impl_linux-64=2.38=h1181459_1
+ - libffi=3.3=he6710b0_2
+ - libgcc-ng=11.2.0=h1234567_1
+ - libgomp=11.2.0=h1234567_1
+ - libstdcxx-ng=11.2.0=h1234567_1
+ - ncurses=6.4=h6a678d5_0
+ - openssl=1.1.1w=h7f8727e_0
+ - pip=24.0=py39h06a4308_0
+ - python=3.9.2=hdb3f193_0
+ - readline=8.2=h5eee18b_0
+ - setuptools=72.1.0=py39h06a4308_0
+ - sqlite=3.45.3=h5eee18b_0
+ - tk=8.6.14=h39e8969_0
+ - tzdata=2024a=h04d1e81_0
+ - wheel=0.43.0=py39h06a4308_0
+ - xz=5.4.6=h5eee18b_1
+ - zlib=1.2.13=h5eee18b_1
+ - pip:
+ - absl-py==2.1.0
+ - asciitree==0.3.3
+ - biopython==1.77
+ - cachetools==5.4.0
+ - captum==0.5.0
+ - cloudpickle==3.0.0
+ - coloredlogs==15.0.1
+ - cyvcf2==0.30.28
+ - dask==2023.12.1
+ - dask-glm==0.3.2
+ - dask-ml==2023.3.24
+ - distributed==2023.12.1
+ - einops==0.6.1
+ - entrypoints==0.4
+ - eugene-tools==0.1.2
+ - fasteners==0.19
+ - google-auth==2.32.0
+ - google-auth-oauthlib==0.4.6
+ - graph-part==0.1.2
+ - grpcio==1.65.4
+ - hdf5plugin==4.4.0
+ - humanfriendly==10.0
+ - idna==3.7
+ - igraph==0.11.6
+ - leidenalg==0.10.2
+ - lightning-utilities==0.11.6
+ - llvmlite==0.43.0
+ - locket==1.0.0
+ - logomaker==0.8
+ - markdown==3.6
+ - modisco-lite==2.2.1
+ - more-itertools==9.1.0
+ - motifdata==0.1.2
+ - msgpack==1.0.8
+ - multimethod==1.12
+ - multipledispatch==1.0.0
+ - natsort==8.4.0
+ - ncls==0.0.68
+ - numba
+ - numcodecs==0.11.0
+ - numpy==1.23.5
+ - oauthlib==3.2.2
+ - packaging==24.1
+ - pandas
+ - pandera==0.14.5
+ - partd==1.4.2
+ - pillow==10.4.0
+ - polars==0.18.15
+ - protobuf==3.20.3
+ - psutil==6.0.0
+ - pyasn1==0.6.0
+ - pyasn1-modules==0.4.0
+ - pybedtools==0.9.1
+ - pybigwig==0.3.23
+ - pyjaspar==2.1.1
+ - pymemesuite==0.1.0a1
+ - pyparsing==3.1.2
+ - pyranges==0.0.120
+ - pyrle==0.0.40
+ - pysam==0.21.0
+ - pytorch-lightning==2.3.3
+ - pytz==2024.1
+ - pyarrow
+ - requests-oauthlib==2.0.0
+ - rsa==4.9
+ - scikit-learn==1.2.0
+ - seaborn==0.12.2
+ - seqdata==0.1.3
+ - seqdatasets==0.1.2
+ - seqexplainer==0.1.2
+ - seqpro==0.1.11
+ - six==1.16.0
+ - sorted-nearest==0.0.39
+ - sortedcontainers==2.4.0
+ - sparse==0.15.4
+ - statsmodels
+ - tabulate==0.9.0
+ - tblib==3.0.0
+ - tensorboard==2.11.2
+ - tensorboard-data-server==0.6.1
+ - tensorboard-plugin-wit==1.8.1
+ - texttable==1.7.0
+ - toolz==0.12.1
+ - torchinfo==1.8.0
+ - torchmetrics==1.4.1
+ - urllib3==2.2.2
+ - werkzeug==3.0.3
+ - wget==3.2
+ - xarray==2023.4.0
+ - yuzu-ism==0.0.1
+ - zarr==2.18.2
+ - zict==3.0.0
+prefix: /tscc/nfs/home/hsher/miniconda3/envs/eugene2
diff --git a/rules/envs/eugene2.yaml b/rules/envs/eugene2.yaml
new file mode 100644
index 0000000..2e2608b
--- /dev/null
+++ b/rules/envs/eugene2.yaml
@@ -0,0 +1,118 @@
+name: eugene2
+channels:
+ - defaults
+dependencies:
+ - _libgcc_mutex=0.1=main
+ - _openmp_mutex=5.1=1_gnu
+ - ca-certificates=2024.7.2=h06a4308_0
+ - ld_impl_linux-64=2.38=h1181459_1
+ - libffi=3.3=he6710b0_2
+ - libgcc-ng=11.2.0=h1234567_1
+ - libgomp=11.2.0=h1234567_1
+ - libstdcxx-ng=11.2.0=h1234567_1
+ - ncurses=6.4=h6a678d5_0
+ - openssl=1.1.1w=h7f8727e_0
+ - pip=24.0=py39h06a4308_0
+ - python=3.9.2=hdb3f193_0
+ - readline=8.2=h5eee18b_0
+ - setuptools=72.1.0=py39h06a4308_0
+ - sqlite=3.45.3=h5eee18b_0
+ - tk=8.6.14=h39e8969_0
+ - tzdata=2024a=h04d1e81_0
+ - wheel=0.43.0=py39h06a4308_0
+ - xz=5.4.6=h5eee18b_1
+ - zlib=1.2.13=h5eee18b_1
+ - pip:
+ - absl-py==2.1.0
+ - asciitree==0.3.3
+ - biopython==1.77
+ - cachetools==5.4.0
+ - captum==0.5.0
+ - cloudpickle==3.0.0
+ - coloredlogs==15.0.1
+ - cyvcf2==0.30.28
+ - dask==2023.12.1
+ - dask-glm==0.3.2
+ - dask-ml==2023.3.24
+ - distributed==2023.12.1
+ - einops==0.6.1
+ - entrypoints==0.4
+ - eugene-tools==0.1.2
+ - fasteners==0.19
+ - google-auth==2.32.0
+ - google-auth-oauthlib==0.4.6
+ - graph-part==0.1.2
+ - grpcio==1.65.4
+ - hdf5plugin==4.4.0
+ - humanfriendly==10.0
+ - idna==3.7
+ - igraph==0.11.6
+ - leidenalg==0.10.2
+ - lightning-utilities==0.11.6
+ - llvmlite==0.43.0
+ - locket==1.0.0
+ - logomaker==0.8
+ - markdown==3.6
+ - modisco-lite==2.2.1
+ - more-itertools==9.1.0
+ - motifdata==0.1.2
+ - msgpack==1.0.8
+ - multimethod==1.12
+ - multipledispatch==1.0.0
+ - natsort==8.4.0
+ - ncls==0.0.68
+ - numba
+ - numcodecs==0.11.0
+ - numpy==1.23.5
+ - oauthlib==3.2.2
+ - packaging==24.1
+ - pandas
+ - pandera==0.14.5
+ - partd==1.4.2
+ - pillow==10.4.0
+ - polars==0.18.15
+ - protobuf==3.20.3
+ - psutil==6.0.0
+ - pyasn1==0.6.0
+ - pyasn1-modules==0.4.0
+ - pybedtools==0.9.1
+ - pybigwig==0.3.23
+ - pyjaspar==2.1.1
+ - pymemesuite==0.1.0a1
+ - pyparsing==3.1.2
+ - pyranges==0.0.120
+ - pyrle==0.0.40
+ - pysam==0.21.0
+ - pytorch-lightning==2.3.3
+ - pytz==2024.1
+ - pyarrow
+ - requests-oauthlib==2.0.0
+ - rsa==4.9
+ - scikit-learn==1.2.0
+ - seaborn==0.12.2
+ - seqdata==0.1.3
+ - seqdatasets==0.1.2
+ - seqexplainer==0.1.2
+ - seqpro==0.1.11
+ - six==1.16.0
+ - sorted-nearest==0.0.39
+ - sortedcontainers==2.4.0
+ - sparse==0.15.4
+ - statsmodels
+ - tabulate==0.9.0
+ - tblib==3.0.0
+ - tensorboard==2.11.2
+ - tensorboard-data-server==0.6.1
+ - tensorboard-plugin-wit==1.8.1
+ - texttable==1.7.0
+ - toolz==0.12.1
+ - torchinfo==1.8.0
+ - torchmetrics==1.4.1
+ - urllib3==2.2.2
+ - werkzeug==3.0.3
+ - wget==3.2
+ - xarray==2023.4.0
+ - yuzu-ism==0.0.1
+ - zarr==2.18.2
+ - zict==3.0.0
+prefix: /tscc/nfs/home/hsher/miniconda3/envs/eugene2
diff --git a/rules/envs/fast.yaml b/rules/envs/fast.yaml
new file mode 100644
index 0000000..1e40f4c
--- /dev/null
+++ b/rules/envs/fast.yaml
@@ -0,0 +1,8 @@
+name: fast
+channels:
+ - conda-forge
+ - bioconda
+ - defaults
+dependencies:
+ - perl-fast
+prefix: /home/hsher/miniconda3/envs/fast
\ No newline at end of file
diff --git a/rules/envs/lsgkm.yaml b/rules/envs/lsgkm.yaml
new file mode 100644
index 0000000..92f52da
--- /dev/null
+++ b/rules/envs/lsgkm.yaml
@@ -0,0 +1,9 @@
+name: ls-gkm
+channels:
+ - conda-forge
+ - bioconda
+ - defaults
+dependencies:
+ - libstdcxx-ng=13.2.0=h7e041cc_3
+ - ls-gkm=0.0.1=h2d50403_1
+prefix: /home/hsher/miniconda3/envs/ls-gkm
diff --git a/rules/envs/metadensity.yaml b/rules/envs/metadensity.yaml
new file mode 100644
index 0000000..e945dd6
--- /dev/null
+++ b/rules/envs/metadensity.yaml
@@ -0,0 +1,24 @@
+name: metadensity
+channels:
+ - anaconda
+ - conda-forge
+ - bioconda
+ - defaults
+dependencies:
+ - deepdish=0.3.6=pyh9f0ad1d_0
+ - matplotlib=3.4.3=py37h89c1867_2
+ - matplotlib-base=3.4.3=py37h1058ff1_1
+ - matplotlib-inline=0.1.3=pyhd8ed1ab_0
+ - numpy=1.21.2=py37h31617e3_0
+ - pandas=1.1.5=py37hdc94413_0
+ - python=3.7.12=hb7a2778_100_cpython
+ - scipy=1.7.1=py37hf2a6cf1_0
+ - setuptools=58.2.0=py37h89c1867_0
+ - statsmodels=0.12.0=py37h7b6447c_0
+ - pybigwig
+ - pysam
+ - biopython
+ - scikit-learn
+ - seaborn
+ - pybedtools
+prefix: /home/hsher/miniconda3/envs/metadensity
diff --git a/rules/envs/mondo.yaml b/rules/envs/mondo.yaml
new file mode 100644
index 0000000..d942e40
--- /dev/null
+++ b/rules/envs/mondo.yaml
@@ -0,0 +1,20 @@
+name: my_metadensity
+channels:
+ - bioconda
+ - anaconda
+ - conda-forge
+ - defaults
+dependencies:
+ - python
+ - networkx=2.6.3
+ - numpy
+ - pandas=1.3.5
+ - pip
+ - scikit-learn
+ - scipy
+ - seaborn
+ - statsmodels=0.12.0=py37h7b6447c_0
+ - pip:
+ - nxontology==0.4.0
+ - venn
+prefix: /tscc/nfs/home/hsher/miniconda3/envs/my_metadensity
diff --git a/rules/envs/pum2model.yaml b/rules/envs/pum2model.yaml
new file mode 100644
index 0000000..277d9ad
--- /dev/null
+++ b/rules/envs/pum2model.yaml
@@ -0,0 +1,44 @@
+name: pum2model
+channels:
+ - conda-forge
+ - defaults
+dependencies:
+ - _libgcc_mutex=0.1=main
+ - _openmp_mutex=5.1=1_gnu
+ - asteval=1.0.1=pyhd8ed1ab_0
+ - blas=1.0=openblas
+ - bzip2=1.0.8=h5eee18b_6
+ - ca-certificates=2024.7.2=h06a4308_0
+ - dill=0.3.8=py312h06a4308_0
+ - expat=2.6.2=h6a678d5_0
+ - future=0.18.3=py312h06a4308_0
+ - ld_impl_linux-64=2.38=h1181459_1
+ - libffi=3.4.4=h6a678d5_1
+ - libgcc-ng=11.2.0=h1234567_1
+ - libgfortran-ng=11.2.0=h00389a5_1
+ - libgfortran5=11.2.0=h1234567_1
+ - libgomp=11.2.0=h1234567_1
+ - libopenblas=0.3.21=h043d6bf_0
+ - libstdcxx-ng=11.2.0=h1234567_1
+ - libuuid=1.41.5=h5eee18b_0
+ - lmfit=1.3.1=pyhd8ed1ab_1
+ - ncurses=6.4=h6a678d5_0
+ - numpy=1.26.4=py312h2809609_0
+ - numpy-base=1.26.4=py312he1a6c75_0
+ - openssl=3.0.14=h5eee18b_0
+ - packaging=24.1=py312h06a4308_0
+ - pip=24.0=py312h06a4308_0
+ - pybind11-abi=5=hd3eb1b0_0
+ - python=3.12.4=h5148396_1
+ - readline=8.2=h5eee18b_0
+ - scipy=1.13.1=py312h2809609_0
+ - setuptools=69.5.1=py312h06a4308_0
+ - setuptools-scm=8.1.0=py312h06a4308_0
+ - sqlite=3.45.3=h5eee18b_0
+ - tk=8.6.14=h39e8969_0
+ - tzdata=2024a=h04d1e81_0
+ - uncertainties=3.2.2=pyhd8ed1ab_1
+ - wheel=0.43.0=py312h06a4308_0
+ - xz=5.4.6=h5eee18b_1
+ - zlib=1.2.13=h5eee18b_1
+prefix: /tscc/nfs/home/hsher/miniconda3/envs/pum2model
diff --git a/rules/envs/skewer.yaml b/rules/envs/skewer.yaml
new file mode 100644
index 0000000..1c01200
--- /dev/null
+++ b/rules/envs/skewer.yaml
@@ -0,0 +1,13 @@
+name: skewer
+channels:
+ - conda-forge
+ - bioconda
+ - defaults
+dependencies:
+ - _libgcc_mutex=0.1=conda_forge
+ - _openmp_mutex=4.5=2_gnu
+ - libgcc-ng=12.2.0=h65d4601_19
+ - libgomp=12.2.0=h65d4601_19
+ - libstdcxx-ng=12.2.0=h46fd767_19
+ - skewer=0.2.2=hc9558a2_3
+prefix: /home/hsher/miniconda3/envs/skewer
diff --git a/rules/finemap.smk b/rules/finemap.smk
new file mode 100644
index 0000000..232fd72
--- /dev/null
+++ b/rules/finemap.smk
@@ -0,0 +1,103 @@
+locals().update(config)
+rule get_nt_coverage:
+ input:
+ windows = "output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz",
+ clip_bams = lambda wildcards: [config['replicate_label_to_bams'][clip_replicate_label] for clip_replicate_label in experiment_to_clip_replicate_labels[wildcards.experiment_label]],
+ input_bams = lambda wildcards: [config['replicate_label_to_bams'][input_replicate_label] for input_replicate_label in experiment_to_input_replicate_labels[wildcards.experiment_label]],
+ output:
+ nt_census = temp("output/finemapping/nt_coverage/{experiment_label}.nt_census.bed"),
+ nt_input_counts = temp("output/finemapping/nt_coverage/{experiment_label}.nt_coverage.input.counts"),
+ nt_clip_counts = temp("output/finemapping/nt_coverage/{experiment_label}.nt_coverage.clip.counts"),
+ nt_coverage = "output/finemapping/nt_coverage/{experiment_label}.nt_coverage.bed"
+ threads: 6
+ resources:
+ mem_mb=64000,
+ runtime="6h",
+ tmpdir=TMPDIR
+ benchmark: "benchmarks/get_nt_coverage/{experiment_label}.all_replicates.reproducible.txt"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17_bedtools_2.31.0"
+ shell:
+ "export TMPDIR={resources.tmpdir};"
+ "zcat {input.windows} | tail -n +2 | sort -k1,1 -k2,2n | awk -v OFS=\"\t\" '{{start = $2-37; if(start < 0) {{start = 0}}; print $1, start, $3+37,$4,$5,$6}}' | "
+ "bedtools merge -i - -s -c 6 -o distinct | awk -v OFS=\"\t\" '{{for(i=$2;i< $3;i++) {{print $1,i,i+1,\"MW:\" NR \":\" i - $2,0,$4, NR}} }}' > {output.nt_census}; "
+ "samtools cat {input.input_bams} | bedtools intersect -s -wa -a - -b {output.nt_census} | "
+ "bedtools bamtobed -i - | awk '($1 != \"chrEBV\") && ($4 !~ \"/{UNINFORMATIVE_READ}$\")' | "
+ "bedtools flank -s -l 1 -r 0 -g {CHROM_SIZES} -i - | "
+ "bedtools shift -p 1 -m -1 -g {CHROM_SIZES} -i - | "
+ "bedtools sort -i - | "
+ "bedtools coverage -counts -s -a {output.nt_census} -b - | awk '{{print $NF}}' > {output.nt_input_counts};"
+ "samtools cat {input.clip_bams} | bedtools intersect -s -wa -a - -b {output.nt_census} | "
+ "bedtools bamtobed -i - | awk '($1 != \"chrEBV\") && ($4 !~ \"/{UNINFORMATIVE_READ}$\")' | "
+ "bedtools flank -s -l 1 -r 0 -g {CHROM_SIZES} -i - | "
+ "bedtools shift -p 1 -m -1 -g {CHROM_SIZES} -i - | "
+ "bedtools sort -i - | "
+ "bedtools coverage -counts -s -a {output.nt_census} -b - | awk '{{print $NF}}' > {output.nt_clip_counts};"
+ "paste {output.nt_census} {output.nt_input_counts} {output.nt_clip_counts} > {output.nt_coverage}"
+
+rule finemap_windows:
+ input:
+ nt_coverage = rules.get_nt_coverage.output.nt_coverage,
+ output:
+ finemapped_windows = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz"
+ threads: 6,
+ resources:
+ mem_mb=45000,
+ runtime="2h"
+ benchmark: "benchmarks/finemap_windows/{experiment_label}.all_replicates.reproducible.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ resources:
+ mem_mb=45000
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/finemap_enriched_windows.R {input.nt_coverage} output/finemapping/mapped_sites/ {wildcards.experiment_label}"
+
+rule annotate_finemap:
+ input:
+ finemapped = rules.finemap_windows.output.finemapped_windows,
+ feature_annotations = FEATURE_ANNOTATIONS,
+ ranking = ACCESSION_RANKINGS,
+ output:
+ "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.annotated.tsv"
+ threads:
+ 1
+ resources:
+ mem_mb=64000,
+ runtime="4h"
+ conda:
+ "envs/metadensity.yaml"
+ shell:
+ """
+ module purge; # fixes weird $PATH issue where python being used is the module python, not the one in conda environment.
+ if [ -s {input.finemapped} ]; then
+ python {TOOL_DIR}/annotate_finemapped_regions.py \
+ {input.finemapped} \
+ {input.ranking} \
+ {input.feature_annotations} \
+ {output}
+ else
+ touch {output}
+ fi
+ """
+
+rule find_both_tested_windows:
+ input:
+ lambda wildcards: expand("output/tested_windows/{{experiment_label}}.{clip_replicate_label}.tested_windows.tsv.gz",
+ clip_replicate_label = experiment_to_clip_replicate_labels[wildcards.experiment_label])
+ output:
+ tested_windows_in_2_rep = "output/finemapping/both_tested_sites/{experiment_label}.both_tested_windows.bed",
+ tested_windows_merged = "output/finemapping/both_tested_sites/{experiment_label}.both_tested_windows.merged.bed"
+ threads: 1
+ resources:
+ mem_mb=45000,
+ runtime="30"
+ conda:
+ "envs/metadensity.yaml"
+ shell:
+ """
+ module purge; # fixes weird $PATH issue where python being used is the module python, not the one in conda environment.
+ python {TOOL_DIR}/find_both_tested_windows.py \
+ "{input}" \
+ {output.tested_windows_in_2_rep} \
+ {output.tested_windows_merged}
+ """
diff --git a/rules/genome_windows.smk b/rules/genome_windows.smk
new file mode 100644
index 0000000..5a4ff9d
--- /dev/null
+++ b/rules/genome_windows.smk
@@ -0,0 +1,195 @@
+locals().update(config)
+rule parse_gff:
+ input:
+ gff = ancient(GFF),
+ rankings = ancient(ACCESSION_RANKINGS),
+ output:
+ partition = PARTITION,
+ feature_annotations = FEATURE_ANNOTATIONS,
+ threads: 1
+ resources:
+ mem_mb=48000,
+ runtime="3h"
+ benchmark: "benchmarks/parse_gff.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/parse_gff.R {input.gff} {input.rankings} {output.partition} {output.feature_annotations}"
+
+rule partition_bam_reads:
+ input:
+ CHROM_SIZES,
+ bam = lambda wildcards: config['replicate_label_to_bams'][wildcards.replicate_label],
+ region_partition = PARTITION,
+ output:
+ counts = "output/counts/genome/vectors/{replicate_label}.counts",
+ resources:
+ mem_mb=lambda wildcards, attempt: 64000 * (1.5 ** (attempt - 1)),
+ runtime="12h"
+ benchmark: "benchmarks/counts/unassigned_experiment.{replicate_label}.partition_bam_reads.txt"
+ container:
+ "docker://howardxu520/skipper:bedtools_2.31.0"
+ shell:
+ "bedtools bamtobed -i {input.bam} | awk '($1 != \"chrEBV\") && ($4 !~ \"/{UNINFORMATIVE_READ}$\")' | "
+ "bedtools flank -s -l 1 -r 0 -g {CHROM_SIZES} -i - | "
+ "bedtools shift -p 1 -m -1 -g {CHROM_SIZES} -i - | "
+ "bedtools sort -i - | "
+ "bedtools coverage -counts -s -a {input.region_partition} -b - | cut -f 7 | "
+ "awk 'BEGIN {{print \"{wildcards.replicate_label}\"}} {{print}}' > {output.counts};"
+
+rule calc_partition_nuc:
+ input:
+ partition = PARTITION,
+ genome = GENOME
+ output:
+ nuc = PARTITION.replace(".bed", ".nuc")
+ resources:
+ mem_mb=16000,
+ runtime="2h"
+ benchmark: "benchmarks/partition_nuc.txt"
+ container:
+ "docker://howardxu520/skipper:bedtools_2.31.0"
+ shell:
+ "bedtools nuc -s -fi {input.genome} -bed {input.partition} | gzip -c > {output.nuc}"
+
+rule make_genome_count_table:
+ input:
+ partition = PARTITION.replace(".bed", ".nuc"),
+ replicate_counts = lambda wildcards: expand("output/counts/genome/vectors/{replicate_label}.counts", replicate_label = experiment_to_replicate_labels[wildcards.experiment_label]),
+ output:
+ count_table = "output/counts/genome/tables/{experiment_label}.tsv.gz",
+ threads: 4
+ resources:
+ mem_mb=1000,
+ runtime=10
+ benchmark: "benchmarks/counts/{experiment_label}.all_replicates.make_genome_count_table.txt"
+ container:
+ "docker://howardxu520/skipper:bedtools_2.31.0"
+ resources:
+ mem_mb=1000
+ shell:
+ "paste <(zcat {input.partition} | awk -v OFS=\"\\t\" 'BEGIN {{print \"chr\\tstart\\tend\\tname\\tscore\\tstrand\\tgc\"}} NR > 1 {{print $1,$2,$3,$4,$5,$6,$8}}' ) {input.replicate_counts} | gzip -c > {output.count_table};"
+
+rule fit_input_betabinomial_model:
+ input:
+ table = rules.make_genome_count_table.output.count_table
+ output:
+ coef = "output/input_model_coef/{experiment_label}.{input_replicate_label}.tsv",
+ # plot = lambda wildcards: expand("output/figures/input_distributions/{{experiment_label}}.{{input_replicate_label}}.{other_label}.input_distribution.pdf", other_label = experiment_to_input_replicate_labels[wildcards.experiment_label][wildcards.Input_replicate_label])
+ threads: 4
+ benchmark: "benchmarks/betabinomial/{experiment_label}.{input_replicate_label}.fit_input.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ resources:
+ mem_mb=48000,
+ runtime="6h"
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/fit_input_betabinom.R {input.table} {wildcards.experiment_label} {wildcards.input_replicate_label}"
+
+rule fit_clip_betabinomial_model:
+ input:
+ table = rules.make_genome_count_table.output.count_table
+ output:
+ coef = "output/clip_model_coef/{experiment_label}.{clip_replicate_label}.tsv",
+ # plot = lambda wildcards: expand("output/figures/clip_distributions/{{experiment_label}}.{{clip_replicate_label}}.{other_label}.clip_distribution.pdf", other_label = experiment_to_input_replicate_labels[wildcards.experiment_label][wildcards.Input_replicate_label])
+ threads: 2
+ benchmark: "benchmarks/fit_clip_betabinomial_model/{experiment_label}.{clip_replicate_label}.fit_clip.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ resources:
+ mem_mb=48000,
+ runtime="6h"
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/fit_clip_betabinom.R {input.table} {wildcards.experiment_label} {wildcards.clip_replicate_label}"
+
+rule call_enriched_windows:
+ input:
+ feature_annotations = ancient(FEATURE_ANNOTATIONS),
+ accession_rankings = ancient(ACCESSION_RANKINGS),
+ replicate = lambda wildcards: "output/counts/genome/vectors/" + re.sub("IP_\d$","IP_2",wildcards.clip_replicate_label) + ".counts",
+ table = rules.make_genome_count_table.output.count_table,
+ parameters = lambda wildcards: "output/" + OVERDISPERSION_MODE + "_model_coef/{experiment_label}." + overdispersion_replicate_lookup[wildcards.clip_replicate_label] + ".tsv",
+ # parameters = lambda wildcards: "output/clip_model_coef/{experiment_label}.{wildcards.clip_replicate_label}.tsv",
+ output:
+ "output/threshold_scan/{experiment_label}.{clip_replicate_label}.threshold_data.tsv",
+ "output/tested_windows/{experiment_label}.{clip_replicate_label}.tested_windows.tsv.gz",
+ "output/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_windows.tsv.gz",
+ "output/enrichment_summaries/{experiment_label}.{clip_replicate_label}.enriched_window_feature_summary.tsv",
+ "output/enrichment_summaries/{experiment_label}.{clip_replicate_label}.enriched_window_transcript_summary.tsv",
+ "output/enrichment_summaries/{experiment_label}.{clip_replicate_label}.enriched_window_gene_summary.tsv",
+ "output/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_fractions_feature_data.tsv",
+ "output/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds_feature_data.tsv",
+ "output/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds_transcript_data.tsv",
+ "output/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds_feature_gc_data.tsv",
+ "output/figures/threshold_scan/{experiment_label}.{clip_replicate_label}.threshold_scan.pdf",
+ "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_coverage.pdf",
+ "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_rates.pdf",
+ "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_counts.linear.pdf",
+ "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_counts.log10.pdf",
+ "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_odds.feature.pdf",
+ "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_odds.all_transcript_types.pdf",
+ "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_odds.select_transcript_types.pdf",
+ "output/figures/enriched_windows/{experiment_label}.{clip_replicate_label}.enriched_window_counts.per_gene_feature.pdf",
+ "output/figures/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_fractions.feature.pdf",
+ "output/figures/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds.feature.pdf",
+ "output/figures/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds.all_transcript_types.pdf",
+ "output/figures/all_reads/{experiment_label}.{clip_replicate_label}.all_reads_odds.feature_gc.pdf"
+ threads: 2
+ benchmark: "benchmarks/call_enriched_windows/{experiment_label}.{clip_replicate_label}.call_enriched_windows.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ params:
+ input_replicate_label = lambda wildcards: clip_to_input_replicate_label[wildcards.clip_replicate_label]
+ resources:
+ mem_mb=48000,
+ runtime="24h"
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/call_enriched_windows.R {input.table} {input.accession_rankings} {input.feature_annotations} {input.parameters} {params.input_replicate_label} {wildcards.clip_replicate_label} {wildcards.experiment_label}.{wildcards.clip_replicate_label}"
+
+rule check_window_concordance:
+ input:
+ windows = lambda wildcards: expand("output/tested_windows/{{experiment_label}}.{clip_replicate_label}.tested_windows.tsv.gz", clip_replicate_label = experiment_to_clip_replicate_labels[wildcards.experiment_label])
+ output:
+ "output/figures/enrichment_reproducibility/{experiment_label}.enrichment_reproducibility.pdf",
+ "output/enrichment_reproducibility/{experiment_label}.enrichment_reproducibility.tsv",
+ "output/enrichment_reproducibility/{experiment_label}.odds_data.tsv"
+ benchmark: "benchmarks/check_window_concordance/{experiment_label}.all_replicates.concordance.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ resources:
+ mem_mb=16000,
+ runtime=15
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/check_window_concordance.R output/tested_windows {wildcards.experiment_label} " + (BLACKLIST if BLACKLIST is not None else "")
+
+rule find_reproducible_enriched_windows:
+ input:
+ windows = lambda wildcards: expand("output/enriched_windows/{{experiment_label}}.{clip_replicate_label}.enriched_windows.tsv.gz", clip_replicate_label = experiment_to_clip_replicate_labels[wildcards.experiment_label])
+ output:
+ reproducible_windows = "output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz",
+ linear_bar = "output/figures/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_window_counts.linear.pdf",
+ log_bar = "output/figures/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_window_counts.log10.pdf"
+ benchmark: "benchmarks/find_reproducible_enriched_windows/{experiment_label}.all_replicates.reproducible.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ resources:
+ mem_mb=8000,
+ runtime=30
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/identify_reproducible_windows.R output/enriched_windows/ {wildcards.experiment_label} " + (BLACKLIST if BLACKLIST is not None else "")
+
+rule sample_background_windows_by_region:
+ input:
+ enriched_windows = rules.find_reproducible_enriched_windows.output.reproducible_windows,
+ all_windows = ancient(FEATURE_ANNOTATIONS),
+ output:
+ variable_windows = "output/homer/region_matched_background/variable/{experiment_label}.sampled_variable_windows.bed.gz",
+ fixed_windows = "output/homer/region_matched_background/fixed/{experiment_label}.sampled_fixed_windows.bed.gz"
+ benchmark: "benchmarks/sample_background_windows_by_region/{experiment_label}.sample_background_windows_by_region.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ resources:
+ mem_mb=16000,
+ runtime=lambda wildcards, attempt: 30 * (2 ** (attempt - 1)),
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/sample_matched_background_by_region.R {input.enriched_windows} {input.all_windows} 75 output/homer/region_matched_background {wildcards.experiment_label};"
diff --git a/rules/meta_analysis.smk b/rules/meta_analysis.smk
new file mode 100644
index 0000000..a5ab041
--- /dev/null
+++ b/rules/meta_analysis.smk
@@ -0,0 +1,147 @@
+locals().update(config)
+rule make_genome_mega_table:
+ input:
+ feature = FEATURE_ANNOTATIONS,
+ replicate_counts = lambda wildcards: expand(
+ "output/counts/genome/vectors/{replicate_label}.counts",
+ replicate_label = replicate_labels),
+ output:
+ "output/counts/genome/megatables/megatable.tsv.gz",
+ threads: 4
+ resources:
+ mem_mb=1000,
+ runtime=75
+ shell:
+ """
+ paste <(zcat {input.feature}) {input.replicate_counts} | gzip > {output}
+ """
+
+rule make_repeat_mega_tables:
+ input:
+ unique_repeats = REPEAT_BED,
+ replicate_counts = lambda wildcards: expand("output/counts/repeats/vectors/{replicate_label}.counts",
+ replicate_label = replicate_labels),
+ output:
+ name_table = "output/counts/repeats/megatables/name.tsv.gz",
+ class_table = "output/counts/repeats/megatables/class.tsv.gz",
+ family_table = "output/counts/repeats/megatables/family.tsv.gz",
+ resources:
+ mem_mb=2000,
+ runtime=90
+ shell:
+ "echo \"repeat_name\" | paste - {input.replicate_counts} | sed -n '1p' | gzip > {output.name_table};"
+ "echo \"repeat_class\" | paste - {input.replicate_counts} | sed -n '1p' | gzip > {output.class_table};"
+ "echo \"repeat_family\" | paste - {input.replicate_counts} | sed -n '1p' | gzip > {output.family_table};"
+ "paste <(zcat {input.unique_repeats} | awk -v OFS=\"\\t\" 'BEGIN {{print \"repeat_name\";}} {{print $7}}') {input.replicate_counts} | "
+ "awk -v OFS=\"\\t\" 'NR > 1 {{for(i = 2; i <= NF; i++) {{tabulation[$1][i] += $i}} }} END {{for(name in tabulation) {{ printf name; for(i = 2; i <= NF; i++) {{printf \"\\t\" tabulation[name][i]}} print \"\";}} }}' | sort -k 1,1 | gzip >> {output.name_table};"
+ "paste <(zcat {input.unique_repeats} | awk -v OFS=\"\\t\" 'BEGIN {{print \"repeat_class\";}} {{print $8}}') {input.replicate_counts} | "
+ "awk -v OFS=\"\\t\" 'NR > 1 {{for(i = 2; i <= NF; i++) {{tabulation[$1][i] += $i}} }} END {{for(name in tabulation) {{ printf name; for(i = 2; i <= NF; i++) {{printf \"\\t\" tabulation[name][i]}} print \"\";}} }}' | sort -k 1,1 | gzip >> {output.class_table};"
+ "paste <(zcat {input.unique_repeats} | awk -v OFS=\"\\t\" 'BEGIN {{print \"repeat_family\";}} {{print $9}}') {input.replicate_counts} | "
+ "awk -v OFS=\"\\t\" 'NR > 1 {{for(i = 2; i <= NF; i++) {{tabulation[$1][i] += $i}} }} END {{for(name in tabulation) {{ printf name; for(i = 2; i <= NF; i++) {{printf \"\\t\" tabulation[name][i]}} print \"\";}} }}' | sort -k 1,1 | gzip >> {output.family_table};"
+
+# Unique fragment per library
+rule join_unique_fragments:
+ input:
+ expand("output/QC/{replicate_label}.uniq_fragments", replicate_label = replicate_labels)
+ output:
+ "output/QC/unique_fragments.csv"
+ resources:
+ mem_mb=2000,
+ runtime=25
+ shell:
+ """
+ awk '{{print FILENAME "," $0}}' {input} > {output}
+ """
+
+rule join_aligned_reads:
+ input:
+ expand("output/QC/{replicate_label}.aligned_reads", replicate_label = replicate_labels)
+ output:
+ "output/QC/aligned_reads.csv"
+ resources:
+ mem_mb=2000,
+ runtime=25
+ shell:
+ """
+ awk '{{print FILENAME "," $0}}' {input} > {output}
+ """
+
+# summarize per transcript type and family type
+rule summarize_genome_megatable:
+ input:
+ "output/counts/genome/megatables/megatable.tsv.gz",
+ output:
+ f="output/counts/genome/megatables/feature_type_top.tsv.gz",
+ t="output/counts/genome/megatables/transcript_type_top.tsv.gz",
+ conda:
+ "envs/metadensity.yaml"
+ resources:
+ mem_mb=2000,
+ runtime=60
+ shell:
+ """
+ module purge; # fixes weird $PATH issue where python being used is the module python, not the one in conda environment.
+ python {TOOL_DIR}/group_genome_megatable.py {input} {output.f} {output.t}
+ """
+# tested windows # with nan ready for analysis?
+# table containing QC statistics
+
+rule join_reproducible_enriched_re:
+ input:
+ expand("output/reproducible_enriched_re/{experiment_label}.reproducible_enriched_re.tsv.gz",
+ experiment_label=experiment_labels)
+ output:
+ binary="output/joined_reproducible_re/binary.csv",
+ l2or="output/joined_reproducible_re/l2or.csv",
+ resources:
+ mem_mb=2000,
+ runtime=25
+ conda:
+ "envs/metadensity.yaml"
+ shell:
+ """
+ module purge; # fixes weird $PATH issue where python being used is the module python, not the one in conda environment.
+ python {TOOL_DIR}/join_reproducible_enriched_re.py . {output.binary} {output.l2or}
+ """
+
+rule join_reproducible_enriched_windows:
+ input:
+ expand("output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz",
+ experiment_label=experiment_labels)
+ output:
+ binary="output/joined_reproducible_windows/binary.{feature_type}.csv",
+ l2or="output/joined_reproducible_windows/l2or.{feature_type}.csv",
+ resources:
+ mem_mb=2000,
+ runtime=25
+ conda:
+ "envs/metadensity.yaml"
+ shell:
+ """
+ module purge; # fixes weird $PATH issue where python being used is the module python, not the one in conda environment.
+ python {TOOL_DIR}/join_reproducible_enriched_windows.py . {wildcards.feature_type} {output.binary} {output.l2or}
+ """
+
+def find_all_tested_windows(experiment_labels, experiment_to_clip_replicate_labels):
+ tested_windows = []
+ for e in experiment_labels:
+ for rep in experiment_to_clip_replicate_labels[e]:
+ tested_windows.append(f"output/tested_windows/{e}.{rep}.tested_windows.tsv.gz")
+ return tested_windows
+rule join_reproducible_enriched_windows:
+ input:
+ expand("output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz",
+ experiment_label=experiment_labels),
+ tested_windows = lambda wildcards: find_all_tested_windows(experiment_labels, experiment_to_clip_replicate_labels)
+ output:
+ binary="output/joined_reproducible_windows/all.csv",
+ resources:
+ mem_mb=2000,
+ runtime=25
+ conda:
+ "envs/metadensity.yaml"
+ shell:
+ """
+ module purge; # fixes weird $PATH issue where python being used is the module python, not the one in conda environment.
+ python {TOOL_DIR}/join_all_reproducible_enriched_windows.py . {output.binary}
+ """
diff --git a/rules/pe_preprocess.smk b/rules/pe_preprocess.smk
new file mode 100644
index 0000000..2fab6d4
--- /dev/null
+++ b/rules/pe_preprocess.smk
@@ -0,0 +1,240 @@
+import pandas as pd
+from functools import reduce
+import re
+import os
+import sys
+import glob
+from time import sleep
+locals().update(config)
+
+
+rule copy_with_umi:
+ input:
+ fq_1 = lambda wildcards: config['replicate_label_to_fastq_1'][wildcards.replicate_label],
+ fq_2 = lambda wildcards: config['replicate_label_to_fastq_2'][wildcards.replicate_label],
+ output:
+ fq_1 = temp("output/fastqs/copy/{replicate_label}-1.fastq.gz"), #SORT OUT!!
+ fq_2 = temp("output/fastqs/copy/{replicate_label}-2.fastq.gz"), #SORT OUT!!
+ threads: 2
+ resources:
+ runtime="8h",
+ mem_mb=8000
+ benchmark: "benchmarks/umi/unassigned_experiment.{replicate_label}.copy_with_umi.txt"
+ shell:
+ "zcat {input.fq_1} | awk 'NR % 4 != 1 {{print}} NR % 4 == 1 {{split($1,header,\":\"); print $1 \":\" substr(header[1],2,length(header[1]) - 1) }}' | gzip > {output.fq_1};"
+ "zcat {input.fq_2} | awk 'NR % 4 != 1 {{print}} NR % 4 == 1 {{split($1,header,\":\"); print $1 \":\" substr(header[1],2,length(header[1]) - 1) }}' | gzip > {output.fq_2};"
+
+rule run_initial_fastqc:
+ input:
+ r1 = rules.copy_with_umi.output.fq_1,
+ r2 = rules.copy_with_umi.output.fq_2
+ output:
+ report_r1 = "output/fastqc/initial/{replicate_label}-1_fastqc.html",
+ zip_file_r1 = "output/fastqc/initial/{replicate_label}-1_fastqc.zip",
+ report_r2 = "output/fastqc/initial/{replicate_label}-2_fastqc.html",
+ zip_file_r2 = "output/fastqc/initial/{replicate_label}-2_fastqc.zip",
+ threads: 2
+ benchmark: "benchmarks/fastqc/unassigned_experiment.{replicate_label}.initial_fastqc.txt"
+ container:
+ "docker://howardxu520/skipper:fastqc_0.12.1"
+ resources:
+ mem_mb=16000,
+ runtime="3h"
+ params:
+ outdir="output/fastqc/initial/"
+ shell:
+ "fastqc {input.r1} --extract --outdir {params.outdir} -t {threads};"
+ "fastqc {input.r2} --extract --outdir {params.outdir} -t {threads};"
+
+rule trim_fastq_encode:
+ input:
+ # fq_1 = lambda wildcards: replicate_label_to_fastq_1[wildcards.replicate_label],
+ # fq_2 = lambda wildcards: replicate_label_to_fastq_2[wildcards.replicate_label],
+ fq_1 = rules.copy_with_umi.output.fq_1,
+ fq_2 = rules.copy_with_umi.output.fq_2,
+ adapter_1 = lambda wildcards: config['replicate_label_to_adapter_1'][wildcards.replicate_label],
+ adapter_2 = lambda wildcards: config['replicate_label_to_adapter_2'][wildcards.replicate_label],
+ output:
+ fq_1_trimmed = temp("output/fastqs/trimmed/{replicate_label}-trimmed-pair1.fastq.gz"),
+ fq_2_trimmed = temp("output/fastqs/trimmed/{replicate_label}-trimmed-pair2.fastq.gz"),
+ metrics = "output/fastqs/trimmed/{replicate_label}-trimmed.log"
+ threads: 8
+ resources:
+ tmpdir=TMPDIR,
+ mem_mb=16000,
+ runtime="3h"
+ benchmark: "benchmarks/trim/unassigned_experiment.{replicate_label}.trim.txt"
+ container:
+ "docker://howardxu520/skipper:skewer_0.2.2"
+ shell:
+ "skewer "
+ "-t {threads} "
+ "-x {input.adapter_1} "
+ "-y {input.adapter_2} "
+ "-o output/fastqs/trimmed/{wildcards.replicate_label} "
+ "-z -r 0.2 -d 0.2 -q 13 -l 20 "
+ "{input.fq_1} {input.fq_2}"
+
+rule run_trimmed_fastqc:
+ input:
+ r1 = rules.trim_fastq_encode.output.fq_1_trimmed,
+ r2 = rules.trim_fastq_encode.output.fq_2_trimmed,
+ output:
+ report_r1 = "output/fastqc/processed/{replicate_label}-trimmed-pair1_fastqc.html",
+ zip_file_r1 = "output/fastqc/processed/{replicate_label}-trimmed-pair1_fastqc.zip",
+ report_r2 = "output/fastqc/processed/{replicate_label}-trimmed-pair2_fastqc.html",
+ zip_file_r2 = "output/fastqc/processed/{replicate_label}-trimmed-pair2_fastqc.zip",
+ threads: 2
+ benchmark: "benchmarks/fastqc/unassigned_experiment.{replicate_label}.trimmed_fastqc.txt"
+ container:
+ "docker://howardxu520/skipper:fastqc_0.12.1"
+ resources:
+ mem_mb=16000,
+ runtime="4h"
+ shell:
+ "fastqc {input.r1} --extract --outdir output/fastqc/processed -t {threads};"
+ "fastqc {input.r2} --extract --outdir output/fastqc/processed -t {threads};"
+
+rule align_reads_encode:
+ input:
+ fq_1 = rules.trim_fastq_encode.output.fq_1_trimmed,
+ fq_2 = rules.trim_fastq_encode.output.fq_2_trimmed
+ output:
+ ubam = temp("output/bams/raw/genome/{replicate_label}.genome.Aligned.out.bam"),
+ # unmapped= "output/bams/raw/genome/{replicate_label}.genome.Unmapped.out.mate1",
+ log= "output/bams/raw/genome/{replicate_label}.genome.Log.final.out",
+ threads: 8
+ params:
+ star_sjdb = STAR_DIR,
+ outprefix = "output/bams/raw/genome/{replicate_label}.genome.",
+ rg = "{replicate_label}"
+ benchmark: "benchmarks/align/unassigned_experiment.{replicate_label}.align_reads_genome.txt"
+ container:
+ "docker://howardxu520/skipper:star_2.7.10b"
+ resources:
+ mem_mb=40000,
+ runtime="2h"
+ shell:
+ "STAR "
+ "--alignEndsType EndToEnd "
+ "--genomeDir {params.star_sjdb} "
+ "--genomeLoad NoSharedMemory "
+ "--outBAMcompression 10 "
+ "--outFileNamePrefix {params.outprefix} "
+ "--winAnchorMultimapNmax 100 "
+ "--outFilterMultimapNmax 100 "
+ "--outFilterMultimapScoreRange 1 "
+ "--outSAMmultNmax 1 "
+ "--outMultimapperOrder Random "
+ "--outFilterScoreMin 10 "
+ "--outFilterType BySJout "
+ "--limitOutSJcollapsed 5000000 "
+ "--outReadsUnmapped None "
+ "--outSAMattrRGline ID:{wildcards.replicate_label} "
+ "--outSAMattributes All "
+ "--outSAMmode Full "
+ "--outSAMtype BAM Unsorted "
+ "--outSAMunmapped Within "
+ "--readFilesCommand zcat "
+ "--outStd Log "
+ "--readFilesIn {input.fq_1} {input.fq_2} "
+ "--runMode alignReads "
+ "--runThreadN {threads}"
+
+rule sort_bam:
+ input:
+ bam="output/bams/raw/{ref}/{replicate_label}.{ref}.Aligned.out.bam",
+ output:
+ sort = "output/bams/raw/{ref}/{replicate_label}.{ref}.Aligned.sort.bam",
+ threads: 2
+ benchmark: "benchmarks/sort/{ref}/unassigned_experiment.{replicate_label}.sort_bam.txt"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17"
+ resources:
+ mem_mb=16000,
+ runteim="1h"
+ shell:
+ "samtools sort -T {wildcards.replicate_label} -@ {threads} -o {output.sort} {input.bam};"
+
+rule index_bams:
+ input:
+ bam = "output/bams/{round}/{ref}/{replicate_label}.Aligned.{mid}.bam"
+ output:
+ ibam = "output/bams/{round}/{ref}/{replicate_label}.Aligned.{mid}.bam.bai"
+ threads: 2
+ benchmark: "benchmarks/index_bam/{round}/{ref}/{mid}/unassigned_experiment.{replicate_label}.index_bam.txt"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17"
+ resources:
+ mem_mb=1000,
+ runtime=20
+ shell:
+ "samtools index -@ {threads} {input.bam};"
+
+rule dedup_umi_encode:
+ input:
+ bam="output/bams/raw/genome/{replicate_label}.genome.Aligned.sort.bam",
+ ibam = "output/bams/raw/genome/{replicate_label}.genome.Aligned.sort.bam.bai"
+ output:
+ bam_dedup="output/bams/dedup/genome/{replicate_label}.genome.Aligned.sort.dedup.bam"
+ params:
+ prefix='output/bams/dedup/genome/{replicate_label}.genome.sort'
+ resources:
+ mem_mb=34000,
+ runtime="8h"
+ benchmark: "benchmarks/dedup/genome/unassigned_experiment.{replicate_label}.dedup_umi.txt"
+ container:
+ "docker://howardxu520/skipper:umicollapse_1.0.0"
+ resources:
+ mem_mb=34000
+ shell:
+ "java -server -Xms32G -Xmx32G -Xss40M -jar /UMICollapse/umicollapse.jar bam "
+ "-i {input.bam} -o {output.bam_dedup} --umi-sep : --two-pass"
+
+rule select_informative_read:
+ input:
+ bam_combined="output/bams/dedup/genome/{replicate_label}.genome.Aligned.sort.dedup.bam"
+ output:
+ bam_informative="output/bams/dedup/genome_R"+str(INFORMATIVE_READ)+"/{replicate_label}.genome.Aligned.sort.dedup.R"+str(INFORMATIVE_READ)+".bam"
+ benchmark: "benchmarks/select/unassigned_experiment.{replicate_label}.select_informative_read.txt"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17"
+ resources:
+ mem_mb=10000,
+ runtime=30
+ shell:
+ "samtools view -bF " + str(64 if UNINFORMATIVE_READ == 1 else 128) + " {input.bam_combined} > {output.bam_informative}"
+
+rule obtain_unique_reads:
+ input:
+ rules.select_informative_read.output.bam_informative
+ output:
+ temp("output/QC/{replicate_label}.uniq_fragments")
+ benchmark:
+ "benchmarks/{replicate_label}.count_uniq_fragments.txt"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17"
+ resources:
+ mem_mb=10000,
+ runtime=30
+ shell:
+ """
+ samtools idxstats {input} | awk -F '\t' '{{s+=$3+$4}}END{{print s}}' > {output}
+ """
+
+rule obtain_aligned_reads:
+ input:
+ rules.align_reads_encode.output.ubam
+ output:
+ "output/QC/{replicate_label}.aligned_reads"
+ benchmark:
+ "benchmarks/{replicate_label}.count_aligned_reads.txt"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17"
+ resources:
+ mem_mb=8000,
+ runtime=30
+ shell:
+ """
+ samtools idxstats {input} | awk -F '\t' '{{s+=$3+$4}}END{{print s}}' > {output}
+ """
diff --git a/rules/prep_ml.smk b/rules/prep_ml.smk
new file mode 100644
index 0000000..68608bf
--- /dev/null
+++ b/rules/prep_ml.smk
@@ -0,0 +1,19 @@
+import pandas as pd
+locals().update(config)
+rule fetch_sequence:
+ input:
+ finemapped_windows = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz",
+ background = "output/homer/region_matched_background/fixed/{experiment_label}.sampled_fixed_windows.bed.gz",
+ output:
+ finemapped_fa = "output/ml/sequence/{experiment_label}.foreground.fa",
+ background_fa = "output/ml/sequence/{experiment_label}.background.fa"
+ container:
+ "docker://howardxu520/skipper:bedtools_2.31.0"
+ resources:
+ mem_mb=2000,
+ runtime=40
+ shell:
+ '''
+ bedtools getfasta -fo {output.finemapped_fa} -fi {GENOME} -bed {input.finemapped_windows} -s
+ bedtools getfasta -fo {output.background_fa} -fi {GENOME} -bed {input.background} -s
+ '''
diff --git a/rules/qc.smk b/rules/qc.smk
new file mode 100644
index 0000000..612a3d8
--- /dev/null
+++ b/rules/qc.smk
@@ -0,0 +1,97 @@
+locals().update(config)
+rule multiqc:
+ input:
+ trimmed_fastqc = lambda wildcards:
+ [f"output/fastqc/processed/{replicate_label}.trimmed.umi_fastqc.zip"
+ for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]
+ ] if config['protocol']=='ENCODE4'
+ else [f"output/fastqc/processed/{replicate_label}-trimmed-pair1_fastqc.zip"
+ for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]
+ ]+[f"output/fastqc/processed/{replicate_label}-trimmed-pair2_fastqc.zip"
+ for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]
+ ],
+ initial_fastqc = lambda wildcards: [f"output/fastqc/initial/{replicate_label}_fastqc.zip"
+ for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]
+ ] if config['protocol'] == 'ENCODE4'
+ else [f"output/fastqc/initial/{replicate_label}-1_fastqc.zip"
+ for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]
+ ]+[f"output/fastqc/initial/{replicate_label}-2_fastqc.zip"
+ for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]
+ ],
+ star_log = lambda wildcards: [f"output/bams/raw/genome/{replicate_label}.genome.Log.final.out" for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]],
+ fastp = lambda wildcards: [f"output/fastp/{replicate_label}.fastp.json" for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]] if config['protocol']=='ENCODE4' else [],
+ trimmed = lambda wildcards: [f"output/fastqs/trimmed/{replicate_label}-trimmed.log" for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]] if config['protocol']=='ENCODE4' else []
+ output:
+ multiqc_results = directory("output/multiqc/{experiment_label}/multiqc_data/"),
+ multiqc_plots = directory("output/multiqc/{experiment_label}/multiqc_plots/"),
+ multiqc_report = "output/multiqc/{experiment_label}/multiqc_report.html"
+ benchmark: "benchmarks/multiqc/{experiment_label}.multiqc.txt"
+ container:
+ "docker://jeltje/multiqc:1.6"
+ resources:
+ mem_mb=8000,
+ runtime=60
+ shell:
+ """
+ ls {input.trimmed_fastqc} {input.initial_fastqc} {input.star_log} {input.fastp} {input.trimmed} > output/multiqc/{wildcards.experiment_label}/files.txt
+ multiqc --outdir output/multiqc/{wildcards.experiment_label} -f --export --data-format json --file-list output/multiqc/{wildcards.experiment_label}/files.txt
+ """
+
+rule quantify_gc_bias:
+ input:
+ "output/counts/genome/tables/{experiment_label}.tsv.gz"
+ output:
+ gc_bias = "output/qc/{experiment_label}.gc_bias.txt"
+ conda:
+ "envs/metadensity.yaml"
+ resources:
+ mem_mb=40000,
+ runtime=5
+ shell:
+ """
+ module purge;
+ python {TOOL_DIR}/quantify_gcbias.py {input} {output}
+ """
+
+def get_bams(wildcards):
+ ''' return a list of final bam for all IP and Input replicate given experiment label '''
+ if config['protocol']=='ENCODE':
+ return [f"output/bams/dedup/genome_R{INFORMATIVE_READ}/{replicate_label}.genome.Aligned.sort.dedup.R{INFORMATIVE_READ}.bam"
+ for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]
+ ]
+ else:
+ return [f"output/bams/dedup/genome/{replicate_label}.genome.Aligned.sort.dedup.bam"
+ for replicate_label in experiment_to_replicate_labels[wildcards.experiment_label]
+ ]
+
+rule nread_in_finemapped_regions:
+ input:
+ bam=lambda wildcards: get_bams(wildcards),
+ bed="output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz"
+ output:
+ sorted_bed="output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.sorted.bed.gz",
+ nread_in_finemapped_regions = "output/qc/{experiment_label}.nread_in_finemapped_regions.txt"
+ container:
+ "docker://howardxu520/skipper:bigwig_1.0"
+ params:
+ genome=os.path.join(config['STAR_DIR'], 'chrNameLength.txt')
+ resources:
+ mem_mb=lambda wildcards, attempt: 32000 * (2 ** (attempt - 1)),
+ runtime=lambda wildcards, attempt: 5 * (2 ** (attempt - 1)),
+ shell:
+ """
+ zcat {input.bed} | bedtools sort -g {params.genome} -i - | gzip -c > {output.sorted_bed};
+ for bam in {input.bam}
+ do
+ nread_in_peak=$(bedtools coverage \
+ -a {output.sorted_bed} \
+ -b $bam \
+ -sorted \
+ -counts \
+ -g {params.genome} \
+ -s | cut -f 10 | awk '{{sum += $NF}} END {{print sum}}')
+ replicate_label=$(basename $bam | cut -d'.' -f1)
+ echo -e "$replicate_label\t$nread_in_peak" >> {output.nread_in_finemapped_regions}
+ done
+ """
+
diff --git a/rules/repeat.smk b/rules/repeat.smk
new file mode 100644
index 0000000..fe5d881
--- /dev/null
+++ b/rules/repeat.smk
@@ -0,0 +1,137 @@
+locals().update(config)
+rule uniq_repeats:
+ input:
+ repeatmasker = ancient(REPEAT_TABLE),
+ genome = ancient(GENOME)
+ output:
+ sorted_bed = temp("repeats.sort.temp.bed.gz"),
+ unique_repeats = REPEAT_BED
+ benchmark: "benchmarks/uniq_repeats.txt"
+ container:
+ "docker://howardxu520/skipper:bedtools_2.31.0"
+ resources:
+ mem_mb=32000,
+ runtime="4h"
+ shell:
+ "zcat {REPEAT_TABLE} | awk -v OFS=\"\\t\" '{{print $6,$7,$8,$11 \":\" name_count[$11]++, $2, $10,$11,$12,$13}} "
+ "$13 == \"L1\" || $13 == \"Alu\" {{$11 = $11 \"_AS\"; $12 = $12 \"_AS\"; $13 = $13 \"_AS\"; "
+ "if($10 == \"+\") {{$10 = \"-\"}} else {{$10 = \"+\"}}; print $6,$7,$8,$11 \":\" name_count[$11]++, $2, $10,$11,$12,$13}}' | "
+ "tail -n +2 | bedtools sort -i - | gzip > {output.sorted_bed}; "
+ "bedtools coverage -s -d -a {output.sorted_bed} -b {output.sorted_bed} | awk -v OFS=\"\\t\" "
+ "'$NF >1 {{print $1,$2+$(NF-1)-1,$2+$(NF-1),$4,$5,$6}}' | "
+ "bedtools sort -i - | "
+ "bedtools merge -c 4,5,6 -o distinct -s -i - | "
+ "bedtools subtract -s -a {output.sorted_bed} -b - | "
+ "bedtools nuc -s -fi {input.genome} -bed - | awk -v OFS=\"\\t\" 'NR > 1 {{print $1,$2,$3,$4,$5,$6,$7,$8,$9,$11}}' | "
+ "gzip -c > {output.unique_repeats}"
+
+
+rule quantify_repeats:
+ input:
+ CHROM_SIZES,
+ bam = lambda wildcards: replicate_label_to_bams[wildcards.replicate_label],
+ repeats = REPEAT_BED
+ output:
+ counts = "output/counts/repeats/vectors/{replicate_label}.counts"
+ benchmark: "benchmarks/repeats/unassigned_experiment.{replicate_label}.quantify_repeats.txt"
+ container:
+ "docker://howardxu520/skipper:bedtools_2.31.0"
+ resources:
+ mem_mb=lambda wildcards, attempt: 32000 * (2 ** (attempt - 1)),
+ runtime=lambda wildcards, attempt: 40 * (2 ** (attempt - 1)),
+ shell:
+ "bedtools bamtobed -i {input.bam} | awk '($1 != \"chrEBV\") && ($4 !~ \"/{UNINFORMATIVE_READ}$\")' | "
+ "bedtools flank -s -l 1 -r 0 -g {CHROM_SIZES} -i - | "
+ "bedtools shift -p 1 -m -1 -g {CHROM_SIZES} -i - | "
+ "bedtools sort -i - | "
+ "bedtools coverage -s -counts -a {input.repeats} -b - | "
+ "awk 'BEGIN {{print \"{wildcards.replicate_label}\"}} {{print $NF}}' > {output.counts}"
+
+rule make_repeat_count_tables:
+ input:
+ unique_repeats = REPEAT_BED,
+ replicate_counts = lambda wildcards: expand("output/counts/repeats/vectors/{replicate_label}.counts", replicate_label = experiment_to_replicate_labels[wildcards.experiment_label]),
+ output:
+ name_table = "output/counts/repeats/tables/name/{experiment_label}.tsv.gz",
+ class_table = "output/counts/repeats/tables/class/{experiment_label}.tsv.gz",
+ family_table = "output/counts/repeats/tables/family/{experiment_label}.tsv.gz",
+ benchmark: "benchmarks/counts/{experiment_label}.all_replicates.make_repeat_count_table.txt"
+ resources:
+ mem_mb=2000,
+ runtime=120
+ shell:
+ "echo \"repeat_name\" | paste - {input.replicate_counts} | sed -n '1p' | gzip > {output.name_table};"
+ "echo \"repeat_class\" | paste - {input.replicate_counts} | sed -n '1p' | gzip > {output.class_table};"
+ "echo \"repeat_family\" | paste - {input.replicate_counts} | sed -n '1p' | gzip > {output.family_table};"
+ "paste <(zcat {input.unique_repeats} | awk -v OFS=\"\\t\" 'BEGIN {{print \"repeat_name\";}} {{print $7}}') {input.replicate_counts} | "
+ "awk -v OFS=\"\\t\" 'NR > 1 {{for(i = 2; i <= NF; i++) {{tabulation[$1][i] += $i}} }} END {{for(name in tabulation) {{ printf name; for(i = 2; i <= NF; i++) {{printf \"\\t\" tabulation[name][i]}} print \"\";}} }}' | sort -k 1,1 | gzip >> {output.name_table};"
+ "paste <(zcat {input.unique_repeats} | awk -v OFS=\"\\t\" 'BEGIN {{print \"repeat_class\";}} {{print $8}}') {input.replicate_counts} | "
+ "awk -v OFS=\"\\t\" 'NR > 1 {{for(i = 2; i <= NF; i++) {{tabulation[$1][i] += $i}} }} END {{for(name in tabulation) {{ printf name; for(i = 2; i <= NF; i++) {{printf \"\\t\" tabulation[name][i]}} print \"\";}} }}' | sort -k 1,1 | gzip >> {output.class_table};"
+ "paste <(zcat {input.unique_repeats} | awk -v OFS=\"\\t\" 'BEGIN {{print \"repeat_family\";}} {{print $9}}') {input.replicate_counts} | "
+ "awk -v OFS=\"\\t\" 'NR > 1 {{for(i = 2; i <= NF; i++) {{tabulation[$1][i] += $i}} }} END {{for(name in tabulation) {{ printf name; for(i = 2; i <= NF; i++) {{printf \"\\t\" tabulation[name][i]}} print \"\";}} }}' | sort -k 1,1 | gzip >> {output.family_table};"
+
+rule fit_clip_betabinomial_re_model:
+ input:
+ table = rules.make_repeat_count_tables.output.name_table,
+ output:
+ coef = "output/clip_model_coef_re/{experiment_label}.{clip_replicate_label}.tsv",
+ # plot = lambda wildcards: expand("output/figures/clip_distributions/{{experiment_label}}.{{clip_replicate_label}}.{other_label}.clip_distribution.pdf", other_label = experiment_to_input_replicate_labels[wildcards.experiment_label][wildcards.Input_replicate_label])
+ benchmark: "benchmarks/fit_clip_betabinomial_re_model/{experiment_label}.{clip_replicate_label}.fit_clip.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ resources:
+ mem_mb=32000,
+ runtime="6h"
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/fit_clip_betabinom_re.R {input.table} {wildcards.experiment_label} {wildcards.clip_replicate_label}"
+
+rule fit_input_betabinomial_re_model:
+ input:
+ table = rules.make_repeat_count_tables.output.name_table,
+ output:
+ coef = "output/input_model_coef_re/{experiment_label}.{input_replicate_label}.tsv",
+ # plot = lambda wildcards: expand("output/figures/input_distributions/{{experiment_label}}.{{input_replicate_label}}.{other_label}.input_distribution.pdf", other_label = experiment_to_input_replicate_labels[wildcards.experiment_label][wildcards.Input_replicate_label])
+ benchmark: "benchmarks/fit_input_betabinomial_re_model/{experiment_label}.{input_replicate_label}.fit_input.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ resources:
+ mem_mb=32000,
+ runtime=60
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/fit_input_betabinom_re.R {input.table} {wildcards.experiment_label} {wildcards.input_replicate_label}"
+
+rule call_enriched_re:
+ input:
+ table = rules.make_repeat_count_tables.output.name_table,
+ replicate = lambda wildcards: "output/counts/repeats/vectors/" + re.sub("IP_\d$","IP_2",wildcards.clip_replicate_label) + ".counts",
+ repeats = REPEAT_BED,
+ parameters = lambda wildcards: "output/" + OVERDISPERSION_MODE + "_model_coef_re/{experiment_label}." + overdispersion_replicate_lookup[wildcards.clip_replicate_label] + ".tsv",
+ threads: 2
+ output:
+ "output/figures/clip_scatter_re/{experiment_label}.{clip_replicate_label}.clip_test_distribution.pdf",
+ "output/enriched_re/{experiment_label}.{clip_replicate_label}.enriched_re.tsv.gz"
+ benchmark: "benchmarks/call_enriched_re/{experiment_label}.{clip_replicate_label}.call_enriched_re.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ params:
+ input_replicate_label = lambda wildcards: clip_to_input_replicate_label[wildcards.clip_replicate_label]
+ resources:
+ mem_mb=48000,
+ runtime="3h"
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/call_enriched_re.R {input.table} {input.repeats} {input.parameters} {params.input_replicate_label} {wildcards.clip_replicate_label} {wildcards.experiment_label}.{wildcards.clip_replicate_label}"
+
+rule find_reproducible_enriched_re:
+ input:
+ windows = lambda wildcards: expand("output/enriched_re/{{experiment_label}}.{clip_replicate_label}.enriched_re.tsv.gz", clip_replicate_label = experiment_to_clip_replicate_labels[wildcards.experiment_label])
+ output:
+ reproducible_windows = "output/reproducible_enriched_re/{experiment_label}.reproducible_enriched_re.tsv.gz",
+ benchmark: "benchmarks/find_reproducible_enriched_re/{experiment_label}.all_replicates.reproducible.txt"
+ container:
+ "docker://howardxu520/skipper:R_4.1.3_1"
+ resources:
+ mem_mb=16000,
+ runtime=120
+ shell:
+ "Rscript --vanilla {TOOL_DIR}/identify_reproducible_re.R output/enriched_re/ {wildcards.experiment_label}"
+
diff --git a/rules/se_preprocess.smk b/rules/se_preprocess.smk
new file mode 100644
index 0000000..c459343
--- /dev/null
+++ b/rules/se_preprocess.smk
@@ -0,0 +1,207 @@
+locals().update(config)
+if config.get('AGGRESSIVE_TRIM', False):
+ skewer_k = 1
+ skewer_m = 'any'
+else:
+ skewer_k = 2
+ skewer_m = 'tail'
+
+rule run_initial_fastqc:
+ input:
+ fq = lambda wildcards: config['replicate_label_to_fastqs'][wildcards.replicate_label].split(" "),
+ output:
+ report = "output/fastqc/initial/{replicate_label}_fastqc.html",
+ zip_file = "output/fastqc/initial/{replicate_label}_fastqc.zip",
+ directory = directory("output/fastqc/initial/{replicate_label}_fastqc")
+ threads: 2
+ benchmark: "benchmarks/fastqc/unassigned_experiment.{replicate_label}.initial_fastqc.txt"
+ container:
+ "docker://howardxu520/skipper:fastqc_0.12.1"
+ resources:
+ mem_mb=16000,
+ runtime="6h"
+ shell:
+ "zcat {input.fq} | fastqc stdin:{wildcards.replicate_label} --extract --outdir output/fastqc/initial -t {threads}"
+
+rule trim_fastq:
+ input:
+ fq = lambda wildcards: config['replicate_label_to_fastqs'][wildcards.replicate_label].split(" "),
+ adapter = lambda wildcards: config['replicate_label_to_adapter'][wildcards.replicate_label],
+ output:
+ fq_trimmed = temp("output/fastqs/trimmed/{replicate_label}-trimmed.fastq.gz"),
+ metrics = "output/fastqs/trimmed/{replicate_label}-trimmed.log"
+ threads: 8
+ params:
+ k = skewer_k,
+ m = skewer_m
+ benchmark: "benchmarks/trim/unassigned_experiment.{replicate_label}.trim.txt"
+ container:
+ "docker://howardxu520/skipper:skewer_0.2.2"
+ resources:
+ mem_mb=16000,
+ runtime="6h"
+ shell:
+ "zcat {input.fq} | skewer "
+ "-t {threads} "
+ "-x {input.adapter} "
+ "-1 -r 0.2 -d 0.2 -q 13 -l 20 -k {params.k} -m {params.m} - "
+ "| skewer -t {threads} "
+ "-x {input.adapter} "
+ "-o output/fastqs/trimmed/{wildcards.replicate_label} "
+ "-z -r 0.2 -d 0.2 -q 13 -l 20 -k {params.k} -m {params.m} -"
+
+rule extract_umi:
+ input:
+ fq = rules.trim_fastq.output.fq_trimmed,
+ output:
+ fq_umi = "output/fastqs/umi/{replicate_label}.trimmed.umi.fq.gz",
+ json = "output/fastp/{replicate_label}.fastp.json",
+ html = "output/fastp/{replicate_label}.fastp.html",
+ threads: 8
+ params:
+ umi_length = config['UMI_SIZE'],
+ benchmark: "benchmarks/umi/unassigned_experiment.{replicate_label}.extract_umi.txt"
+ container:
+ "docker://howardxu520/skipper:fastp_0.23.4"
+ resources:
+ mem_mb=8000,
+ runtime=45
+ shell:
+ "fastp "
+ "-i {input.fq} "
+ "-o {output.fq_umi} "
+ "-A "
+ "-U "
+ "--umi_len={params.umi_length} "
+ "--umi_loc=read1 "
+ "-j output/fastp/{wildcards.replicate_label}.fastp.json "
+ "-h output/fastp/{wildcards.replicate_label}.fastp.html "
+ "-w {threads}"
+
+
+rule run_trimmed_fastqc:
+ input:
+ rules.extract_umi.output.fq_umi,
+ output:
+ report = "output/fastqc/processed/{replicate_label}.trimmed.umi_fastqc.html",
+ zip_file = "output/fastqc/processed/{replicate_label}.trimmed.umi_fastqc.zip",
+ threads: 2
+ benchmark: "benchmarks/fastqc/unassigned_experiment.{replicate_label}.trimmed_fastqc.txt"
+ container:
+ "docker://howardxu520/skipper:fastqc_0.12.1"
+ resources:
+ mem_mb=16000,
+ runtime="3h"
+ shell:
+ "fastqc {input} --extract --outdir output/fastqc/processed -t {threads}"
+
+rule align_reads:
+ input:
+ fq= rules.extract_umi.output.fq_umi,
+ output:
+ ubam = temp("output/bams/raw/genome/{replicate_label}.genome.Aligned.out.bam"),
+ # unmapped= "output/bams/raw/genome/{replicate_label}.genome.Unmapped.out.mate1",
+ log= "output/bams/raw/genome/{replicate_label}.genome.Log.final.out",
+ threads: 8
+ params:
+ star_sjdb = config['STAR_DIR'],
+ outprefix = "output/bams/raw/genome/{replicate_label}.genome.",
+ rg = "{replicate_label}"
+ benchmark: "benchmarks/align/unassigned_experiment.{replicate_label}.align_reads_genome.txt"
+ container:
+ "docker://howardxu520/skipper:star_2.7.10b"
+ resources:
+ mem_mb=40000,
+ runtime=240
+ shell:
+ "STAR "
+ "--alignEndsType EndToEnd "
+ "--genomeDir {params.star_sjdb} "
+ "--genomeLoad NoSharedMemory "
+ "--outBAMcompression 10 "
+ "--outFileNamePrefix {params.outprefix} "
+ "--winAnchorMultimapNmax 100 "
+ "--outFilterMultimapNmax 100 "
+ "--outFilterMultimapScoreRange 1 "
+ "--outSAMmultNmax 1 "
+ "--outMultimapperOrder Random "
+ "--outFilterScoreMin 10 "
+ "--outFilterType BySJout "
+ "--limitOutSJcollapsed 10000000 "
+ "--outReadsUnmapped None "
+ "--outSAMattrRGline ID:{wildcards.replicate_label} "
+ "--outSAMattributes All "
+ "--outSAMmode Full "
+ "--outSAMtype BAM Unsorted "
+ "--outSAMunmapped Within "
+ "--readFilesCommand zcat "
+ "--outStd Log "
+ "--readFilesIn {input.fq} "
+ "--runMode alignReads "
+ "--runThreadN {threads}"
+
+rule sort_bam:
+ input:
+ bam="output/bams/raw/{ref}/{replicate_label}.{ref}.Aligned.out.bam",
+ output:
+ sort="output/bams/raw/{ref}/{replicate_label}.{ref}.Aligned.sort.bam",
+ threads: 4
+ benchmark: "benchmarks/sort/{ref}/unassigned_experiment.{replicate_label}.sort_bam.txt"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17"
+ resources:
+ mem_mb=16000,
+ runtime="2h"
+ shell:
+ "samtools sort -T {wildcards.replicate_label} -@ {threads} -o {output.sort} {input.bam};"
+
+
+rule index_bams:
+ input:
+ bam = "output/bams/{round}/{ref}/{replicate_label}.Aligned.{mid}.bam"
+ output:
+ ibam = "output/bams/{round}/{ref}/{replicate_label}.Aligned.{mid}.bam.bai"
+ threads: 2
+ benchmark: "benchmarks/index_bam/{round}/{ref}/{mid}/unassigned_experiment.{replicate_label}.index_bam.txt"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17"
+ resources:
+ mem_mb=1000,
+ runtime=10
+ shell:
+ "samtools index -@ {threads} {input.bam};"
+
+
+rule dedup_umi:
+ input:
+ bam="output/bams/raw/genome/{replicate_label}.genome.Aligned.sort.bam",
+ ibam = "output/bams/raw/genome/{replicate_label}.genome.Aligned.sort.bam.bai"
+ output:
+ bam_dedup="output/bams/dedup/genome/{replicate_label}.genome.Aligned.sort.dedup.bam",
+ benchmark: "benchmarks/dedup/genome/unassigned_experiment.{replicate_label}.dedup_umi.txt"
+ container:
+ "docker://howardxu520/skipper:umicollapse_1.0.0"
+ resources:
+ mem_mb=34000,
+ runtime="3h",
+ tmpdir=TMPDIR
+ shell:
+ "java -server -Xms32G -Xmx32G -Xss40M -Djava.io.tmpdir={resources.tmpdir} -jar /UMICollapse/umicollapse.jar bam "
+ "-i {input.bam} -o {output.bam_dedup} --umi-sep : --two-pass"
+
+rule obtain_unique_reads:
+ input:
+ rules.dedup_umi.output.bam_dedup
+ output:
+ "output/QC/{replicate_label}.uniq_fragments"
+ benchmark:
+ "benchmarks/{replicate_label}.count_uniq_fragments.txt"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17"
+ resources:
+ mem_mb=8000,
+ runtime=30
+ shell:
+ """
+ samtools idxstats {input} | awk -F '\t' '{{s+=$3+$4}}END{{print s}}' > {output}
+ """
diff --git a/rules/train_rbpnet.smk b/rules/train_rbpnet.smk
new file mode 100644
index 0000000..d255b93
--- /dev/null
+++ b/rules/train_rbpnet.smk
@@ -0,0 +1,101 @@
+import pandas as pd
+locals().update(config)
+
+rule prepare_data:
+ input:
+ finemapped_windows = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz",
+ ip_bigwigs = lambda wildcards: expand("output/bigwigs/unscaled/plus/{replicate_label}.unscaled.plus.bw",
+ replicate_label = experiment_to_replicate_labels[wildcards.experiment_label]),
+ output:
+ "output/ml/rbpnet_data/{experiment_label}/prep_done"
+ # container:
+ # "docker://brianyee/eugene-tools:0.1.2" # THIS DOCKER IS NOT UPDATED WITH PYAROOW YET? NO SPACE LEFT ON DEVICE PROBLEM. PLUS CHARLENE CAN NEVER PULL CORRECTLY
+ resources:
+ mem_mb=80000,
+ runtime=40
+ container:
+ "/tscc/nfs/home/bay001/eugene-tools_0.1.2.sif"
+ shell:
+ """
+ export NUMBA_CACHE_DIR=/tscc/lustre/ddn/scratch/${{USER}} # TODO: HARCODED IS BAD
+ export MPLCONFIGDIR=/tscc/lustre/ddn/scratch/${{USER}}
+ python {RBPNET_PATH}/prep_data.py {CONFIG_PATH} {wildcards.experiment_label} \
+ output/ml/rbpnet_data/{wildcards.experiment_label}
+ """
+
+rule train_model:
+ input:
+ "output/ml/rbpnet_data/{experiment_label}/prep_done"
+ output:
+ model = "output/ml/rbpnet_model/{experiment_label}/training_done",
+ resources:
+ mem_mb=lambda wildcards, attempt: 64000 * (2 ** (attempt - 1)),
+ runtime=lambda wildcards, attempt: 240 * (2 ** (attempt - 1)),
+ slurm_partition="rtx3090",
+ slurm_account="csd792",
+ slurm_extra="'--qos=condo-gpu' '--gpus=1'",
+ container:
+ "/tscc/nfs/home/bay001/eugene-tools_0.1.2.sif"
+ # container:
+ # "docker://brianyee/eugene-tools:0.1.2" #NO SPACE LEFT ON DEVICE PROBLEM
+ shell:
+ """
+ export NUMBA_CACHE_DIR=/tscc/lustre/ddn/scratch/${{USER}} # TODO: HARCODED IS BAD
+ export MPLCONFIGDIR=/tscc/lustre/ddn/scratch/${{USER}}
+ python {RBPNET_PATH}/train.py output/ml/rbpnet_data/{wildcards.experiment_label} \
+ output/ml/rbpnet_model/{wildcards.experiment_label}
+ """
+
+rule validation:
+ input:
+ model = "output/ml/rbpnet_model/{experiment_label}/training_done",
+ zarr = "output/ml/rbpnet_data/{experiment_label}/prep_done"
+ output:
+ validation = "output/ml/rbpnet_model/{experiment_label}/valid/test_data_metric.csv",
+ resources:
+ mem_mb=lambda wildcards, attempt: 64000 * (2 ** (attempt - 1)),
+ runtime=lambda wildcards, attempt: 180 * (2 ** (attempt - 1)),
+ slurm_partition="rtx3090",
+ slurm_account="csd792",
+ slurm_extra="'--qos=condo-gpu' '--gpus=1'",
+ container:
+ "/tscc/nfs/home/bay001/eugene-tools_0.1.2.sif"
+ # container:
+ # "docker://brianyee/eugene-tools:0.1.2"
+ shell:
+ """
+ module load gpu
+ export NUMBA_CACHE_DIR=/tscc/lustre/ddn/scratch/${{USER}}
+ export MPLCONFIGDIR=/tscc/lustre/ddn/scratch/${{USER}}
+ python {RBPNET_PATH}/validate.py {CONFIG_PATH} \
+ {wildcards.experiment_label} \
+ output/ml/rbpnet_data/{wildcards.experiment_label} \
+ output/ml/rbpnet_model/{wildcards.experiment_label}
+ """
+
+rule seqlet:
+ input:
+ validation = "output/ml/rbpnet_model/{experiment_label}/valid/test_data_metric.csv",
+ model = "output/ml/rbpnet_model/{experiment_label}/training_done",
+ zarr = "output/ml/rbpnet_data/{experiment_label}/prep_done"
+ output:
+ validation = "output/ml/rbpnet_model/{experiment_label}/motif_done",
+ resources:
+ mem_mb=lambda wildcards, attempt: 64000 * (2 ** (attempt - 1)),
+ runtime=lambda wildcards, attempt: 60 * (2 ** (attempt - 1)),
+ slurm_partition="rtx3090",
+ slurm_account="csd792",
+ slurm_extra="'--qos=condo-gpu' '--gpus=1'",
+ container:
+ "/tscc/nfs/home/bay001/eugene-tools_0.1.2.sif"
+ # container:
+ # "docker://brianyee/eugene-tools:0.1.2"
+ shell:
+ """
+ module load gpu
+ export NUMBA_CACHE_DIR=/tscc/lustre/ddn/scratch/${{USER}}
+ export MPLCONFIGDIR=/tscc/lustre/ddn/scratch/${{USER}}
+ python {RBPNET_PATH}/seqlet.py \
+ output/ml/rbpnet_data/{wildcards.experiment_label} \
+ output/ml/rbpnet_model/{wildcards.experiment_label}
+ """
diff --git a/rules/variants_rbpnet.smk b/rules/variants_rbpnet.smk
new file mode 100644
index 0000000..b8b6f98
--- /dev/null
+++ b/rules/variants_rbpnet.smk
@@ -0,0 +1,348 @@
+
+import pandas as pd
+from pathlib import Path
+locals().update(config)
+VEP_CACHEDIR = config['VEP_CACHEDIR']
+VEP_CACHE_VERSION = config['VEP_CACHE_VERSION']
+
+rule filter_roulette_for_high:
+ input:
+ vcf=Path(ROULETTE_DIR)/'{chr_number}_rate_v5.2_TFBS_correction_all.vcf.bgz',
+ header=HEADER
+ output:
+ reheader = temp(Path(ROULETTE_DIR)/'{chr_number}_rate_v5.2_TFBS_correction_all.header.vcf'),
+ filtered = temp(Path(ROULETTE_DIR)/'{chr_number}_rate_v5.2_TFBS_correction_all.header.filtered.vcf'),
+ container:
+ "docker://brianyee/bcftools:1.17"
+ threads: 1
+ resources:
+ mem_mb=40000,
+ runtime="1h"
+ shell:
+ """
+ bcftools reheader -h {input.header} \
+ {input.vcf} > {output.reheader}
+ bcftools filter -O z -o {output.filtered} -i 'FILTER=="high"' {output.reheader}
+ """
+
+rule annotate_roulette_w_gnomAD:
+ input:
+ rename=RENAME,
+ gnomad=Path(GNOMAD_DIR) / 'gnomad.genomes.v4.1.sites.chr{chr_number}.vcf.bgz',
+ roulette=Path(ROULETTE_DIR)/'{chr_number}_rate_v5.2_TFBS_correction_all.header.filtered.vcf'
+ output:
+ rename=temp(Path(ROULETTE_DIR)/'{chr_number}_rate_v5.2_TFBS_correction_all.header.filtered.rename.vcf.gz'),
+ annotated=Path(ROULETTE_DIR)/'{chr_number}_rate_v5.2_TFBS_correction_all.header.filtered.rename.annotated.vcf.gz'
+ threads: 1
+ resources:
+ mem_mb=80000,
+ runtime="6h"
+ container:
+ "docker://brianyee/bcftools:1.17"
+ shell:
+ """
+ bcftools annotate --rename-chrs {input.rename} \
+ {input.roulette} -Oz -o {output.rename}
+ bcftools index {output.rename}
+ bcftools annotate -a {input.gnomad} \
+ -c INFO/AC,INFO/AN \
+ -k {output.rename} \
+ -o {output.annotated}
+ bcftools index {output.annotated}
+ """
+
+rule fetch_SNP_from_gnomAD_and_roulette:
+ ''' fetch gnomAD variants from database '''
+ input:
+ vcf=Path(ROULETTE_DIR)/'{chr_number}_rate_v5.2_TFBS_correction_all.header.filtered.rename.annotated.vcf.gz',
+ finemapped_windows = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz"
+ output:
+ "output/variants/gnomAD_roulette/{experiment_label}.chr{chr_number}.vcf"
+ threads: 2
+ resources:
+ runtime=lambda wildcards, attempt: 480 * (1.5 ** (attempt - 1)),
+ mem_mb=40000,
+ container:
+ "docker://brianyee/bcftools:1.17"
+ shell:
+ """
+ if [ -s {input.finemapped_windows} ]; then
+ bcftools query -R {input.finemapped_windows} -f '%CHROM\t%POS\t%ID\t%REF\t%ALT\t%INFO/AC\t%INFO/AN\t%INFO/MR\t%INFO/AR\t%INFO/MG\t%INFO/MC\n' \
+ {input.vcf} > {output}
+ else
+ touch {output}
+ fi
+ """
+
+rule slop_finemap:
+ input:
+ finemapped_windows = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz"
+ output:
+ "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.slop.bed.gz"
+ threads: 2
+ resources:
+ mem_mb=40000,
+ runtime="6h"
+ container:
+ "docker://howardxu520/skipper:bigwig_1.0"
+ shell:
+ """
+ bedtools slop -i {input.finemapped_windows} -g {CHROM_SIZES} -b 100 | gzip -c > {output}
+ """
+
+rule fetch_peak_sequence:
+ input:
+ finemapped_windows = rules.slop_finemap.output,
+ output:
+ finemapped_fa = "output/ml/sequence/{experiment_label}.foreground.slop.fa",
+ resources:
+ mem_mb=2000,
+ runtime=40
+ container:
+ "docker://howardxu520/skipper:bedtools_2.31.0"
+ shell:
+ '''
+ bedtools getfasta -fo {output.finemapped_fa} -fi {GENOME} -bed {input.finemapped_windows} -s
+ '''
+
+rule fetch_variant_sequence:
+ input:
+ subset_vcf="output/variants/{subset}/{experiment_label_thing}.vcf",
+ seq_fa = lambda wildcards: f"output/ml/sequence/{wildcards.experiment_label_thing}.foreground.slop.fa"
+ if 'chr' not in wildcards.experiment_label_thing
+ else "output/ml/sequence/"+wildcards.experiment_label_thing.split('.')[0]+".foreground.slop.fa",
+ finemapped_windows = lambda wildcards: f"output/finemapping/mapped_sites/{wildcards.experiment_label_thing}.finemapped_windows.slop.bed.gz"
+ if 'chr' not in wildcards.experiment_label_thing
+ else "output/finemapping/mapped_sites/"+wildcards.experiment_label_thing.split('.')[0]+".finemapped_windows.slop.bed.gz"
+ output:
+ ref_fa = temp("output/variants/{subset}/{experiment_label_thing}.ref.fa"),
+ alt_fa = temp("output/variants/{subset}/{experiment_label_thing}.alt.fa"),
+ csv = "output/variants/{subset}/{experiment_label_thing}.csv"
+ threads: 2
+ resources:
+ mem_mb=80000,
+ runtime="1h"
+ params:
+ out_prefix = lambda wildcards, output: output.csv.replace('.csv', ''),
+ tmpdir = config["TMPDIR"],
+ conda:
+ "envs/metadensity.yaml"
+ shell:
+ """
+ module purge;
+ export TMPDIR={params.tmpdir};
+ if [ -s {input.subset_vcf} ]; then
+ python {TOOL_DIR}/generate_variant_sequence.py \
+ {input.subset_vcf} \
+ {input.seq_fa} \
+ {input.finemapped_windows} \
+ {params.out_prefix}
+ else
+ touch {output.csv}
+ touch {output.ref_fa}
+ touch {output.alt_fa}
+ fi
+ """
+
+rule score_fa:
+ input:
+ model=lambda wildcards: "output/ml/rbpnet_model/{experiment_label_thing}/training_done" if 'chr' not in wildcards.experiment_label_thing
+ else "output/ml/rbpnet_model/"+wildcards.experiment_label_thing.split('.')[0]+"/training_done",
+ fa = "output/variants/{subset}/{experiment_label_thing}.{type}.fa"
+ output:
+ score=temp("output/variants/{subset}/{experiment_label_thing}.{type}.score.csv"),
+ fai=temp("output/variants/{subset}/{experiment_label_thing}.{type}.fa.fai")
+ threads: 1
+ resources:
+ mem_mb=lambda wildcards, attempt: 25000 * (2 ** (attempt - 1)),
+ runtime=lambda wildcards, attempt: 180 * (2 ** (attempt - 1)),
+ params:
+ exp =lambda wildcards: wildcards.experiment_label_thing.split('.')[0],
+ container:
+ "/tscc/nfs/home/bay001/eugene-tools_0.1.2.sif"
+ shell:
+ """
+ export NUMBA_CACHE_DIR=/tscc/lustre/ddn/scratch/${{USER}} # TODO: HARCODED IS BAD
+ export MPLCONFIGDIR=/tscc/lustre/ddn/scratch/${{USER}}
+
+ if [ -s {input.fa} ]; then
+ samtools faidx {input.fa};
+ python {RBPNET_PATH}/score_fa.py \
+ output/ml/rbpnet_model/{params.exp}/ \
+ {input.fa} \
+ {output.score} \
+ /tscc/lustre/ddn/scratch/${{USER}}
+ else
+ touch {output.score}
+ touch {output.fai}
+ fi
+ """
+
+rule join_gnomAD_info:
+ input:
+ scores = expand("output/variants/gnomAD_roulette/{experiment_label}.chr{chr_number}.{type}.score.csv",
+ experiment_label = ["{experiment_label}"],
+ chr_number = list(range(1,23)),
+ type = ["ref", "alt"]),
+ vcf = expand("output/variants/gnomAD_roulette/{experiment_label}.chr{chr_number}.vcf",
+ experiment_label = ["{experiment_label}"],
+ chr_number = list(range(1,23))),
+ output:
+ "output/variants/gnomAD_roulette/{experiment_label}.total.csv"
+ threads: 1
+ resources:
+ mem_mb=80000,
+ runtime=20
+ run:
+ indir = Path('output/variants/gnomAD_roulette/')
+ scores = []
+ exp = wildcards.experiment_label
+ for chr in range(1,23):
+ if os.stat(indir / f'{exp}.chr{chr}.vcf').st_size == 0:
+ continue
+ print(f'handling chrom{chr}')
+ alt_score = pd.read_csv(indir / f'{exp}.chr{chr}.alt.score.csv', index_col = 0)
+ ref_score = pd.read_csv(indir / f'{exp}.chr{chr}.ref.score.csv', index_col = 0)
+
+
+ vcf = pd.read_csv(indir / f'{exp}.chr{chr}.vcf', sep = '\t',
+ names = ['CHROM', 'POS', '.', 'REF', 'ALT',
+ 'INFO/AC', 'INFO/AN', 'INFO/MR', 'INFO/AR',
+ 'INFO/MG', 'INFO/MC'])
+
+ ref_score[['CHROM', 'POS', 'REF', 'name']]=ref_score['ID'].str.split('-', expand = True)
+ alt_score[['CHROM', 'POS', 'ALT', 'name']]=alt_score['ID'].str.split('-', expand = True)
+ score = alt_score.drop('ID', axis = 1).merge(ref_score.drop('ID', axis = 1),
+ left_on = ['CHROM', 'POS', 'name'],
+ right_on = ['CHROM', 'POS', 'name'],
+ suffixes = ('_ALT', '_REF')
+ )
+ score['delta_score'] = score['dlogodds_pred_ALT']-score['dlogodds_pred_REF']
+ score['POS'] = score['POS'].astype(int)
+ score_m = score.merge(vcf.drop('.', axis = 1), left_on = ['CHROM', 'POS', 'REF', 'ALT'],
+ right_on = ['CHROM', 'POS', 'REF', 'ALT']
+ )
+ scores.append(score_m)
+ try:
+ scores = pd.concat(scores, axis = 0)
+ scores.to_csv(output[0], index = False)
+ except:
+ print('no gnomAD variants found')
+ open(output[0], 'w').close()
+
+rule fetch_Clinvar_SNP:
+ input:
+ finemapped_windows = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.bed.gz",
+ vcf = CLINVAR_VCF.replace('.vcf.gz', '.rename.vcf.gz')
+ output:
+ "output/variants/clinvar/{experiment_label}.vcf"
+ threads: 2
+ resources:
+ mem_mb=50000,
+ runtime="3h"
+ container:
+ "docker://brianyee/bcftools:1.17"
+ shell:
+ """
+ if [ -s {input.finemapped_windows} ]; then
+ bcftools query -R {input.finemapped_windows} \
+ -f '%CHROM\t%POS\t%ID\t%REF\t%ALT\t%INFO/CLNDN\t%INFO/CLNVC\t%INFO/CLNSIG\t%INFO/CLNDISDB\t%INFO/AF_ESP\t%INFO/AF_EXAC\t%INFO/AF_TGP\t%INFO/ALLELEID\n' \
+ {input.vcf} > {output}
+ else
+ touch {output}
+ fi
+ """
+
+rule download_vep_cache:
+ output:
+ Path(VEP_CACHEDIR) / f'homo_sapiens/{VEP_CACHE_VERSION}_GRCh38/1/all_vars.gz'
+ threads: 1
+ resources:
+ mem_mb=2000,
+ runtime="1h"
+ container:
+ "docker://ensemblorg/ensembl-vep:release_113.4"
+ shell:
+ """
+ mkdir -p {VEP_CACHEDIR}/ && cd {VEP_CACHEDIR}/;
+ curl -o {VEP_CACHEDIR}/homo_sapiens_vep_{VEP_CACHE_VERSION}_GRCh38.tar.gz https://ftp.ensembl.org/pub/release-113/variation/indexed_vep_cache/homo_sapiens_vep_{VEP_CACHE_VERSION}_GRCh38.tar.gz;
+ tar xzf homo_sapiens_vep_{VEP_CACHE_VERSION}_GRCh38.tar.gz
+ """
+rule vep:
+ input:
+ vcf = "output/variants/clinvar/{experiment_label}.vcf",
+ vars = Path(VEP_CACHEDIR) / f'homo_sapiens/{VEP_CACHE_VERSION}_GRCh38/1/all_vars.gz'
+ output:
+ "output/variants/clinvar/{experiment_label}.vep.tsv"
+ threads: 2
+ resources:
+ mem_mb=40000,
+ runtime="1h"
+ container:
+ "docker://ensemblorg/ensembl-vep:release_113.4"
+ shell:
+ """
+ if [ -s {input.vcf} ]; then
+ vep \
+ -i {input.vcf} \
+ --force_overwrite \
+ -o {output} -offline --cache {VEP_CACHEDIR}
+ else
+ touch {output}
+ fi
+ """
+
+rule variant_analysis:
+ input:
+ clinvar = "output/variants/clinvar/{experiment_label}.vep.tsv",
+ gnomAD = "output/variants/gnomAD_roulette/{experiment_label}.total.csv",
+ annotated = "output/finemapping/mapped_sites/{experiment_label}.finemapped_windows.annotated.tsv"
+ output:
+ "output/variant_analysis/{experiment_label}.clinvar_variants.csv",
+ "output/variant_analysis/{experiment_label}.annotated.csv.gz",
+ "output/variant_analysis/{experiment_label}.feature_type_top.oe_stat.lofbins.csv",
+ "output/variant_analysis/{experiment_label}.transcript_type_top.MAPS_stat.csv",
+ "output/variant_analysis/{experiment_label}.feature_type_top_spectrum_enrichment.csv",
+ "output/variant_analysis/{experiment_label}.transcript_type_top.MAPS.csv",
+ "output/variant_analysis/{experiment_label}.feature_type_top.oe.csv",
+ "output/variant_analysis/{experiment_label}.transcript_type_top_spectrum_enrichment.csv",
+ "output/variant_analysis/{experiment_label}.transcript_type_top.oe_stat.csv",
+ "output/variant_analysis/{experiment_label}.transcript_type_top.oe.csv",
+ "output/variant_analysis/{experiment_label}.clinvar_CLINSIC_counts.csv",
+ "output/variant_analysis/{experiment_label}.global_MAPS.csv",
+ "output/variant_analysis/{experiment_label}.global_spectrum_enrichment.csv",
+ "output/variant_analysis/{experiment_label}.feature_type_top.MAPS.lofbins.csv",
+ "output/variant_analysis/{experiment_label}.feature_type_top.oe_stat.csv",
+ "output/variant_analysis/{experiment_label}.clinvar_impact_counts.pdf",
+ "output/variant_analysis/{experiment_label}.clinvar_CLINSIC_counts.pdf",
+ "output/variant_analysis/{experiment_label}.global_MAPS_stat.csv",
+ "output/variant_analysis/{experiment_label}.feature_type_top.MAPS_stat.lofbins.csv",
+ "output/variant_analysis/{experiment_label}.global_oe.csv",
+ "output/variant_analysis/{experiment_label}.feature_type_top.MAPS_stat.csv",
+ "output/variant_analysis/{experiment_label}.clinvar_impact_counts.csv",
+ "output/variant_analysis/{experiment_label}.clinvar_variants_exploded.csv",
+ "output/variant_analysis/{experiment_label}.global_oe_stat.csv",
+ "output/variant_analysis/{experiment_label}.feature_type_top.MAPS.csv",
+ "output/variant_analysis/{experiment_label}.feature_type_top.oe.lofbins.csv",
+ threads: 1
+ resources:
+ mem_mb=40000,
+ runtime=20
+ conda:
+ "envs/metadensity.yaml"
+ shell:
+ """
+ module purge;
+ if [ -s {input.gnomAD} ]; then
+ python {TOOL_DIR}/mega_variant_analysis.py \
+ . \
+ {wildcards.experiment_label} \
+ {SINGLETON_REFERENCE} \
+ {OE_RATIO_REFERENCE} \
+ {GNOMAD_CONSTRAINT} \
+
+ else
+ touch {output}
+ fi
+ """
+
diff --git a/rules/visualize_ml.smk b/rules/visualize_ml.smk
new file mode 100644
index 0000000..3bf6f3a
--- /dev/null
+++ b/rules/visualize_ml.smk
@@ -0,0 +1,226 @@
+workdir: config['WORKDIR']
+import pandas as pd
+"""
+snakemake -kps rules/visualize_ml.smk \
+ --configfile /tscc/nfs/home/hsher/projects/skipper/encode_configs/encode_pe_rules_config_K562_20230929.yaml \
+ --profile profiles/tscc2_single -n
+
+# making splice site predictions
+snakemake -kps rules/visualize_ml.smk \
+ --configfile /tscc/nfs/home/hsher/projects/skipper/encode_configs/encode_pe_rules_config_HepG2_20230620.yaml \
+ --profile profiles/tscc2_single output/gkmsvm_explain/windows/{AQR_HepG2_ENCSR018WPY,BUD13_HepG2_ENCSR830BSQ,U2AF2_HepG2_ENCSR202BFN,SUGP2_HepG2_ENCSR506UPY,HNRNPC_HepG2_ENCSR550DVK,PTBP1_HepG2_ENCSR384KAN,HLTF_HepG2_ENCSR647HOX,GTF2F1_HepG2_ENCSR265ZIS,BCLAF1_HepG2_ENCSR876EYA,SF3A3_HepG2_ENCSR331MIC,QKI_HepG2_ENCSR570WLM,MATR3_HepG2_ENCSR290VLT,SF3B4_HepG2_ENCSR279UJF,ILF3_HepG2_ENCSR786TSC,HNRNPK_HepG2_ENCSR828ZID,UCHL5_HepG2_ENCSR490IEE,RBFOX2_HepG2_ENCSR987FTF,TAF15_HepG2_ENCSR841EQA,CSTF2_HepG2_ENCSR384MWO,DDX59_HepG2_ENCSR214BZA,FAM120A_HepG2_ENCSR987NYS,PRPF8_HepG2_ENCSR121NVA,HNRNPM_HepG2_ENCSR267UCX,KHSRP_HepG2_ENCSR366DGX,NKRF_HepG2_ENCSR277DEO,PCBP2_HepG2_ENCSR339FUY,AGGF1_HepG2_ENCSR543TPH,CSTF2T_HepG2_ENCSR919HSE,GRWD1_HepG2_ENCSR893NWB,EFTUD2_HepG2_ENCSR527DXF,HNRNPL_HepG2_ENCSR724RDN,SFPQ_HepG2_ENCSR965DLL,PPIG_HepG2_ENCSR097NEE,XPO5_HepG2_ENCSR921SXC,PRPF4_HepG2_ENCSR977OXG}.709.705.gkmexplain.txt \
+ -n
+# compare with dlogodds
+snakemake -kps rules/visualize_ml.smk \
+ --configfile /tscc/nfs/home/hsher/projects/skipper/encode_configs/encode_pe_rules_config_HepG2_20230620.yaml \
+ --profile profiles/tscc2_single output/ml/sequence/reproducible_enriched_windows/{RBFOX2_HepG2_ENCSR987FTF,AQR_HepG2_ENCSR018WPY,BUD13_HepG2_ENCSR830BSQ,U2AF2_HepG2_ENCSR202BFN,PRPF4_HepG2_ENCSR977OXG}.txt \
+ -n
+# compare with dlogodds
+snakemake -kps rules/visualize_ml.smk \
+ --configfile /tscc/nfs/home/hsher/projects/skipper/encode_configs/encode_pe_rules_config_HepG2_20230620.yaml \
+ --profile profiles/tscc2 \
+ -n
+
+# compare with biophysical
+snakemake -kps rules/visualize_ml.smk \
+ --configfile /tscc/nfs/home/hsher/projects/skipper/encode_configs/encode_pe_rules_config_K562_20230929.yaml \
+ --profile profiles/tscc2_single output/ml/PUM_biophysical_model_enriched/{PUM1_K562_ENCSR308YNT,PUM2_K562_ENCSR661ICQ}/individual_scores.0.tsv \
+ -n
+"""
+locals().update(config)
+auprc = pd.read_csv('output/ml/gkmsvm/AUPRC.txt', index_col = 0)
+pass_exp = auprc.loc[auprc['mean AUPRC']>0.65, 'Experiment'].tolist()
+
+
+rule all:
+ input:
+ lambda wildcards: [f"output/gkmsvm_explain/{experiment_label}.foreground.{experiment_label}.gkmexplain.txt"
+ for experiment_label in pass_exp
+ ],
+ expand("output/ml/sequence/reproducible_enriched_windows/{experiment_label}.txt",
+ experiment_label = pass_exp)
+
+
+rule fix_model:
+ input:
+ model = "output/ml/gkmsvm/{experiment_label}.model.txt"
+ output:
+ "output/ml/gkmsvm/{experiment_label}.model.fix.txt"
+ params:
+ error_file = "stderr/{experiment_label}.fix_model.err",
+ out_file = "stdout/{experiment_label}.fix_model.out",
+ run_time = "10:00",
+ memory = "1000",
+ job_name = "run_homer",
+ shell:
+ """
+ sed -e 's/norc/gamma/g' {input.model} > {output}
+ """
+
+
+rule gkmsvm_explain:
+ input:
+ fa="output/ml/sequence/{something}.fa",
+ model = "output/ml/gkmsvm/{experiment_label}.model.fix.txt"
+ output:
+ output="output/gkmsvm_explain/{something}.{experiment_label}.gkmexplain.txt"
+ container:
+ "docker://kundajelab/lsgkm:latest"
+ params:
+ error_file = "stderr/{experiment_label}.{something}.gkmexplain.err",
+ out_file = "stdout/{experiment_label}.{something}.gkmexplin.out",
+ run_time = "10:00",
+ memory = "40000",
+ job_name = "gkmsvm_explain",
+ shell:
+ """
+ head -n 40 {input.fa} > {input.fa}.head
+ /opt/lsgkm/src/gkmexplain \
+ {input.fa}.head \
+ {input.model} \
+ {output.output}
+ """
+
+rule window_to_sequence_with_range:
+ input:
+ config['PARTITION']
+ output:
+ "output/gkmsvm_explain/windows/sequence/{max}.{min}.fa"
+ params:
+ error_file = "stderr/{max}.{min}.fa.err",
+ out_file = "stdout/{max}.{min}.fa.out",
+ run_time = "10:00",
+ memory = "1000",
+ job_name = "window_to_sequence",
+ container:
+ "docker://howardxu520/skipper:samtools_1.17_bedtools_2.31.0"
+ shell:
+ """
+ zcat {input} | awk '{{ if ($4 >= {wildcards.min} && $4 <= {wildcards.max}) print}}' |\
+ bedtools getfasta -fi {GENOME} -bed stdin -s -fo {output}
+ """
+
+rule gkmsvm_explain_windows:
+ input:
+ fa="output/gkmsvm_explain/windows/sequence/{max}.{min}.fa",
+ model = "output/ml/gkmsvm/{experiment_label}.model.fix.txt"
+ output:
+ output="output/gkmsvm_explain/windows/{experiment_label}.{max}.{min}.gkmexplain.txt"
+ params:
+ error_file = "stderr/{experiment_label}.{max}.{min}.gkmexplain.err",
+ out_file = "stdout/{experiment_label}.{max}.{min}.gkmexplin.out",
+ run_time = "10:00",
+ memory = "40000",
+ job_name = "gkmsvm_explain",
+ container:
+ "docker://kundajelab/lsgkm:latest"
+ shell:
+ """
+ /opt/lsgkm/src/gkmexplain \
+ {input.fa} \
+ {input.model} \
+ {output.output}
+ """
+
+rule sequence_from_reproducible_enriched_windows:
+ input:
+ "output/reproducible_enriched_windows/{experiment_label}.reproducible_enriched_windows.tsv.gz"
+ output:
+ temp("output/ml/sequence/reproducible_enriched_windows/{experiment_label}.fa")
+ params:
+ error_file = "stderr/{experiment_label}.fa.err",
+ out_file = "stdout/{experiment_label}.fa.out",
+ run_time = "10:00",
+ memory = "1000",
+ job_name = "sequence_from_reproducible_enriched_windows"
+ container:
+ "docker://howardxu520/skipper:samtools_1.17_bedtools_2.31.0"
+ shell:
+ """
+ zcat {input} | tail -n +2 | bedtools getfasta -fi {GENOME} -bed stdin -s -fo {output}
+ """
+
+rule predict_enriched_windows:
+ input:
+ fa = "output/ml/sequence/reproducible_enriched_windows/{experiment_label}.fa",
+ model = "output/ml/gkmsvm/{experiment_label}.model.txt",
+ output:
+ score = "output/ml/sequence/reproducible_enriched_windows/{experiment_label}.txt",
+ threads: 2
+ params:
+ error_file = "stderr/score_ref_variants.{experiment_label}",
+ out_file = "stdout/score_ref_variants.{experiment_label}",
+ run_time = "4:00:00",
+ cores = 1,
+ memory = 40000, #320000 is ceiling of gold slow
+ container:
+ "docker://shl198/lsgkm:0.1.1"
+ shell:
+ """
+ /bin/gkmpredict -T 16 {input.fa} {input.model} {output.score}
+ """
+
+rule cleaup_fa:
+ input:
+ "{anything}.fa"
+ output:
+ replacet=temp("{anything}.U.fa"),
+ removen=temp("{anything}.UN.fa")
+ threads: 1
+ params:
+ error_file = "stderr/score_ref_variants.{anything}",
+ out_file = "stdout/score_ref_variants.{anything}",
+ run_time = "00:00:05",
+ cores = 1,
+ memory = 40000, #320000 is ceiling of gold slow
+ conda: "envs/metadensity.yaml"
+ shell:
+ """
+ sed '/^[^>]/s/T/U/g' {input} > {output.replacet}
+ python /tscc/nfs/home/hsher/projects/PUM2_biophysical/removeN.py {output.replacet} {output.removen}
+ """
+
+BIOPHYSICAL_MODEL_PATH='/tscc/nfs/home/hsher/projects/PUM2_biophysical/final_pum2_w_flips_coupling_eab_predict.py'
+rule pum2_biophysical_model:
+ input:
+ foreground="output/ml/sequence/{experiment_label}.foreground.UN.fa",
+ background="output/ml/sequence/{experiment_label}.background.UN.fa",
+ output:
+ "output/ml/PUM_biophysical_model/{experiment_label}/individual_scores.0.tsv",
+ threads: 1
+ params:
+ error_file = "stderr/pum_biophysical.{experiment_label}",
+ out_file = "stdout/pum_biophysical.{experiment_label}",
+ run_time = "00:20:00",
+ cores = 1,
+ memory = 40000, #320000 is ceiling of gold slow
+ conda: "envs/pum2model.yaml"
+ shell:
+ """
+ python {BIOPHYSICAL_MODEL_PATH} \
+ --pos_fasta {input.foreground} \
+ --neg_fasta {input.background} \
+ --out_dir output/ml/PUM_biophysical_model/{wildcards.experiment_label}
+ """
+
+rule pum2_biophysical_model_enriched_windows:
+ input:
+ foreground="output/ml/sequence/reproducible_enriched_windows/{experiment_label}.UN.fa",
+ background="output/ml/sequence/{experiment_label}.background.UN.fa",
+ output:
+ "output/ml/PUM_biophysical_model_enriched/{experiment_label}/individual_scores.0.tsv",
+ threads: 1
+ params:
+ error_file = "stderr/pum_biophysical.{experiment_label}",
+ out_file = "stdout/pum_biophysical.{experiment_label}",
+ run_time = "00:20:00",
+ cores = 1,
+ memory = 40000, #320000 is ceiling of gold slow
+ conda: "envs/pum2model.yaml"
+ shell:
+ """
+ python {BIOPHYSICAL_MODEL_PATH} \
+ --pos_fasta {input.foreground} \
+ --neg_fasta {input.background} \
+ --out_dir output/ml/PUM_biophysical_model_enriched/{wildcards.experiment_label}
+ """
\ No newline at end of file
diff --git a/tools/annotate_clinvar.py b/tools/annotate_clinvar.py
new file mode 100644
index 0000000..9c7e16a
--- /dev/null
+++ b/tools/annotate_clinvar.py
@@ -0,0 +1,224 @@
+import pandas as pd
+from pathlib import Path
+from pybedtools import BedTool
+import sys
+import matplotlib.pyplot as plt
+import seaborn as sns
+from scipy.stats import norm
+import numpy as np
+import statsmodels.api as sm
+import statsmodels.formula.api as smf
+from statsmodels.stats.multitest import fdrcorrection
+from scipy.stats import fisher_exact
+from scipy.stats import chisquare
+
+def sigmoid(z):
+ return 1/(1 + np.exp(-z))
+
+def testing(df):
+ ''' perform fisher exact or chisq given contingency table'''
+ if df.shape != (2,2):
+ # some outcomes are unobserved
+ print(df.shape)
+ print('no binding is observed in')
+ return 1, np.nan
+ if df.le(5).any().any():
+ odds_ratio, pvalue = fisher_exact(df)
+ else:
+ chi, pvalue = chisquare(df.loc[True], (df.loc[True].sum())*df.loc[False].div(df.loc[False].sum()))
+ odds_ratio = (df.loc[True, True]/df.loc[True, False])/(df.loc[False, True]/df.loc[False, False])
+ return pvalue, odds_ratio
+
+if __name__ == '__main__':
+ indir = Path(sys.argv[1])
+ exp = sys.argv[2]
+ finemap_annotation = indir / 'output/finemapping/mapped_sites/' / f'{exp}.finemapped_windows.annotated.tsv'
+
+ # load all files
+ ref = pd.read_csv(indir / 'output/variants' / 'clinvar' / f'{exp}.ref.score.txt', sep = '\t', names = ['ID', 'score'])
+ ref.rename({'score': 'score_ref'}, axis = 1, inplace = True)
+ ref.drop('ID', axis = 1, inplace = True)
+ alt = pd.read_csv(indir / 'output/variants' / 'clinvar' / f'{exp}.alt.score.txt', sep = '\t', names = ['ID', 'score'])
+ alt.rename({'score': 'score_alt'}, axis = 1, inplace = True)
+ alt.drop('ID', axis = 1, inplace = True)
+ annot = pd.read_csv(indir / 'output/variants' / 'clinvar' / f'{exp}.csv', index_col = 0)
+ site_annotation_uniq = pd.read_csv(finemap_annotation, sep = '\t')
+
+ # load gnomAD test
+ gnomAD_test = pd.read_csv(indir / 'output/variants/gnomAD_analysis' / f'{exp}.test_stats.csv', index_col = 0)
+ # calculate binning
+ df = pd.concat([ref,alt, annot], axis = 1)
+ df.rename({'5': 'INFO/CLNDN', '6': 'INFO/CLNVC', '7': 'INFO/CLNSIG',
+ '8': 'INFO/CLNDISDB', '9': 'INFO/AF_ESP', '10': 'INFO/AF_EXAC',
+ '11': 'INFO/AF_TGP', '12': 'INFO/ALLELEID'}
+ , axis = 1, inplace = True)
+
+ # clean up CLNSIG
+ df['INFO/CLNSIG']=df['INFO/CLNSIG'].replace(
+ 'Likely_benign', 'Benign/Likely_benign').replace(
+ 'Benign', 'Benign/Likely_benign').replace(
+ 'Pathogenic', 'Pathogenic/Likely_pathogenic').replace(
+ 'Likely_pathogenic', 'Pathogenic/Likely_pathogenic').replace(
+ 'Conflicting_interpretations_of_pathogenicity', 'Uncertain_significance')
+ df['INFO/CLNSIG'] = pd.Categorical(df['INFO/CLNSIG'],
+ categories = ['Benign/Likely_benign', 'Uncertain_significance', 'Pathogenic/Likely_pathogenic'],
+ ordered = True)
+ df['CLNSIG_rank'] = df['INFO/CLNSIG'].rank(method = 'dense')
+ df['score_alt'] = sigmoid(df['score_alt'])
+ df['score_ref'] = sigmoid(df['score_ref'])
+ df['delta_score']=df['score_alt']-df['score_ref']
+
+
+ # now annotate, all variants should be derived from finemapped windows
+ assert df['name'].isin(site_annotation_uniq['name']).all()
+ df['feature_type_top'] = df['name'].map(site_annotation_uniq.set_index('name')['feature_type_top'])
+ df['transcript_type_top'] = df['name'].map(site_annotation_uniq.set_index('name')['transcript_type_top'])
+ df['gene_name']=df['name'].map(site_annotation_uniq.set_index('name')['gene_name'])
+ df['strand'] = df['name'].map(site_annotation_uniq.set_index('name')['strand'])
+
+ # annotate variant type
+ df.loc[(df['REF'].str.len()==1)&(df['ALT'].str.len()==1), 'TYPE']='SNV'
+ df.loc[(df['REF'].str.len()!=1)|(df['ALT'].str.len()!=1), 'TYPE']='INDEL'
+
+ # annotat impact
+ # annotate LoF and GoF based on gnomAD std
+ with open(indir / 'output/variants' / 'gnomAD' / f'{exp}.variants_scores.tsv', 'r') as f:
+ for line in f:
+ std=float(line.split(':')[1].rstrip())
+ break
+ df.loc[df['delta_score']<-2*std, 'impact'] = 'LoF'
+ df.loc[df['delta_score']>2*std, 'impact'] = 'GoF'
+ df['impact'].fillna('neutral', inplace = True)
+
+ # Summary statistics: Distribution of delta
+ with open(indir / 'output/variants' / 'gnomAD' / f'{exp}.variants_scores.tsv', 'r') as f:
+ for line in f:
+ if 'std' in line:
+ std=float(line.split(':')[1].rstrip())
+ elif 'mean' in line:
+ mean=float(line.split(':')[1].rstrip())
+ else:
+ break
+
+ df.loc[df['delta_score']<-2*std, 'impact'] = 'LoF'
+ df.loc[df['delta_score']>2*std, 'impact'] = 'GoF'
+ df['impact'].fillna('neutral', inplace = True)
+ df['impact'] = pd.Categorical(df['impact'], categories = ['LoF', 'neutral', 'GoF'], ordered = True)
+
+ df['zscore'] = df['delta_score']/std
+ df['zscore_bins'] = pd.cut(df['zscore'], bins = (-np.inf, -2,-1,0,1,2, np.inf))
+
+ impact_per_sig = pd.pivot_table(df, index = 'impact', columns = 'INFO/CLNSIG',aggfunc = 'size').T
+ impact_per_sig.div(impact_per_sig.sum(axis = 0)).T.plot.bar(
+ cmap = 'coolwarm', figsize = (3,3))
+ plt.ylabel('Fraction of variants')
+ plt.legend(bbox_to_anchor = (1.5,1))
+ sns.despine()
+ plt.savefig(indir / 'output/variants' / 'clinvar_analysis' / f'{exp}.impact_CLNSIG.pdf')
+
+ # parse by disease
+ df['CLNDN_parsed'] = df['INFO/CLNDN'].str.split('|').tolist()
+ df_exploded = df.explode('CLNDN_parsed')
+ disease_impact_count = pd.pivot_table(df_exploded, index = 'CLNDN_parsed',
+ columns = 'INFO/CLNSIG', aggfunc = 'size').fillna(0)
+ disease_impact_count.loc[disease_impact_count.sum(axis = 1).sort_values(ascending = False).index
+ ].iloc[:10,:].plot.barh(stacked = True, figsize = (3,3), cmap = 'coolwarm')
+ plt.xlabel('# variants overlapping \n binding site')
+ plt.legend(title = 'CLNSIG', bbox_to_anchor = (1, 0.5))
+ sns.despine()
+ plt.savefig(indir / 'output/variants' / 'clinvar_analysis' / f'{exp}.variants_overlapping_binding.pdf')
+
+ disease_impact_count = pd.pivot_table(df_exploded.loc[df['INFO/CLNSIG']!='Benign/Likely_benign'], index = 'CLNDN_parsed',
+ columns = 'zscore_bins', aggfunc = 'size').fillna(0)
+ disease_impact_count.loc[disease_impact_count.sum(axis = 1).sort_values(ascending = False).index
+ ].iloc[:10,:].plot.barh(stacked = True, figsize = (3,3), cmap = 'coolwarm')
+ plt.xlabel('# non-benign variants')
+ plt.legend(title = 'delta score bins')
+ sns.despine()
+ plt.savefig(indir / 'output/variants' / 'clinvar_analysis' / f'{exp}.non_benign_impact_cnt.pdf')
+
+ disease_impact_count = pd.pivot_table(df_exploded.loc[df['INFO/CLNSIG']!='Benign/Likely_benign'], index = 'CLNDN_parsed',
+ columns = 'zscore_bins', aggfunc = 'size').fillna(0)
+ disease_impact_count.div(disease_impact_count.sum(axis = 1
+ ), axis = 0).loc[disease_impact_count.sum(axis = 1).sort_values(ascending = False).index
+ ].iloc[:10,:].sort_values(by = pd.Interval(-np.inf, -2, closed='right')).plot.barh(stacked = True, figsize = (3,3), cmap = 'coolwarm')
+ sns.despine()
+ plt.legend(bbox_to_anchor = (1.5,1))
+ plt.xlabel('Fraction non-benign variants')
+ plt.savefig(indir / 'output/variants' / 'clinvar_analysis' / f'{exp}.non_benign_impact_distribution.pdf')
+
+ # # test for singificance for constrained genes
+ gnomAD_test['is_ClinVar']=gnomAD_test.index.isin(df['gene_name'])
+ gnomAD_test['sig_against_0'] = gnomAD_test['FDR_against_0']<0.2
+ gnomAD_test['sig_against_mean'] = gnomAD_test['FDR_against_mean']<0.2
+
+ cont = pd.pivot_table(gnomAD_test, index = 'is_ClinVar', columns = 'sig_against_0', aggfunc = 'size').fillna(0)
+ pv, odds = testing(cont)
+ with open(indir / 'output/variants' / 'clinvar_analysis' / f'{exp}.constrain_disease_test', 'w') as f:
+ f.write(f'{exp},{pv},{odds}\n')
+
+ # prioritize disease variants in constrained genes
+ f, ax = plt.subplots(1,2, sharex = True, figsize = (12,3))
+ lof_count = df.loc[(df['gene_name'].isin(gnomAD_test.loc[gnomAD_test['sig_against_0']].index))
+ & (df['impact']=='LoF')
+ ].explode('CLNDN_parsed')['CLNDN_parsed'].value_counts()
+ if not lof_count.empty:
+ lof_count.plot.barh(
+ stacked = True, by = 'z_score_bins',
+ ax = ax[0],
+ color = 'royalblue'
+ )
+ ax[0].set_xlabel('# LoF variants in constrained genes')
+
+
+ gof_count = df.loc[(df['gene_name'].isin(gnomAD_test.loc[gnomAD_test['sig_against_0']].index))
+ & (df['impact']=='GoF')
+ ].explode('CLNDN_parsed')['CLNDN_parsed'].value_counts()
+ if not gof_count.empty:
+ gof_count.plot.barh(
+ stacked = True, by = 'z_score_bins',
+ ax = ax[1],
+ color = 'tomato'
+ )
+ ax[1].set_xlabel('# GoF variants in constrained genes')
+ plt.tight_layout()
+ sns.despine()
+
+ disease_count = pd.concat([lof_count, gof_count], axis = 1).fillna(0)
+ disease_count.columns = ['LoF', 'GoF']
+ disease_count.to_csv(indir/'output/variants' / 'clinvar_analysis' / f'{exp}.constrain_disease_count.csv')
+
+
+ f, ax = plt.subplots(1,2, sharex = True, figsize = (12,3))
+ lof_count = df.loc[(df['gene_name'].isin(gnomAD_test.loc[gnomAD_test['sig_against_0']].index))
+ & (df['impact']=='LoF')
+ ]['gene_name'].value_counts()
+ if not lof_count.empty:
+ lof_count.plot.barh(
+ stacked = True, by = 'z_score_bins',
+ ax = ax[0],
+ color = 'royalblue'
+ )
+ ax[0].set_xlabel('# LoF variants in constrained genes')
+
+ gof_count = df.loc[(df['gene_name'].isin(gnomAD_test.loc[gnomAD_test['sig_against_0']].index))
+ & (df['impact']=='GoF')
+ ]['gene_name'].value_counts()
+ if not gof_count.empty:
+ gof_count.plot.barh(
+ stacked = True, by = 'z_score_bins',
+ ax = ax[1],
+ color = 'tomato'
+ )
+ ax[1].set_xlabel('# GoF variants in constrained genes')
+ plt.tight_layout()
+ sns.despine()
+
+ gene_count = pd.concat([lof_count, gof_count], axis = 1).fillna(0)
+ gene_count.columns = ['LoF', 'GoF']
+ disease_count.to_csv(indir/'output/variants' / 'clinvar_analysis' / f'{exp}.constrain_disease_gene_count.csv')
+
+ # save file
+ df.to_csv(indir/'output/variants' / 'clinvar' / f'{exp}.variants_scores.csv')
+
+
diff --git a/tools/annotate_cosmic.py b/tools/annotate_cosmic.py
new file mode 100644
index 0000000..e8dee1d
--- /dev/null
+++ b/tools/annotate_cosmic.py
@@ -0,0 +1,196 @@
+import pandas as pd
+from pathlib import Path
+import matplotlib.pyplot as plt
+import seaborn as sns
+import numpy as np
+import sys
+from scipy.stats import chisquare
+from scipy.stats import binom
+def test_positive_selection(
+ df,
+ rate_index = ('neutral', '.')
+ ):
+ stat = []
+ p_null = df.loc[rate_index, 'COSMIC_SAMPLE_MUTATED']/df.loc[rate_index, 'COSMIC_SAMPLE_TESTED']
+
+ for index, row in df.iterrows():
+ if index != rate_index:
+ # probability of getting something
+ pvalue = 1-binom.cdf(n = row['COSMIC_SAMPLE_TESTED'], k = row['COSMIC_SAMPLE_MUTATED'],
+ p = p_null)
+ fc = (row['COSMIC_SAMPLE_MUTATED']/row['COSMIC_SAMPLE_TESTED'])/p_null
+
+ stat.append(list(index)+[pvalue, fc])
+
+ stat = pd.DataFrame(stat, columns = df.index.names + ['pvalue', 'fc'])
+ return stat
+
+def test_association(df,
+ categories = ['LoF', 'GoF'],
+ groups = ['1','2'],
+ null_group = '.'
+ ):
+ '''
+ impact_per_sig: categories by groups
+ '''
+
+ # get frequency of categories in group
+ freq = df.div(df.sum(axis = 0), axis = 1)
+
+
+ stats = []
+ null_frequency = freq[null_group]
+ for category in categories: # LoF or GoF
+ # s = maf_vs_impact.sum()
+ for index in groups: # Tier 1,2
+ try:
+ row = df[index]
+ total_cnt = row.sum()
+ # expected count from tier '
+ p = null_frequency.loc[category] #
+
+ f = np.array([p, 1-p])
+ exp = total_cnt*f
+
+ # observed
+ cnt = row.loc[category]
+
+
+ chi, p = chisquare(np.array([cnt, total_cnt-cnt]), exp)
+ odds_ratio = (cnt/(total_cnt-cnt))/(p/(1-p))
+ stats.append([index, category, p, odds_ratio])
+ except Exception as e:
+ print(e, index, category)
+
+ stats = pd.DataFrame(stats, columns = ['group', 'category', 'pvalue', 'odds_ratio'])
+ return stats
+
+def sigmoid(z):
+ return 1/(1 + np.exp(-z))
+
+if __name__ == '__main__':
+ indir = Path(sys.argv[1])
+ exp = Path(sys.argv[2])
+ mutation_census_file = '/tscc/nfs/home/hsher/ps-yeolab5/cosmic_data/CancerMutationCensus_AllData_v98_GRCh38.tsv.gz'
+ gene_census_file = '/tscc/nfs/home/hsher/ps-yeolab5/cosmic_data/CancerGeneCensus.tsv'
+
+ # load cancer gene annotations
+ cancer_mut_census = pd.read_csv(mutation_census_file,
+ sep = '\t')
+ cancer_gene_census = pd.read_csv(gene_census_file,
+ sep = '\t')
+ cancer_gene_census['Role'] = cancer_gene_census['Role in Cancer'].str.split(', ')
+ cancer_gene_census_exploded=cancer_gene_census.explode('Role')
+
+ # calculate binding score
+ ref = pd.read_csv(indir / 'output/variants' / 'cosmic_noncoding' / f'{exp}.ref.score.txt', sep = '\t', names = ['ID', 'score'])
+ ref.rename({'score': 'score_ref'}, axis = 1, inplace = True)
+ ref.drop('ID', axis = 1, inplace = True)
+ alt = pd.read_csv(indir / 'output/variants' / 'cosmic_noncoding' / f'{exp}.alt.score.txt', sep = '\t', names = ['ID', 'score'])
+ alt.rename({'score': 'score_alt'}, axis = 1, inplace = True)
+ alt.drop('ID', axis = 1, inplace = True)
+ annot = pd.read_csv(indir / 'output/variants' / 'cosmic_noncoding' / f'{exp}.csv', index_col = 0)
+
+ df = pd.concat([ref,alt, annot], axis = 1)
+
+ df.rename({'5': 'INFO/SAMPLE_COUNT', '6': 'INFO/TIER'}
+ , axis = 1, inplace = True)
+
+ df['score_alt'] = sigmoid(df['score_alt'])
+ df['score_ref'] = sigmoid(df['score_ref'])
+ df['delta_score']=df['score_alt']-df['score_ref']
+
+ # annotate
+ finemap_annotation = indir / 'output/finemapping/mapped_sites/' / f'{exp}.finemapped_windows.annotated.tsv'
+ site_annotation_uniq = pd.read_csv(finemap_annotation, sep = '\t')
+
+
+ # now annotate, all variants should be derived from finemapped windows
+ assert df['name'].isin(site_annotation_uniq['name']).all()
+ df['feature_type_top'] = df['name'].map(site_annotation_uniq.set_index('name')['feature_type_top'])
+ df['transcript_type_top'] = df['name'].map(site_annotation_uniq.set_index('name')['transcript_type_top'])
+ df['gene_name']=df['name'].map(site_annotation_uniq.set_index('name')['gene_name'])
+ df['strand'] = df['name'].map(site_annotation_uniq.set_index('name')['strand'])
+
+ # annotate variant type
+ df.loc[(df['REF'].str.len()==1)&(df['ALT'].str.len()==1), 'TYPE']='SNV'
+ df.loc[(df['REF'].str.len()!=1)|(df['ALT'].str.len()!=1), 'TYPE']='INDEL'
+ df = df.merge(cancer_mut_census, left_on = 'ID', right_on = 'GENOMIC_MUTATION_ID')
+
+ # annotate impact
+ with open(indir / 'output/variants' / 'gnomAD' / f'{exp}.variants_scores.tsv', 'r') as f:
+ for line in f:
+ if 'std' in line:
+ std=float(line.split(':')[1].rstrip())
+ elif 'mean' in line:
+ mean=float(line.split(':')[1].rstrip())
+ else:
+ break
+
+ df.loc[df['delta_score']<-2*std, 'impact'] = 'LoF'
+ df.loc[df['delta_score']>2*std, 'impact'] = 'GoF'
+ df['impact'].fillna('neutral', inplace = True)
+ df['impact'] = pd.Categorical(df['impact'], categories = ['LoF', 'neutral', 'GoF'], ordered = True)
+
+
+ df['zscore'] = df['delta_score']/std
+ df['zscore_bins'] = pd.cut(df['zscore'], bins = (-np.inf, -2,-1,0,1,2, np.inf))
+
+ # annotate gene role
+ gene2role = pd.pivot_table(cancer_gene_census_exploded,
+ index = 'Gene Symbol',
+ columns = ['Role'], aggfunc = 'size').fillna(0).applymap(lambda x: True if x==1 else False)
+ df = df.merge(gene2role, left_on = 'gene_name', right_index = True, how = 'left')
+ df[['oncogene', 'TSG']] = df[['oncogene', 'TSG']].fillna(False)
+
+ # test association RBP impact with gene tier
+ impact_per_sig = pd.pivot_table(df, index = 'impact', columns = 'INFO/TIER',aggfunc = 'size')
+ impact_per_sig.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.impact_vs_tier.csv')
+
+ stats_tier = test_association(impact_per_sig)
+ stats_tier.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.impact_vs_tier_stat.csv')
+
+ # test association RBP impact with gene role
+ role_to_variants = df.merge(cancer_gene_census_exploded[['Gene Symbol', 'Role']],
+ left_on = 'gene_name',
+ right_on = 'Gene Symbol',
+ how = 'left')
+ role_to_variants['Role'].fillna('No established role', inplace = True)
+ impact_per_role = pd.pivot_table(role_to_variants, index = 'impact', columns = 'Role',aggfunc = 'size')
+ impact_per_role.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.impact_vs_role.csv')
+
+ stats_role = test_association(impact_per_role, groups = ['TSG', 'fusion', 'oncogene'],
+ null_group = 'No established role')
+ stats_role.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.impact_vs_role_stat.csv')
+
+ # test positive selection in various groups
+ saf_by_impact = df.groupby(by = ['impact', 'INFO/TIER'])['COSMIC_SAMPLE_MUTATED', 'COSMIC_SAMPLE_TESTED'].sum()
+ saf_by_impact.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.SAF_by_tier.csv')
+ try:
+ stat_positive_selection_by_tier = test_positive_selection(saf_by_impact,
+ rate_index = ('neutral', '2'))
+
+
+ except Exception as e:
+ stat_positive_selection_by_tier = pd.DataFrame()
+
+ stat_positive_selection_by_tier.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.pos_selection_tier.csv')
+
+ try:
+ saf_by_oncogene = df.groupby(by = ['impact', 'oncogene'])['COSMIC_SAMPLE_MUTATED', 'COSMIC_SAMPLE_TESTED'].sum()
+ saf_by_oncogene.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.SAF_by_oncogene.csv')
+ stat_positive_selection_by_oncogene = test_positive_selection(saf_by_oncogene, rate_index = ('neutral', True))
+
+ except Exception as e:
+ stat_positive_selection_by_oncogene = pd.DataFrame()
+ stat_positive_selection_by_oncogene.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.pos_selection_oncogene.csv')
+
+ try:
+ saf_by_tsg = df.groupby(by = ['impact', 'TSG'])['COSMIC_SAMPLE_MUTATED', 'COSMIC_SAMPLE_TESTED'].sum()
+ saf_by_tsg.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.SAF_by_oncogene.csv')
+ stat_positive_selection_by_tsg = test_positive_selection(saf_by_tsg, rate_index = ('neutral', True))
+ except Exception as e:
+ stat_positive_selection_by_tsg = pd.DataFrame()
+ stat_positive_selection_by_tsg.to_csv(indir/'output/variants' / 'cosmic_analysis' / f'{exp}.pos_selection_tsg.csv')
+
+ df.to_csv((indir/'output/variants' / 'cosmic_noncoding' / f'{exp}.variants_scores.csv'))
diff --git a/tools/annotate_finemapped_regions.py b/tools/annotate_finemapped_regions.py
new file mode 100644
index 0000000..38d0051
--- /dev/null
+++ b/tools/annotate_finemapped_regions.py
@@ -0,0 +1,36 @@
+# to annotate finemapped regions back to window's annotations
+import pandas as pd
+from pybedtools import BedTool
+import sys
+
+
+if __name__ == '__main__':
+ finemapped_sites = BedTool(sys.argv[1])
+ finemapped_sites_df = finemapped_sites.to_dataframe()
+
+ ranking = pd.read_csv(sys.argv[2], sep = '\t')
+
+ # read annotation
+ window = pd.read_csv(sys.argv[3],
+ sep = '\t')
+ window_bed = BedTool.from_dataframe(window)
+
+ outf = sys.argv[4]
+
+ # find windows overlapping finemapped regions
+ site_annotation_finemap = finemapped_sites.intersect(
+ window_bed,s = True, wb = True).to_dataframe(
+ names = finemapped_sites_df.columns.tolist()+[
+ i+'.1' for i in window.columns.tolist()[:6]]+window.columns.tolist()[6:])
+
+ site_annotation_finemap['overlap_size'] = site_annotation_finemap['end']-site_annotation_finemap['start']
+ site_annotation_finemap['gene_types_rank'] = site_annotation_finemap['gene_types'].map(ranking.set_index('accession')['rank'])
+
+ # get unique 1-to-1 mapping, priorize by ranking and overlap size (middle of peak)
+ site_annotation_uniq = site_annotation_finemap.sort_values(
+ by = 'overlap_size', ascending = False
+ ).sort_values(by = 'gene_types_rank', ascending = True
+ ).drop_duplicates(subset = 'name', keep = 'first')
+
+ # save to file
+ site_annotation_uniq.to_csv(outf, sep = '\t', index = False)
\ No newline at end of file
diff --git a/tools/annotate_gnomAD.py b/tools/annotate_gnomAD.py
new file mode 100644
index 0000000..8213834
--- /dev/null
+++ b/tools/annotate_gnomAD.py
@@ -0,0 +1,187 @@
+import pandas as pd
+from pathlib import Path
+from pybedtools import BedTool
+import sys
+import matplotlib.pyplot as plt
+plt.rcParams['pdf.fonttype'] = 42
+plt.rcParams["image.cmap"] = "Dark2"
+plt.rcParams['axes.prop_cycle'] = plt.cycler(color=plt.cm.Dark2.colors)
+import seaborn as sns
+from scipy.stats import norm
+import numpy as np
+import statsmodels.api as sm
+import statsmodels.formula.api as smf
+from statsmodels.stats.multitest import fdrcorrection
+
+def sigmoid(z):
+ return 1/(1 + np.exp(-z))
+
+def test_selection_in_subset(df, groupby = 'feature_type_top'):
+ stats = []
+ for name, group in df.groupby(by = groupby):
+ results = smf.ols('delta_score ~ MAF_bin_rank', data=group).fit()
+ stats.append([name, results.pvalues['MAF_bin_rank'], results.params['MAF_bin_rank'], group.shape[0]])
+ stats = pd.DataFrame(stats, columns = [groupby,'pvalue', 'coef', '# SNP in binding site'])
+ stats['sig'], stats['FDR']= fdrcorrection(stats['pvalue'], alpha = 0.2)
+
+ return stats
+
+if __name__ == '__main__':
+ indir = Path(sys.argv[1])
+ exp = sys.argv[2]
+ finemap_annotation = indir / 'output/finemapping/mapped_sites/' / f'{exp}.finemapped_windows.annotated.tsv'
+
+ # load all files
+ ref = pd.read_csv(indir / 'output/variants' / 'gnomAD' / f'{exp}.ref.score.txt', sep = '\t', names = ['ID', 'score'])
+ ref.rename({'score': 'score_ref'}, axis = 1, inplace = True)
+ ref.drop('ID', axis = 1, inplace = True)
+ alt = pd.read_csv(indir / 'output/variants' / 'gnomAD' / f'{exp}.alt.score.txt', sep = '\t', names = ['ID', 'score'])
+ alt.rename({'score': 'score_alt'}, axis = 1, inplace = True)
+ alt.drop('ID', axis = 1, inplace = True)
+ annot = pd.read_csv(indir / 'output/variants' / 'gnomAD' / f'{exp}.csv', index_col = 0)
+ site_annotation_uniq = pd.read_csv(finemap_annotation, sep = '\t')
+
+ # assure they are the same length
+ assert ref.shape[0] == alt.shape[0]
+ assert ref.shape[0] == annot.shape[0]
+
+ # calculate MAF and binning
+ df = pd.concat([ref,alt, annot], axis = 1)
+ df.rename({'5': 'INFO/AC', '6': 'INFO/AN'}, axis = 1, inplace = True)
+ df = df.loc[df['INFO/AC']>0]
+ # calculate scoring: sigmoid transformation into probability
+ df['score_alt'] = sigmoid(df['score_alt'])
+ df['score_ref'] = sigmoid(df['score_ref'])
+
+ df['delta_score']=df['score_alt']-df['score_ref']
+ df ['log_ratio'] = np.log(df['score_alt'])-np.log(df['score_ref'])
+
+ df['MAF']=df['INFO/AC']/df['INFO/AN']
+ df['MAF_bin'] = pd.cut(df['MAF'], bins = [0, 1e-3, 1e-2, 1])
+ bins = df['MAF_bin'].cat.categories.tolist()
+ bins = ['singleton']+bins
+ df['MAF_bin']=pd.Categorical(df['MAF_bin'], categories = bins, ordered = True)
+ df.loc[df['INFO/AC']==1, 'MAF_bin']='singleton'
+ df['MAF_bin_rank']=df['MAF_bin'].rank(method = 'dense')
+
+
+
+ # now annotate, all variants should be derived from finemapped windows
+ assert df['name'].isin(site_annotation_uniq['name']).all()
+ df['feature_type_top'] = df['name'].map(site_annotation_uniq.set_index('name')['feature_type_top'])
+ df['transcript_type_top'] = df['name'].map(site_annotation_uniq.set_index('name')['transcript_type_top'])
+ df['gene_name']=df['name'].map(site_annotation_uniq.set_index('name')['gene_name'])
+ df['strand'] = df['name'].map(site_annotation_uniq.set_index('name')['strand'])
+
+ # annotate variant type
+ df.loc[(df['REF'].str.len()==1)&(df['ALT'].str.len()==1), 'TYPE']='SNV'
+ df.loc[(df['REF'].str.len()!=1)|(df['ALT'].str.len()!=1), 'TYPE']='INDEL'
+
+ # Summary statistics: Distribution of delta
+ std=df['delta_score'].std()
+ mean = df['delta_score'].mean()
+ rv = norm(loc = 0, scale = std)
+
+ f, ax = plt.subplots(2,1, sharex = True)
+
+ x = np.linspace(df['delta_score'].min(),
+ df['delta_score'].max(), 100)
+
+ ax[0].plot(x, rv.pdf(x), 'r-', lw=2, label='Normal distribution\n(mean=0)')
+ df['delta_score'].plot.hist(bins = 100, color = 'lightgrey', density = True, ax = ax[0])
+ ax[0].vlines(x = std*2, ymin = 0, ymax = 1, color = 'black',
+ linestyle='dashed')
+ ax[0].vlines(x = -std*2, ymin = 0, ymax = 1, color = 'black',
+ linestyle='dashed', label = f'2 STDEV(std={std:.2f})')
+ ax[0].set_xlabel('delta score')
+ ax[0].legend()
+
+ sns.histplot(data = df, x = 'delta_score',
+ bins = 100, color = 'lightgrey', hue = 'TYPE',
+ stat = 'frequency', ax = ax[1])
+ plt.savefig(indir / 'output/variants' / 'gnomAD_analysis' / f'{exp}.delta_score_distribution.pdf')
+
+ # annotate impact
+ df.loc[df['delta_score']<-2*std, 'impact'] = 'LoF'
+ df.loc[df['delta_score']>2*std, 'impact'] = 'GoF'
+ df['impact'].fillna('neutral', inplace = True)
+
+ # Summary statistics: MAF vs impact
+ maf_vs_impact = pd.pivot_table(df,
+ index = 'MAF_bin',
+ columns = 'impact',
+ aggfunc = 'size')
+
+
+ maf_vs_impact.div(maf_vs_impact.sum(axis = 0)).T.plot.bar(
+ cmap = 'coolwarm', figsize = (3,3))
+ plt.ylabel('Fraction of variants')
+ plt.legend(bbox_to_anchor = (1.5,1))
+ sns.despine()
+ plt.savefig(indir / 'output/variants' / 'gnomAD_analysis' / f'{exp}.MAF_vs_impact.pdf')
+
+ # some summary statistics about MAF-bin and feature type
+ # feature_by_bin = pd.pivot_table(df,
+ # index = 'MAF_bin_rank',
+ # columns = 'feature_type_top',
+ # aggfunc = 'size').fillna(0).T
+
+ # transcript_by_bin = pd.pivot_table(df,
+ # index = 'MAF_bin_rank',
+ # columns = 'transcript_type_top',
+ # aggfunc = 'size').fillna(0)
+
+ # site_count = pd.pivot_table(df,
+ # index = 'MAF_bin_rank',
+ # columns = 'name',
+ # aggfunc = 'size').fillna(0).T
+
+ # gene_count = pd.pivot_table(df,
+ # index = 'MAF_bin_rank',
+ # columns = 'gene_name',
+ # aggfunc = 'size').fillna(0).T
+
+ # test by subset
+ feature_stat = test_selection_in_subset(df, groupby = 'feature_type_top')
+ transcript_stat = test_selection_in_subset(df, groupby = 'transcript_type_top')
+
+ feature_stat.to_csv(indir / 'output/variants' / 'gnomAD_analysis' / f'{exp}.feature_stat.tsv',
+ sep = '\t')
+ transcript_stat.to_csv(indir / 'output/variants' / 'gnomAD_analysis' / f'{exp}.transcript_stat.tsv',
+ sep = '\t')
+
+ # plot by subset
+ f, ax = plt.subplots(1,2, figsize = (16,8), sharey = True)
+
+ sns.boxplot(data = df,
+ hue = 'MAF_bin',
+ y = 'delta_score',
+ x = 'feature_type_top',
+ showfliers=False,
+ ax = ax[0],
+ palette="Blues"
+ )
+ ax[0].tick_params(axis='x', rotation=90)
+ ax[0].get_legend().remove()
+
+ sns.boxplot(data = df,
+ hue = 'MAF_bin',
+ y = 'delta_score',
+ x = 'transcript_type_top',
+ showfliers=False,
+ ax = ax[1],
+ palette="Blues"
+ )
+ ax[1].tick_params(axis='x', rotation=90)
+ sns.move_legend(ax[1], "upper left", bbox_to_anchor=(1, 0.5), title='MAF')
+ plt.tight_layout()
+ sns.despine()
+ plt.savefig(indir / 'output/variants' / 'gnomAD_analysis' / f'{exp}.subset_selection_boxplot.pdf')
+
+ # save results
+ outf = indir / 'output/variants' / 'gnomAD' / f'{exp}.variants_scores.tsv'
+
+ with open(outf, 'w') as f:
+ f.write(f'#std:{std}\n#mean: {mean}\n')
+ df.to_csv(f, sep = '\t')
+
diff --git a/tools/annotate_roulette.py b/tools/annotate_roulette.py
new file mode 100644
index 0000000..2a08f8c
--- /dev/null
+++ b/tools/annotate_roulette.py
@@ -0,0 +1,96 @@
+import sys
+import pandas as pd
+from pathlib import Path
+import numpy as np
+def sigmoid(z):
+ return 1/(1 + np.exp(-z))
+
+def join_data(indir, exp, chrom):
+ ref = pd.read_csv(indir / 'output/variants' / 'roulette' / f'{exp}.{chrom}.ref.score.txt', sep = '\t', names = ['ID', 'score'])
+ ref.rename({'score': 'score_ref'}, axis = 1, inplace = True)
+ ref.drop('ID', axis = 1, inplace = True)
+ alt = pd.read_csv(indir / 'output/variants' / 'roulette' / f'{exp}.{chrom}.alt.score.txt', sep = '\t', names = ['ID', 'score'])
+ alt.rename({'score': 'score_alt'}, axis = 1, inplace = True)
+ alt.drop('ID', axis = 1, inplace = True)
+ annot = pd.read_csv(indir / 'output/variants' / 'roulette' / f'{exp}.{chrom}.csv',
+ index_col = 0
+ ).rename({'5': 'FILTER',
+ '6': 'INFO/MR',
+ '7': 'INFO/AR',
+ '8': 'INFO/MG',
+ '9': 'INFO/MC'
+ }, axis = 1
+ )
+ print(chrom)
+ assert ref.shape[0]==alt.shape[0]
+ assert annot.shape[0]==ref.shape[0]
+
+ # calculate MAF and binning
+ df = pd.concat([ref,alt, annot], axis = 1)
+
+ df['score_alt'] = sigmoid(df['score_alt'])
+ df['score_ref'] = sigmoid(df['score_ref'])
+ df['delta_score']=df['score_alt']-df['score_ref']
+
+ return df
+
+if __name__ =='__main__':
+
+ indir = Path(sys.argv[1])
+ exp = sys.argv[2]
+
+ # read all roulette data
+ alldf = []
+ for n in range(1,23):
+ try:
+ alldf.append(join_data(indir, exp, f'chr{n}'))
+ except Exception as e:
+ print(e, 'chr{n}')
+ df = pd.concat(alldf, axis = 0)
+
+ # merge with annotation
+ site_annotation = pd.read_csv(indir / 'output/finemapping/mapped_sites/' / f'{exp}.finemapped_windows.annotated.tsv',
+ sep = '\t')
+
+ df = df.merge(site_annotation[['name', 'feature_type_top',
+ 'feature_types', 'gene_name', 'gene_id', 'transcript_ids',
+ 'gene_type_top', 'transcript_type_top', 'gene_types',
+ 'transcript_types']], left_on = 'name', right_on = 'name')
+
+ # add gnomAD annotation
+ gnomad = pd.read_csv(indir / 'output/variants' / 'gnomAD' / f'{exp}.csv',
+ index_col = 0).rename({'5': 'INFO/AC', '6':'INFO/AN'}, axis = 1)
+ gnomad['MAF']=gnomad['INFO/AC']/gnomad['INFO/AN']
+ gnomad['MAF_bin'] = pd.cut(gnomad['MAF'], bins = [0, 1e-3, 1e-2, 1])
+ bins = gnomad['MAF_bin'].cat.categories.tolist()
+ bins = ['singleton']+bins
+ gnomad['MAF_bin']=pd.Categorical(gnomad['MAF_bin'], categories = bins, ordered = True)
+ gnomad.loc[gnomad['INFO/AC']==1, 'MAF_bin']='singleton'
+ gnomad['MAF_bin_rank']=gnomad['MAF_bin'].rank(method = 'dense')
+ df = df.merge(gnomad[['CHROM', 'POS', 'ID', 'REF', 'ALT', 'MAF', 'MAF_bin', 'MAF_bin_rank',
+ 'INFO/AC', 'INFO/AN']], how = 'left',
+ left_on = ['CHROM', 'POS', 'REF', 'ALT'],
+ right_on = ['CHROM', 'POS', 'REF', 'ALT']
+ )
+ df['MAF_bin']=pd.Categorical(df['MAF_bin'], categories = ['unobserved']+bins, ordered = True)
+ df['MAF_bin'].fillna('unobserved', inplace = True)
+
+ # add gene-wise election constrain
+ gnomad_constrain = pd.read_csv('/tscc/projects/ps-yeolab5/hsher/gnomAD/gnomad.v4.0.constraint_metrics.tsv', sep = '\t')
+ gnomad_constrain = gnomad_constrain.loc[(gnomad_constrain['mane_select'])&(gnomad_constrain['transcript'].str.contains('ENST'))]
+ gnomad_constrain['lof.pLI bins']=pd.cut(gnomad_constrain['lof.pLI'], bins = [0,0.1,0.9,1],
+ labels = ['LoF tolerant', 'Ambiguous', 'LoF intolerant'])
+ df['lof.pLI bins']=df['gene_name'].map(gnomad_constrain.dropna().set_index('gene')['lof.pLI bins'])
+ df['mis_pphen.oe']=df['gene_name'].map(gnomad_constrain.dropna().set_index('gene')['mis_pphen.oe'])
+ df['syn.oe']=df['gene_name'].map(gnomad_constrain.dropna().set_index('gene')['syn.oe'])
+
+ # filter for good quality stuffs
+ filtered_df = df.loc[df['FILTER']!='low']
+
+ df.to_csv(indir / 'output/variants/roulette' / f'{exp}.full.csv.gz',compression='gzip')
+ filtered_df.to_csv(indir / 'output/variants/roulette' / f'{exp}.filtered.csv.gz',compression='gzip')
+
+
+
+
+
diff --git a/tools/bedGraphToBigWig b/tools/bedGraphToBigWig
old mode 100644
new mode 100755
diff --git a/tools/compare_ukbb_dr.py b/tools/compare_ukbb_dr.py
new file mode 100644
index 0000000..798ed4a
--- /dev/null
+++ b/tools/compare_ukbb_dr.py
@@ -0,0 +1,60 @@
+import pandas as pd
+from pathlib import Path
+import sys
+from pybedtools import BedTool
+from scipy.stats import fisher_exact,chisquare
+import numpy as np
+
+def testing(df):
+ ''' perform fisher exact or chisq given contingency table'''
+ if df.shape != (2,2):
+ # some outcomes are unobserved
+ print(df.shape)
+ print('no binding is observed in')
+ return 1, np.nan
+ if df.le(5).any().any():
+ odds_ratio, pvalue = fisher_exact(df)
+ else:
+ chi, pvalue = chisquare(df.loc[True], (df.loc[True].sum())*df.loc[False].div(df.loc[False].sum()))
+ odds_ratio = (df.loc[True, True]/df.loc[True, False])/(df.loc[False, True]/df.loc[False, False])
+ return pvalue, odds_ratio
+
+if __name__ == '__main__':
+ indir = Path(sys.argv[1])
+ exp = sys.argv[2]
+ finemap_annotation = indir / 'output/finemapping/mapped_sites/' / f'{exp}.finemapped_windows.annotated.tsv'
+ finemap = pd.read_csv(finemap_annotation, sep = '\t')
+ tested = list((indir / 'output/tested_windows').glob(f'{exp}*'))
+ annotation = pd.read_csv(sys.argv[3],
+ sep = '\t')
+ outf = sys.argv[4]
+
+ ## additional files required
+ top1 = BedTool('/tscc/nfs/home/hsher/ps-yeolab5/ukbb_dr_score/top1.bed')
+ top5 = BedTool('/tscc/nfs/home/hsher/ps-yeolab5/ukbb_dr_score/top5.bed')
+
+ # find all tested windows
+ all_tested = set(annotation['name'])
+ for f in tested:
+ df = pd.read_csv(f, sep = '\t')
+ all_tested = all_tested.intersection(set(df['name']))
+
+ # gather information
+ all_info = annotation.loc[annotation['name'].isin(all_tested)]
+ all_info['is_binding'] = all_info['name'].isin(finemap['name.1'])
+
+ # find top DR
+ tested_bed = BedTool.from_dataframe(all_info[['chrom', 'start', 'end', 'name']])
+ all_info['is_top1_DR_percentile'] = all_info['name'].isin(tested_bed.intersect(top1, f=1).to_dataframe()['name'].tolist())
+ all_info['is_top5_DR_percentile'] = all_info['name'].isin(tested_bed.intersect(top5, f=1).to_dataframe()['name'].tolist())
+
+ # test for enrichment of RBP binding site in topDR
+ stats = []
+ for col in ['is_top5_DR_percentile','is_top1_DR_percentile']:
+ for name, group in all_info.groupby(by = 'feature_type_top'):
+ pv = pd.pivot_table(group, index = 'is_binding', columns = col, aggfunc = 'size').fillna(0)
+ pval, odds_ratio = testing(pv)
+ stats.append([pval, odds_ratio, name, col])
+ stats = pd.DataFrame(stats, columns = ['p-value', 'odds ratio', 'feature_type_top', 'subset'])
+
+ stats.to_csv(outf)
\ No newline at end of file
diff --git a/tools/find_both_tested_windows.py b/tools/find_both_tested_windows.py
new file mode 100644
index 0000000..7eca173
--- /dev/null
+++ b/tools/find_both_tested_windows.py
@@ -0,0 +1,19 @@
+import sys
+import pandas as pd
+from pybedtools import BedTool
+
+if __name__ == '__main__':
+ tested_windows = pd.concat([pd.read_csv(f, sep = '\t') for f in sys.argv[1].split(' ')],
+ axis = 0)
+ tested_times = tested_windows['name'].value_counts()
+ both_tested = tested_times[tested_times >= 2].index
+
+ cols = ['chr', 'start', 'end', 'name', 'gc', 'strand']
+ annot = tested_windows[cols].drop_duplicates()
+ both_tested_windows = annot.loc[annot['name'].isin(both_tested)]
+ both_tested_windows.to_csv(sys.argv[2], sep = '\t', index = False, header = False)
+ print(both_tested_windows.shape)
+
+ bed = BedTool.from_dataframe(both_tested_windows)
+ # merge adjacent ones
+ bed.sort().merge(c = [4,5,6], s = True, o = ['distinct', 'mean', 'distinct']).saveas(sys.argv[3])
diff --git a/tools/gather_gkmsvm_auprc.py b/tools/gather_gkmsvm_auprc.py
new file mode 100644
index 0000000..05f02b4
--- /dev/null
+++ b/tools/gather_gkmsvm_auprc.py
@@ -0,0 +1,30 @@
+import pandas as pd
+import numpy as np
+from sklearn.metrics import precision_recall_curve, auc
+import matplotlib.pyplot as plt
+from pathlib import Path
+import sys
+def mean_auprc(df):
+ aucs = []
+ for name, group in df.groupby(by = 'CV-set'):
+ precision, recall, thresholds = precision_recall_curve(group['label'], group['SVM score'])
+
+ aucs.append(auc(recall, precision))
+
+ mean_auprc, std_auprc = np.mean(aucs), np.std(aucs)
+ return mean_auprc, std_auprc
+
+if __name__=='__main__':
+ output = sys.argv[1]
+
+ data = []
+ for f in Path('output/ml/gkmsvm/').glob('*cvpred.txt'):
+ df = pd.read_csv(f,
+ sep = '\t',
+ names = ['sequence_id', 'SVM score', 'label', 'CV-set'])
+ mean, std = mean_auprc(df)
+
+ data.append([f.name.split('.')[0],mean, std])
+ data = pd.DataFrame(data, columns = ['Experiment', 'mean AUPRC', 'std AUPRC'])
+
+ data.to_csv(output)
\ No newline at end of file
diff --git a/tools/generate_variant_sequence.py b/tools/generate_variant_sequence.py
new file mode 100644
index 0000000..f01b135
--- /dev/null
+++ b/tools/generate_variant_sequence.py
@@ -0,0 +1,155 @@
+import pandas as pd
+from pybedtools import BedTool
+import sys
+from Bio import SeqIO
+from Bio.SeqRecord import SeqRecord
+from Bio.Seq import Seq
+
+def generate_variant_sequence(row):
+ ref = row['REF']
+ seq = row['seq']
+
+
+
+ start = row['POS']-row['start']-1
+ end = row['POS']-row['start']-1+len(ref)
+
+ to_replace = seq[start:end]
+ if to_replace != ref:
+ print(to_replace, 'ref=', ref, start, end, len(seq))
+ return None
+ else:
+ print('reference correct')
+ new_seq = seq[:start]+row['ALT']+seq[end:]
+ return new_seq
+
+def reverse_complement(string):
+ newstr = ''
+ mapper={'A':'T',
+ 'T':'A',
+ 'C': 'G',
+ 'G':'C',
+ '.': '',
+ 'N': 'N'}
+ for s in string[::-1]:
+ newstr+=mapper[s]
+ return newstr
+
+def generate_variant_sequence_neg(row):
+ ref = row['REF']
+ seq = row['seq']
+
+
+
+ start = row['end']-row['POS']
+ end = row['end']-row['POS']-len(ref)
+
+ to_replace = reverse_complement(seq[end+1:start+1])
+ if to_replace != ref:
+ print(to_replace, 'ref=',ref, start, end, len(seq))
+ return None
+ else:
+ print('reference correct')
+ new_seq = seq[:end+1]+reverse_complement(row['ALT'])+seq[start+1:]
+ return new_seq
+
+
+
+def make_SeqRecord(df, seq_col):
+ ''' convert df to a list of SeqRecord '''
+ records = []
+ for index, row in df.iterrows():
+ if seq_col == 'seq':
+ # WT
+ id_ = str(row['CHROM'])+'-'+str(row['POS'])+'-'+row['REF']+'-'+str(row['name'])
+ elif seq_col == 'variant_seq':
+ id_ = str(row['CHROM'])+'-'+str(row['POS'])+'-'+row['ALT']+'-'+str(row['name'])
+ else:
+ id_ = row['ID']
+ records.append(
+ SeqRecord(
+ Seq(row[seq_col]),
+ id=id_
+ )
+ )
+ return records
+
+if __name__ == '__main__':
+ vcf_file = sys.argv[1]
+ fa = sys.argv[2]
+ bed = sys.argv[3]
+ out_prefix = sys.argv[4]
+
+ # read vcf
+ variants_df = pd.read_csv(vcf_file,
+ sep = '\t',
+ header = None).drop_duplicates(subset = [0,1,2,3,4])
+ variants = variants_df.iloc[:, :5]
+ variants.columns = ['CHROM','POS','ID','REF', 'ALT']
+
+ sequences = {}
+ for record in SeqIO.parse(fa, format = 'fasta'):
+ sequences[record.id]=str(record.seq)
+
+ # get sequence of binding sites
+ window_bed = BedTool(bed)
+ window_df = window_bed.to_dataframe()
+ window_df['seq']=sequences.values()
+ window_bed = BedTool.from_dataframe(window_df)
+
+
+if variants.empty:
+ variant_df = pd.DataFrame(columns = ['CHROM', 'POS', 'ID', 'REF', 'ALT', 'name', 'variant_seq','feature_type_top', 'feature_types', 'gene_name',
+ 'transcript_types', 'transcript_type_top', 'INFO/AC', 'INFO/AN'])
+ variant_df.to_csv(outf)
+else:
+ # finding window name
+ variants['POS-1']=variants['POS']-1
+
+ # get variants in window
+ df = BedTool.from_dataframe(variants[['CHROM','POS-1','POS','ID','REF', 'ALT']]).intersect(
+ window_bed, wb = True).to_dataframe(names = ['CHROM','POS-1', 'POS','ID','REF', 'ALT']+window_df.columns.tolist())
+
+ df.dropna(subset = ['seq'],inplace = True)
+ df=df.loc[df['REF'].str.len()<10]
+
+ pos = df.loc[df['strand']=='+']
+ neg = df.loc[df['strand']=='-']
+
+ # generate variant sequence
+ if not pos.empty:
+ pos['variant_seq']=pos.apply(generate_variant_sequence, axis = 1)
+ else:
+ pos['variant_seq']=None
+ if not neg.empty:
+ neg['variant_seq']=neg.apply(generate_variant_sequence_neg, axis = 1)
+ else:
+ neg['variant_seq']=None
+
+ cols = ['CHROM', 'POS', 'ID', 'REF', 'ALT', 'seq', 'variant_seq','name', 'strand']
+ variant_seq_df = pd.concat([pos[cols],
+ neg[cols]],
+ axis = 0)
+ variant_seq_df.dropna(subset = ['variant_seq'], inplace = True)
+ variant_seq_df = variant_seq_df.merge(variants_df,
+ left_on = ['CHROM', 'POS', 'ID', 'REF', 'ALT'],
+ right_on = [0,1,2,3,4]).drop([0,1,2,3,4], axis = 1)
+
+ # drop duplicates in variant_seq_df, find the centering window
+ rows = []
+ for name, group in variant_seq_df.groupby(by = ['CHROM', 'POS', 'REF', 'ALT']):
+ selected = group.sort_values(by = 'name').iloc[int(group.shape[0]/2)]
+ rows.append(selected)
+ variant_seq_df = pd.concat(rows, axis = 1).T
+
+ print('writing csv')
+ variant_seq_df.to_csv(f'{out_prefix}.csv')
+
+ # make fasta
+ ref_seq = make_SeqRecord(variant_seq_df, 'seq')
+ alt_seq = make_SeqRecord(variant_seq_df, 'variant_seq')
+
+ SeqIO.write(ref_seq, f'{out_prefix}.ref.fa', format = 'fasta')
+ SeqIO.write(alt_seq, f'{out_prefix}.alt.fa', format = 'fasta')
+ all_seq = sorted(ref_seq+alt_seq, key=lambda x: x.id)
+ SeqIO.write(all_seq,f'{out_prefix}.all.fa', format = 'fasta')
diff --git a/tools/get_R.sh b/tools/get_R.sh
deleted file mode 100644
index 01a1a07..0000000
--- a/tools/get_R.sh
+++ /dev/null
@@ -1,50 +0,0 @@
-# ensure gcc is installed
-# create rskipper conda environment from rskipper.yml
-# conda env create -f rskipper.yml
-# set paths
-CONDA_DIR=$1 # CONDA_DIR=/home/eboyle/miniconda3
-R_DIR=$2 # R_DIR=/projects/ps-yeolab3/eboyle/encode/pipeline/gran
-
-# load conda environment for R dependencies
-conda activate rskipper
-
-# make directories for downloading source
-mkdir -p ${R_DIR}/src
-mkdir ${R_DIR}/texlive
-
-# set library files based on conda directory
-export LD_LIBRARY_PATH=${CONDA_DIR}/envs/rskipper/lib/:$LD_LIBRARY_PATH
-
-cd ${R_DIR}/src # working directory of your choice
-wget --no-check-certificate https://mirror.ctan.org/systems/texlive/tlnet/install-tl-unx.tar.gz # or curl instead of wget
-zcat < install-tl-unx.tar.gz | tar xf -
-cd install-tl-*
-export TEXLIVE_INSTALL_PREFIX=${R_DIR}/texlive
-perl ./install-tl --no-interaction # as root or with writable destination
-# Finally, prepend /usr/local/texlive/YYYY/bin/PLATFORM to your PATH,
-export PATH=${R_DIR}/texlive:$PATH # e.g., /usr/local/texlive/2023/bin/x86_64-linux
-
-# install curl into conda directory manually (version on conda forge does not work)
-cd ${R_DIR}/src
-git clone https://github.com/curl/curl.git
-cd curl
-git checkout tags/curl-7_86_0
-./buildconf
-./configure --with-openssl --prefix=${CONDA_DIR}/envs/rskipper/
-make
-make install
-cd ..
-
-# install base R
-curl https://cran.r-project.org/src/base/R-4/R-4.1.3.tar.gz > R-4.1.3.tar.gz
-tar -xf R-4.1.3.tar.gz
-cd R-4.1.3
-./configure --enable-R-shlib --enable-shared --with-x=no --prefix=${R_DIR} CPPFLAGS="-I${CONDA_DIR}/envs/rskipper/include/" LDFLAGS="-L${CONDA_DIR}/envs/rskipper/lib/" && \
-make && \
-make install
-
-# install versioned R packages using groundhog and BiocManager
-cd ${R_DIR}/bin
-./R -e "dir.create(Sys.getenv('R_LIBS_USER'),recursive=TRUE)"
-./R -e "install.packages('groundhog',repos = 'http://cran.us.r-project.org')"
-./R -e "groundhog::groundhog.library(c('tidyverse', 'VGAM', 'viridis', 'ggrepel', 'RColorBrewer', 'Rtsne', 'ggupset', 'ggdendro', 'cowplot', 'BiocManager'), '2022-03-11', force.install=TRUE)" && ./R -q -e "BiocManager::install(c('GenomicRanges','fgsea','rtracklayer'))"
diff --git a/tools/gkmsvm_explain.py b/tools/gkmsvm_explain.py
new file mode 100644
index 0000000..7362791
--- /dev/null
+++ b/tools/gkmsvm_explain.py
@@ -0,0 +1,24 @@
+import numpy as np
+import matplotlib.pyplot as plt
+import vizsequence
+import sys
+from pathlib import Path
+
+
+
+if __name__ == '__main__':
+ in_file = sys.argv[1]
+ outdir = Path(sys.argv[2])
+ impscores = [
+ np.array( [[float(z) for z in y.split(",")] for y in x.rstrip().split("\t")[2].split(";")])
+ for x in open(in_file)
+ ]
+
+
+
+ #visualize importance scores on a couple of sequences, as a sanity check
+
+ for i,score in enumerate(impscores):
+ vizsequence.viz_sequence.plot_weights(score)
+ plt.savefig(outdir/f'{i}.pdf')
+
diff --git a/tools/group_genome_megatable.py b/tools/group_genome_megatable.py
new file mode 100644
index 0000000..d2dea56
--- /dev/null
+++ b/tools/group_genome_megatable.py
@@ -0,0 +1,25 @@
+import pandas as pd
+import sys
+
+if __name__=='__main__':
+ input = sys.argv[1]
+ feature_output = sys.argv[2]
+ transcript_output = sys.argv[3]
+
+
+ fcounts = []
+ tcounts = []
+ for df in pd.read_csv(input, sep = '\t',
+ chunksize = 1000):
+ sample_col = df.columns[17:]
+ fcount = df.groupby(by = 'feature_type_top')[sample_col].sum().reset_index()
+ tcount = df.groupby(by = 'transcript_type_top')[sample_col].sum().reset_index()
+
+
+ fcounts.append(fcount)
+ tcounts.append(tcount)
+ fcounts = pd.concat(fcounts, axis = 0).groupby(by = 'feature_type_top').sum()
+ tcounts = pd.concat(tcounts, axis = 0).groupby(by = 'transcript_type_top').sum()
+
+ fcounts.to_csv(feature_output, sep = '\t')
+ tcounts.to_csv(transcript_output, sep = '\t')
\ No newline at end of file
diff --git a/tools/identify_reproducible_windows.R b/tools/identify_reproducible_windows.R
index cd1d56b..1faa7a1 100644
--- a/tools/identify_reproducible_windows.R
+++ b/tools/identify_reproducible_windows.R
@@ -20,6 +20,30 @@ enriched_window_data = enriched_window_files %>%
Filter(function(x) nrow(x) > 0, .) %>%
bind_rows(.id = "clip_replicate_label")
+# handle both replicate has 0 enriched windows, there is no name
+if (nrow(enriched_window_data) == 0){
+ pdf(paste0('output/figures/reproducible_enriched_windows/', prefix, '.reproducible_enriched_window_counts.linear.pdf'), height = 1, width = 2)
+ print(ggplot() + annotate("text", x = 1, y = 1, label = "No data") + theme_void())
+ dev.off()
+ pdf(paste0('output/figures/reproducible_enriched_windows/', prefix, '.reproducible_enriched_window_counts.log10.pdf'), height = 1, width = 2)
+ print(ggplot() + annotate("text", x = 1, y = 1, label = "No data") + theme_void())
+ dev.off()
+
+ columns= c("chr","start","end","name","score","strand","gc",
+ "gc_bin","chrom","feature_id","feature_bin","feature_type_top","feature_types",
+ "gene_name","gene_id","transcript_ids","gene_type_top","transcript_type_top",
+ "gene_types","transcript_types", "input_sum","clip_sum","enrichment_n",
+ "enrichment_l2or_min","enrichment_l2or_mean","enrichment_l2or_max","p_max","p_min",
+ "q_max","q_min")
+
+ # pass this vector length to ncol parameter
+ # and nrow with 0
+ reproducible_enriched_window_data = data.frame(matrix(nrow = 0, ncol = length(columns)))
+ colnames(reproducible_enriched_window_data) = columns
+ write_tsv(reproducible_enriched_window_data, paste0("output/reproducible_enriched_windows/", prefix, ".reproducible_enriched_windows.tsv.gz"))
+ quit()
+}
+
if(nrow(enriched_window_data %>% group_by(name) %>% filter(n() > 1)) == 0) {
pdf(paste0('output/figures/reproducible_enriched_windows/', prefix, '.reproducible_enriched_window_counts.linear.pdf'), height = 1, width = 2)
print(ggplot() + annotate("text", x = 1, y = 1, label = "No data") + theme_void())
diff --git a/tools/join_all_reproducible_enriched_windows.py b/tools/join_all_reproducible_enriched_windows.py
new file mode 100644
index 0000000..6e8b516
--- /dev/null
+++ b/tools/join_all_reproducible_enriched_windows.py
@@ -0,0 +1,29 @@
+from pathlib import Path
+import pandas as pd
+import sys
+from pathlib import Path
+
+if __name__ == '__main__':
+ indir = Path(sys.argv[1])
+ outf = sys.argv[2]
+
+ all_data = []
+
+ for f in (indir/'output/reproducible_enriched_windows').glob('*.tsv.gz'):
+ rbp = f.name.split('.')[0]
+ reproducible_windows = pd.read_csv(f, sep = '\t')
+
+ tested_windows_1 = pd.read_csv(indir / 'output/tested_windows'/ f'{rbp}.{rbp}_IP_1.tested_windows.tsv.gz', sep = '\t')
+ tested_windows_2 = pd.read_csv(indir / 'output/tested_windows'/ f'{rbp}.{rbp}_IP_2.tested_windows.tsv.gz', sep = '\t')
+
+ both_tested = set(tested_windows_1['name']).intersection(set(tested_windows_2['name']))
+
+ value = pd.Series(index = both_tested)
+ value[reproducible_windows['name']]=True
+ value.fillna(False, inplace = True)
+ value.name = rbp
+ all_data.append(value)
+
+
+ all_data = pd.concat(all_data, axis = 1)
+ all_data.to_csv(outf)
\ No newline at end of file
diff --git a/tools/join_reproducible_enriched_re.py b/tools/join_reproducible_enriched_re.py
new file mode 100644
index 0000000..c6bbf12
--- /dev/null
+++ b/tools/join_reproducible_enriched_re.py
@@ -0,0 +1,38 @@
+from pathlib import Path
+import pandas as pd
+import sys
+
+def reproducible_re(input_path):
+ ''' given Skipper output/ path, count how many reproducible enriched windows per feature_type and transcript_type '''
+ families = [] # save full data
+ families_l2or = []
+
+ for f in (Path(input_path) / 'output/reproducible_enriched_re').glob('*tsv.gz'):
+ df = pd.read_csv(f, sep = '\t', index_col = 0)
+
+ # append binary matrix
+ name = f.name.split('.')[0]
+ df[name]=True
+ families.append(df[name])
+
+ # append l2or matrix
+ l2or = df['enrichment_l2or_mean']
+ l2or.name = name
+ families_l2or.append(l2or)
+
+
+ families = pd.concat(families, axis = 1).fillna(False)
+ families_l2or = pd.concat(families_l2or, axis = 1)
+
+ return families, families_l2or
+
+
+if __name__ == '__main__':
+ input_path = sys.argv[1]
+ binary_output = sys.argv[2]
+ l2or_output = sys.argv[3]
+
+ binary_matrix, l2or_matrix = reproducible_re(input_path)
+
+ binary_matrix.to_csv(binary_output)
+ l2or_matrix.to_csv(l2or_output)
\ No newline at end of file
diff --git a/tools/join_reproducible_enriched_windows.py b/tools/join_reproducible_enriched_windows.py
new file mode 100644
index 0000000..ba6e0a3
--- /dev/null
+++ b/tools/join_reproducible_enriched_windows.py
@@ -0,0 +1,43 @@
+from pathlib import Path
+import pandas as pd
+import sys
+def reproducible_windows(input_path, feature_type):
+ ''' given Skipper output/ path, count how many reproducible enriched windows per feature_type and transcript_type '''
+ families = [] # save full data
+ families_l2or = []
+
+ for f in (Path(input_path) / 'output/reproducible_enriched_windows').glob('*tsv.gz'):
+ df = pd.read_csv(f, sep = '\t')
+ df = df.loc[df['feature_type_top'].isin(feature_type)]
+
+ # join binary
+ name = f.name.split('.')[0]
+ df[name]=True
+ families.append(df.set_index('name')[name])
+
+ # join l2or
+ try:
+ l2or = df.set_index('name')['enrichment_l2or_mean']
+ l2or.name = name
+ families_l2or.append(l2or)
+ except KeyError as e:
+ # some don't have any reproducible enrichment
+ pass
+
+
+
+ families = pd.concat(families, axis = 1).fillna(False)
+ families_l2or = pd.concat(families_l2or, axis = 1)
+ return families, families_l2or
+
+if __name__=='__main__':
+ input_path = sys.argv[1]
+ feature_type = sys.argv[2].split(',')
+ binary_output = sys.argv[3]
+ l2or_output = sys.argv[4]
+
+ binary_matrix, l2or_matrix = reproducible_windows(input_path, feature_type)
+
+ binary_matrix.to_csv(binary_output)
+ l2or_matrix.to_csv(l2or_output)
+
\ No newline at end of file
diff --git a/tools/mega_variant_analysis.py b/tools/mega_variant_analysis.py
new file mode 100644
index 0000000..d3c237c
--- /dev/null
+++ b/tools/mega_variant_analysis.py
@@ -0,0 +1,404 @@
+import pandas as pd
+from pathlib import Path
+import pybedtools
+import sys
+import matplotlib.pyplot as plt
+import seaborn as sns
+import numpy as np
+from scipy.stats import fisher_exact,chisquare,mannwhitneyu
+import statsmodels.api as sm
+import math
+from statsmodels.stats.multitest import fdrcorrection
+import warnings
+
+def make_MAF_bins(gnomad):
+ """ create MAF bins"""
+ gnomad['MAF']=gnomad['INFO/AC']/gnomad['INFO/AN']
+ gnomad['MAF_bin'] = pd.cut(gnomad['MAF'], bins = [0, 1e-3, 1e-2, 1])
+
+
+ bins = gnomad['MAF_bin'].cat.categories.tolist()
+ bins = ['singleton']+bins
+ gnomad['MAF_bin']=pd.Categorical(gnomad['MAF_bin'], categories = bins, ordered = True)
+ gnomad.loc[gnomad['INFO/AC']==1, 'MAF_bin']='singleton'
+ gnomad['MAF_bin_rank']=gnomad['MAF_bin'].rank(method = 'dense')
+
+ return gnomad
+
+def testing(df):
+ ''' perform fisher exact or chisq given contingency table'''
+ if df.shape != (2,2):
+ # some outcomes are unobserved
+ print(df.shape)
+ print('no binding is observed in')
+ return 1, np.nan
+ if df.le(5).any().any():
+ odds_ratio, pvalue = fisher_exact(df)
+ else:
+ chi, pvalue = chisquare(df.loc[True], (df.loc[True].sum())*df.loc[False].div(df.loc[False].sum()))
+ odds_ratio = (df.loc[True, True]/df.loc[True, False])/(df.loc[False, True]/df.loc[False, False])
+ return pvalue, odds_ratio
+
+def test_enrichment_outliers(maf_vs_impact):
+ ''' test enrichment to LoB and GoB variants'''
+ stats = []
+ background = 'neutral'
+ for impact in ['GoB', 'LoB']:
+ for index in maf_vs_impact.index:
+ try:
+ total = maf_vs_impact[background].sum()
+ is_category = maf_vs_impact.loc[index, background]
+
+ total_group = maf_vs_impact[impact].sum()
+ is_category_group = maf_vs_impact.loc[index, impact]
+ pv = pd.DataFrame(np.array([[is_category_group, total_group-is_category_group],
+ [is_category, total-is_category]]
+ ),
+ index = [True, False],
+ columns = [True, False],
+ )
+ if pv.ge(1).all().all():
+ pvalue, odds_ratio = testing(pv)
+ stats.append([index, impact, pvalue, odds_ratio])
+ except Exception as e:
+ print(e)
+
+ stats = pd.DataFrame(stats, columns = ['MAF_bin', 'impact', 'pvalue', 'odds_ratio'])
+ return stats
+
+def test_subset(df, col = 'feature_type_top'):
+ ''' test enrichment in subgroups based on columns'''
+ all_stats = []
+
+ for name, group in df.groupby(by = col):
+ if group.shape[0]>100:
+ maf_vs_impact = pd.pivot_table(group,
+ index = 'MAF_bin',
+ columns = 'impact',
+ aggfunc = 'size')
+ maf_vs_impact.div(maf_vs_impact.sum(axis = 0)).T.plot.bar(
+ cmap = 'coolwarm', figsize = (3,3))
+ plt.ylabel('Fraction of variants')
+ plt.legend(bbox_to_anchor = (1.5,1))
+ plt.title(name)
+ sns.despine()
+ plt.show()
+ stats_local = test_enrichment_outliers(maf_vs_impact)
+ stats_local[col]=name
+ all_stats.append(stats_local)
+ all_stats = pd.concat(all_stats,axis = 0)
+ all_stats['sig'], all_stats['FDR']= fdrcorrection(all_stats['pvalue'], alpha = 0.2)
+ return all_stats
+def convert_pvalue_to_asterisks(pvalue):
+ if pvalue <= 0.0001:
+ return "****"
+ elif pvalue <= 0.001:
+ return "***"
+ elif pvalue <= 0.01:
+ return "**"
+ elif pvalue <= 0.05:
+ return "*"
+ return "ns"
+def sigmoid(x):
+ return 1 / (1 + np.exp(-x))
+if __name__ == '__main__':
+ indir = Path(sys.argv[1])
+ exp = sys.argv[2]
+ outdir = indir / 'output/variant_analysis'
+
+ # read gnomAD and roulette
+ df = pd.read_csv(indir / f'output/variants/gnomAD_roulette/{exp}.total.csv',
+ na_values = '.')
+ annotation = pd.read_csv(indir / f'output/finemapping/mapped_sites/{exp}.finemapped_windows.annotated.tsv', sep = '\t')
+
+ # define std from commmon variants
+ df = make_MAF_bins(df)
+ std = df.loc[df['MAF']>0.01, 'delta_score'].std()
+ if math.isnan(std):
+ warnings.warn('No common variants observed in binding site. Using all variant to estimate std')
+ std = df['delta_score'].std()
+ df.loc[df['delta_score']<-2*std, 'impact'] = 'LoB'
+ df.loc[df['delta_score']>2*std, 'impact'] = 'GoB'
+ df['impact'].fillna('neutral', inplace = True)
+
+ # annotate
+ df['feature_type_top'] = df['name'].map(annotation.set_index('name')['feature_type_top'])
+ df['transcript_type_top'] = df['name'].map(annotation.set_index('name')['transcript_type_top'])
+ df['gene_name'] = df['name'].map(annotation.set_index('name')['gene_name'])
+
+ gnomad_constrain = pd.read_csv('/tscc/projects/ps-yeolab5/hsher/gnomAD/gnomad.v4.0.constraint_metrics.tsv', sep = '\t')
+ gnomad_constrain = gnomad_constrain.loc[(gnomad_constrain['mane_select'])&(gnomad_constrain['transcript'].str.contains('ENST'))]
+ gnomad_constrain['lof.pLI bins']=pd.cut(gnomad_constrain['lof.pLI'], bins = [0,0.1,0.9,1],
+ labels = ['LoF tolerant', 'Ambiguous', 'LoF intolerant']
+
+ )
+ df['lof.pLI bins']=df['gene_name'].map(gnomad_constrain.dropna().set_index('gene')['lof.pLI bins'])
+ df['mis_pphen.oe']=df['gene_name'].map(gnomad_constrain.dropna().set_index('gene')['mis_pphen.oe'])
+ df['syn.oe']=df['gene_name'].map(gnomad_constrain.dropna().set_index('gene')['syn.oe'])
+
+ # test enrichment of rare variants
+ maf_vs_impact = pd.pivot_table(df,
+ index = 'MAF_bin',
+ columns = 'impact',
+ aggfunc = 'size')
+ test_enrichment_outliers(maf_vs_impact).to_csv(outdir / f'{exp}.global_spectrum_enrichment.csv')
+
+ for col in ['feature_type_top', 'transcript_type_top']:
+ test_subset(df, col = col).to_csv(outdir / f'{exp}.{col}_spectrum_enrichment.csv')
+
+ # load mutation rate expected singleton ratio for MAPs
+ # singleton_model = sm.load('/tscc/nfs/home/hsher/projects/ENCODE/3_CLIP_ML_RBPNet/singleton_adjust.pickle') ## TODO: this has to be outside
+ singleton_coef = pd.read_csv('/tscc/nfs/home/hsher/projects/ENCODE/3_CLIP_ML_RBPNet/singleton_adjust.coef.csv', index_col = 0)['0']
+ df['expected_singleton']=sigmoid(df['INFO/MR']*singleton_coef['MR']+singleton_coef['Intercept'])
+ df['is_singleton']=df['MAF_bin'].eq('singleton')
+
+
+ # o/e ratio reference
+ reference_df= pd.read_csv('/tscc/nfs/home/hsher/ps-yeolab5/gnomAD_reference_set/v4/vep.csv', ## TODO: this has to be outside
+ index_col = 0
+ )
+ selected = ['3_prime_UTR_variant',
+ '5_prime_UTR_variant',
+ 'intron_variant',
+ 'missense_variant',
+ 'splice_acceptor_variant',
+ 'splice_donor_variant',
+ 'stop_gained',
+ 'synonymous_variant']
+
+ reference_df.loc[selected].sort_values(by = 'o/e').reset_index().plot.scatter(
+ x = 'Consequence_splitted', y = 'o/e', color = 'grey', figsize = (3,3))
+ plt.xticks(rotation = 90)
+ sns.despine()
+
+ # scale to intergenic o/e
+ scaling_rate = reference_df.loc['intergenic_variant']['o/e']
+
+ # delta_score
+ df['delta_score_bins'] = pd.cut(df['delta_score'], bins = [-np.inf,-2*std, 2*std, np.inf],)
+ df['delta_zscore'] = pd.cut(df['delta_score'], bins = [-np.inf,-2*std,-std, std ,2*std, np.inf],
+ labels = ['LoB', 'weak LoB', 'Neutral', 'weak GoB', 'GoB']
+ )
+ print(df['delta_score_bins'].value_counts())
+ bin2name = dict(zip(df['delta_score_bins'].cat.categories,
+ ['Weakening', 'No change', 'Strengthening']
+ )
+ )
+ reference_category = df['delta_score_bins'].cat.categories[1]
+ sub_df = df.loc[~df['INFO/AN'].isnull()]
+ df.to_csv(outdir / f'{exp}.annotated.csv.gz')
+
+ # bootstraping function for o/e and MAPS
+ def groupby_bootstrap(df, by = ['delta_score_bins'], n = 30, ratio = 0.8, scaling_rate = scaling_rate):
+ ''' groupby bootstrap for o/e '''
+
+ data = []
+ for name, group in df.groupby(by = by):
+ sample_size = group.shape[0]
+ for i in range(n):
+ n_sampled = int(sample_size*ratio)
+ sampled = group.sample(n_sampled)
+ sampled_oe = (sampled['MAF'].count()/sampled['INFO/MR'].sum())/scaling_rate
+ if type(name)==tuple:
+ data.append(list(name)+[sampled_oe, n_sampled, sample_size])
+ else:
+ data.append([name, sampled_oe, n_sampled, sample_size])
+ data = pd.DataFrame(data, columns = by+['o/e','n', 'total'])
+ return data
+ def groupby_bootstrap_maps(df, by = ['delta_score_bins'], n = 30, ratio = 0.8):
+ data = []
+ for name, group in df.groupby(by = by):
+ sample_size = group.shape[0]
+ for i in range(n):
+ n_sampled = int(sample_size*ratio)
+ sampled = group.sample(n_sampled)
+ n_singleton=sampled['is_singleton'].sum()
+ expected_singleton=sampled['expected_singleton'].sum()
+ maps = (n_singleton-expected_singleton)/n_sampled
+
+
+ if type(name)==tuple:
+ data.append(list(name)+[maps, n_sampled, sample_size])
+ else:
+ data.append([name, maps, n_sampled, sample_size])
+ data = pd.DataFrame(data, columns = by+['MAPS','n', 'total'])
+ return data
+ import math
+ def oe_test_for_negative_selection(bydelta_ind, x, y, reference = reference_category,
+ n_variants = 30):
+ n_category = bydelta_ind[x].unique().shape[0]
+ if reference is None:
+ #use the middle class as reference
+ reference = bydelta_ind[x].unique()[math.floor(n_category/2)]
+
+ if y == 'o/e':
+ alternative = 'less'
+ elif y == 'MAPS':
+ alternative = 'greater'
+ stat_results = []
+ for i, (name, group) in enumerate(bydelta_ind.groupby(by = x)):
+
+ if name != reference and group['total'].max()>n_variants: # require at least 30 variants to test
+ try:
+ _, p = mannwhitneyu(group[y], bydelta_ind.loc[bydelta_ind[x]==reference, y],
+ alternative = alternative) # test for negative selection only
+ median_diff = group[y].median()-bydelta_ind.loc[bydelta_ind[x]==reference, y].median()
+ stat_results.append([bin2name[name], p, median_diff, group['total'].max()])
+ except Exception as e:
+ print(e)
+ return pd.DataFrame(stat_results, columns = [x, 'p-value', 'median difference', 'n'])
+
+
+ ### global MAPS ###
+ global_maps = groupby_bootstrap_maps(sub_df)
+ global_result = oe_test_for_negative_selection(global_maps, 'delta_score_bins', 'MAPS')
+ global_maps.to_csv(outdir / f'{exp}.global_MAPS.csv')
+ global_result.to_csv(outdir / f'{exp}.global_MAPS_stat.csv')
+
+ ### global o/e ###
+ global_oe = groupby_bootstrap(df, by = ['delta_score_bins'])
+ global_oe_result = oe_test_for_negative_selection(global_oe, 'delta_score_bins', 'o/e')
+ global_oe.to_csv(outdir / f'{exp}.global_oe.csv')
+ global_oe_result.to_csv(outdir / f'{exp}.global_oe_stat.csv')
+
+ ### testing subset of variants ###
+ def groupby_test_results(df, by, y = 'o/e', n = 30):
+ if type(by) == str:
+ by = [by]
+
+ if y == 'o/e':
+ bydelta = groupby_bootstrap(df, by = ['delta_score_bins']+by, n=n)
+ elif y == 'MAPS':
+ bydelta = groupby_bootstrap_maps(df, by = ['delta_score_bins']+by, n=n)
+ stats = []
+
+ for fname, fgroup in bydelta.groupby(by = by):
+ test_result = oe_test_for_negative_selection(fgroup, x='delta_score_bins', y=y)
+ if type(fname) == tuple:
+ for i, col in enumerate(by):
+ test_result[col]=fname[i]
+ else:
+ test_result[by]=fname
+ stats.append(test_result)
+ results = pd.concat(stats, axis = 0)
+
+ return bydelta, results
+
+ ### feature/transcript types ###
+ for col in ['feature_type_top', 'transcript_type_top']:
+ for metric, metric_name in zip(['o/e', 'MAPS'], ['oe', 'MAPS']):
+ if metric == 'MAPS':
+ bootstrap_data, test_results = groupby_test_results(sub_df, col, y = metric)
+ else:
+ bootstrap_data, test_results = groupby_test_results(df, col, y = metric)
+ bootstrap_data.to_csv(outdir / f'{exp}.{col}.{metric_name}.csv')
+ test_results.to_csv(outdir / f'{exp}.{col}.{metric_name}_stat.csv')
+
+ ### stratify by LoF bins ###
+ col = 'feature_type_top'
+ for metric, metric_name in zip(['o/e', 'MAPS'], ['oe', 'MAPS']):
+ if metric == 'MAPS':
+ bootstrap_data, test_results = groupby_test_results(sub_df, ['lof.pLI bins', col],
+ y = metric)
+ else:
+ bootstrap_data, test_results = groupby_test_results(df, ['lof.pLI bins', col],
+ y = metric)
+ bootstrap_data.to_csv(outdir / f'{exp}.{col}.{metric_name}.lofbins.csv')
+ test_results.to_csv(outdir / f'{exp}.{col}.{metric_name}_stat.lofbins.csv')
+
+ ### ClinVar ###
+ clinvar_vcf = pd.read_csv(indir / 'output/variants' / 'clinvar' / f'{exp}.vcf', index_col = 0,
+ names = ['CHROM', 'POS', 'Clinvar_ID', 'REF', 'ALT',
+ 'INFO/CLNDN','INFO/CLNVC','INFO/CLNSIG',
+ 'INFO/CLNDISDB','INFO/AF_ESP','INFO/AF_EXAC',
+ 'INFO/AF_TGP','INFO/ALLELEID'],
+ sep = '\t'
+ )
+
+ clinvar_df = df.merge(clinvar_vcf, left_on = ['CHROM', 'POS', 'REF', 'ALT'],
+ right_on = ['CHROM', 'POS', 'REF', 'ALT']
+ )
+ clinvar_df.drop_duplicates(subset = ['CHROM', 'POS', 'REF', 'ALT'],
+ inplace = True)
+
+ clinvar_vep = pd.read_csv(indir / 'output/variants' / 'clinvar' / f'{exp}.vep.tsv', sep = '\t',
+ comment = '#',
+ names = ['Uploaded_variation','Location','Allele',
+ 'Gene','Feature',
+ 'Feature_type','Consequence','cDNA_position','CDS_position',
+ 'Protein_position','Amino_acids','Codons','Existing_variation',
+ 'Extra'])
+ #clinvar_dedup = clinvar_vep[['Uploaded_variation', 'Location', 'Allele']].drop_duplicates()
+ clinvar_df['Consequence'] = clinvar_df['Clinvar_ID'].map(
+ clinvar_vep.groupby(by = 'Uploaded_variation')['Consequence'].agg(
+ lambda x: ','.join(list(set(','.join(x).split(',')))))
+ )
+
+ # clean up CLNSIG
+ clinvar_df['INFO/CLNSIG']=clinvar_df['INFO/CLNSIG'].replace(
+ 'Likely_benign', 'Benign/Likely_benign').replace(
+ 'Benign', 'Benign/Likely_benign').replace(
+ 'Pathogenic', 'Pathogenic/Likely_pathogenic').replace(
+ 'Likely_pathogenic', 'Pathogenic/Likely_pathogenic').replace(
+ 'Conflicting_interpretations_of_pathogenicity', 'Uncertain_significance')
+ clinvar_df['INFO/CLNSIG'] = pd.Categorical(clinvar_df['INFO/CLNSIG'],
+ categories = ['Benign/Likely_benign', 'Uncertain_significance', 'Pathogenic/Likely_pathogenic'],
+ ordered = True)
+ clinvar_df['CLNSIG_rank'] = clinvar_df['INFO/CLNSIG'].rank(method = 'dense')
+
+ clinvar_df['INFO/CLNDN'] = clinvar_df['INFO/CLNDN'].str.replace('not_provided', 'not_specified')
+ def is_coding(s):
+ if 'missense' in s or 'stop' in s or 'start' in s or 'frame' in s:
+ return True
+ else:
+ return False
+ clinvar_df['is_coding']=clinvar_df['Consequence'].apply(lambda x: is_coding(x))
+ clinvar_df['CLNDN_parsed'] = clinvar_df['INFO/CLNDN'].str.split('|').tolist()
+ clinvar_df.to_csv(outdir / f'{exp}.clinvar_variants.csv')
+
+ # perform counting
+ clinvar_df_exploded = clinvar_df.explode('CLNDN_parsed')
+ clinvar_df_exploded.to_csv(outdir / f'{exp}.clinvar_variants_exploded.csv')
+
+ def plot_sorted(counts, ax, n=10, **kwargs):
+ counts.loc[counts.sum(axis = 1).sort_values(ascending = False).index
+ ].iloc[:n,:].plot.barh(stacked = True, **kwargs, ax = ax)
+
+ clinsig_count = pd.pivot_table(clinvar_df_exploded, index = 'CLNDN_parsed',
+ columns = ['is_coding','INFO/CLNSIG'],
+ aggfunc = 'size')
+
+ f, ax = plt.subplots(2,1, figsize = (6,6))
+ try:
+ plot_sorted(clinsig_count[True], ax = ax[0], cmap = 'coolwarm', legend = False)
+ except KeyError as e:
+ print(e)
+ ax[0].set_title('coding')
+ try:
+ plot_sorted(clinsig_count[False], ax = ax[1], cmap = 'coolwarm')
+ except KeyError as e:
+ print(e)
+ ax[1].set_title('non-coding')
+ ax[1].set_xlabel('Number Variants')
+ sns.despine()
+ plt.savefig(outdir / f'{exp}.clinvar_CLINSIC_counts.pdf', bbox_inches='tight')
+ clinsig_count.to_csv(outdir / f'{exp}.clinvar_CLINSIC_counts.csv')
+
+ impact_count = pd.pivot_table(clinvar_df_exploded, index = 'CLNDN_parsed',
+ columns = ['is_coding','delta_zscore'],
+ aggfunc = 'size')
+ f, ax = plt.subplots(2,1, figsize = (6,6))
+ try:
+ plot_sorted(impact_count[True], ax = ax[0], cmap = 'coolwarm', legend = False)
+ except KeyError as e:
+ print(e)
+ ax[0].set_title('coding')
+ try:
+ plot_sorted(impact_count[False], ax = ax[1], cmap = 'coolwarm')
+ except KeyError as e:
+ print(e)
+ ax[1].set_title('non-coding')
+ ax[1].set_xlabel('Number Variants')
+ sns.despine()
+ plt.savefig(outdir / f'{exp}.clinvar_impact_counts.pdf', bbox_inches='tight')
+ impact_count.to_csv(outdir / f'{exp}.clinvar_impact_counts.csv')
diff --git a/tools/permutation_gnomAD.py b/tools/permutation_gnomAD.py
new file mode 100644
index 0000000..5f7b68c
--- /dev/null
+++ b/tools/permutation_gnomAD.py
@@ -0,0 +1,109 @@
+import pandas as pd
+import statsmodels.formula.api as smf
+import concurrent.futures
+import random
+import time
+import multiprocessing
+import sys
+import matplotlib.pyplot as plt
+from scipy.stats import norm, uniform
+from statsmodels.stats.multitest import fdrcorrection
+import statsmodels.api as sm
+
+
+def lm(subset_df):
+ ''' test association between delta score and MAF'''
+ results = smf.ols('delta_score ~ MAF_bin_rank', data=subset_df).fit()
+ return results.params['MAF_bin_rank']
+
+def groupby_lm(df, by = 'gene_name', max_workers = 8):
+ ''' for each "by", test association '''
+ keys = [name for name, group in df.groupby(by = by)]
+ with concurrent.futures.ProcessPoolExecutor(max_workers=max_workers) as executor:
+ result = list(executor.map(lm, [group for name, group in df.groupby(by = by)]))
+
+ return dict(zip(keys, result))
+
+def permute(group, value_to_permute = 'delta_score'):
+ val = group[value_to_permute].tolist()
+ random.shuffle(val)
+ group[value_to_permute] = val
+ return group
+
+def groupby_permute(df, groupby_col = 'MAF_bin_rank', max_workers = 8, by = 'gene_name'):
+ ''' permute to get null distribution per "by" column '''
+
+ print('start permuting')
+ grouped = df.groupby(groupby_col)
+
+ with concurrent.futures.ProcessPoolExecutor(max_workers=max_workers) as executor:
+ resultlist = list(executor.map(permute, [group for name, group in grouped]))
+ permuted_df = pd.concat(resultlist, axis = 0)
+ print('end permuting, starting lm')
+
+ result = groupby_lm(permuted_df, max_workers = max_workers, by = by)
+ print('done')
+ del permuted_df
+ return result
+
+if __name__ == '__main__':
+
+ n_cpu = multiprocessing.cpu_count()
+ print(f'Number of CPU available: {n_cpu}')
+ col_to_test = sys.argv[2]
+ n_iter=100
+
+ # input must have 'MAF_bin_rank', 'delta_score' and 'TYPE'
+ df = pd.read_csv(sys.argv[1],
+ sep = '\t', comment = '#',
+ usecols=['MAF_bin_rank', 'delta_score', col_to_test, 'TYPE'])
+
+ out_prefix = sys.argv[3]
+
+ # only test for SNV
+ df = df.loc[df['TYPE']=='SNV']
+ df.drop('TYPE', inplace = True, axis = 1)
+
+ assert not df['delta_score'].isnull().any()
+
+ # generate null distribution
+ with concurrent.futures.ThreadPoolExecutor(max_workers=4) as executor:
+ result = executor.map(groupby_permute, [df]*n_iter)
+
+ null = pd.DataFrame(list(result))
+ null.to_csv(f'{out_prefix}.null.csv')
+
+ # do the test
+ mean_null, std_null = null.mean(axis = 0),null.std(axis = 0)
+ stat = pd.concat([mean_null, std_null], axis = 1)
+ stat.columns = ['mean', 'std']
+
+ # get slope
+ slope = groupby_lm(df)
+ stat['slope'] = stat.index.map(slope)
+
+ # test against slope>0
+ stat['pvalue_against_0'] = stat.apply(lambda row:
+ 1-norm(loc = 0, scale = row['std']).cdf(row['slope']),
+ axis = 1)
+ _, stat['FDR_against_0'] = fdrcorrection(stat['pvalue_against_0'], alpha = 0.2)
+
+ # test against slope>avg (transcriptome wide mean)
+ stat['pvalue_against_mean'] = stat.apply(lambda row:
+ 1-norm(loc = row['mean'], scale = row['std']).cdf(row['slope']),
+ axis = 1)
+ _, stat['FDR_against_mean'] = fdrcorrection(stat['pvalue_against_mean'], alpha = 0.2)
+
+ # plott qq plot
+ f, ax = plt.subplots(1,2)
+ p = sm.qqplot(stat['pvalue_against_mean'], line ='45',
+ dist=uniform, ax = ax[0])
+ ax[0].set_title('H1: slope > transcriptome-wide)')
+
+ p = sm.qqplot(stat['pvalue_against_0'], line ='45',
+ dist=uniform, ax = ax[1])
+ ax[1].set_title('H1: slope > 0)')
+ plt.savefig(f'{out_prefix}.qqplot.pdf')
+
+ # savefile
+ stat.to_csv(f'{out_prefix}.test_stats.csv')
diff --git a/utils/download_gnomAD_v4.sh b/utils/download_gnomAD_v4.sh
new file mode 100644
index 0000000..e0df016
--- /dev/null
+++ b/utils/download_gnomAD_v4.sh
@@ -0,0 +1,7 @@
+
+cd /tscc/nfs/home/hsher/ps-yeolab5/gnomAD/v4
+for c in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
+do
+wget https://storage.googleapis.com/gcp-public-data--gnomad/release/4.1/vcf/genomes/gnomad.genomes.v4.1.sites.chr${c}.vcf.bgz.tbi
+wget https://storage.googleapis.com/gcp-public-data--gnomad/release/4.1/vcf/genomes/gnomad.genomes.v4.1.sites.chr${c}.vcf.bgz
+done
\ No newline at end of file
diff --git a/utils/download_roulette.sh b/utils/download_roulette.sh
new file mode 100644
index 0000000..0563fc0
--- /dev/null
+++ b/utils/download_roulette.sh
@@ -0,0 +1,7 @@
+mkdir ~/ps-yeolab5/roulette
+cd ~/ps-yeolab5/roulette
+for c in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
+do
+wget http://genetics.bwh.harvard.edu/downloads/Vova/Roulette/${c}_rate_v5.2_TFBS_correction_all.vcf.bgz
+wget http://genetics.bwh.harvard.edu/downloads/Vova/Roulette/${c}_rate_v5.2_TFBS_correction_all.vcf.bgz.csi
+done
\ No newline at end of file
diff --git a/utils/envs/java.yaml b/utils/envs/java.yaml
new file mode 100644
index 0000000..b774ac7
--- /dev/null
+++ b/utils/envs/java.yaml
@@ -0,0 +1,73 @@
+name: jvarkit
+channels:
+ - conda-forge
+ - defaults
+dependencies:
+ - _libgcc_mutex=0.1=conda_forge
+ - _openmp_mutex=4.5=2_gnu
+ - alsa-lib=1.2.12=h4ab18f5_0
+ - bzip2=1.0.8=h4bc722e_7
+ - ca-certificates=2024.7.4=hbcca054_0
+ - cairo=1.18.0=hebfffa5_3
+ - expat=2.6.2=h59595ed_0
+ - font-ttf-dejavu-sans-mono=2.37=hab24e00_0
+ - font-ttf-inconsolata=3.000=h77eed37_0
+ - font-ttf-source-code-pro=2.038=h77eed37_0
+ - font-ttf-ubuntu=0.83=h77eed37_2
+ - fontconfig=2.14.2=h14ed4e7_0
+ - fonts-conda-ecosystem=1=0
+ - fonts-conda-forge=1=0
+ - freetype=2.12.1=h267a509_2
+ - giflib=5.2.2=hd590300_0
+ - graphite2=1.3.13=h59595ed_1003
+ - harfbuzz=9.0.0=hda332d3_1
+ - icu=75.1=he02047a_0
+ - keyutils=1.6.1=h166bdaf_0
+ - krb5=1.21.3=h659f571_0
+ - lcms2=2.16=hb7c19ff_0
+ - lerc=4.0.0=h27087fc_0
+ - libcups=2.3.3=h4637d8d_4
+ - libdeflate=1.20=hd590300_0
+ - libedit=3.1.20191231=he28a2e2_2
+ - libexpat=2.6.2=h59595ed_0
+ - libffi=3.4.2=h7f98852_5
+ - libgcc-ng=14.1.0=h77fa898_0
+ - libglib=2.80.3=h8a4344b_1
+ - libgomp=14.1.0=h77fa898_0
+ - libiconv=1.17=hd590300_2
+ - libjpeg-turbo=3.0.0=hd590300_1
+ - libpng=1.6.43=h2797004_0
+ - libstdcxx-ng=14.1.0=hc0a3c3a_0
+ - libtiff=4.6.0=h1dd3fc0_3
+ - libuuid=2.38.1=h0b41bf4_0
+ - libwebp-base=1.4.0=hd590300_0
+ - libxcb=1.16=hd590300_0
+ - libzlib=1.3.1=h4ab18f5_1
+ - ncurses=6.5=h59595ed_0
+ - openjdk=22.0.1=h8651b0f_1
+ - openssl=3.3.1=h4bc722e_2
+ - pcre2=10.44=h0f59acf_0
+ - pixman=0.43.2=h59595ed_0
+ - pthread-stubs=0.4=h36c2ea0_1001
+ - xorg-fixesproto=5.0=h7f98852_1002
+ - xorg-inputproto=2.3.2=h7f98852_1002
+ - xorg-kbproto=1.0.7=h7f98852_1002
+ - xorg-libice=1.1.1=hd590300_0
+ - xorg-libsm=1.2.4=h7391055_0
+ - xorg-libx11=1.8.9=hb711507_1
+ - xorg-libxau=1.0.11=hd590300_0
+ - xorg-libxdmcp=1.1.3=h7f98852_0
+ - xorg-libxext=1.3.4=h0b41bf4_2
+ - xorg-libxfixes=5.0.3=h7f98852_1004
+ - xorg-libxi=1.7.10=h4bc722e_1
+ - xorg-libxrender=0.9.11=hd590300_0
+ - xorg-libxt=1.3.0=hd590300_1
+ - xorg-libxtst=1.2.4=h4bc722e_0
+ - xorg-recordproto=1.14.2=h7f98852_1002
+ - xorg-renderproto=0.11.1=h7f98852_1002
+ - xorg-xextproto=7.3.0=h0b41bf4_1003
+ - xorg-xproto=7.0.31=h7f98852_1007
+ - xz=5.2.6=h166bdaf_0
+ - zlib=1.3.1=h4ab18f5_1
+ - zstd=1.5.6=ha6fb4c9_0
+prefix: /tscc/nfs/home/hsher/miniconda3/envs/jvarkit
diff --git a/utils/expected_singleton_rate_regression.py b/utils/expected_singleton_rate_regression.py
new file mode 100644
index 0000000..659ee30
--- /dev/null
+++ b/utils/expected_singleton_rate_regression.py
@@ -0,0 +1,95 @@
+import pandas as pd
+import sys
+import numpy as np
+import matplotlib.pyplot as plt
+from pathlib import Path
+import statsmodels.api as sm
+import statsmodels.formula.api as smf
+def make_MAF_bins(gnomad):
+ gnomad['MAF']=gnomad['INFO/AC']/gnomad['INFO/AN']
+ gnomad['MAF_bin'] = pd.cut(gnomad['MAF'], bins = [0, 1e-3, 1e-2, 1],
+ labels = ['ultrarare',
+ 'rare',
+ 'common']
+
+ )
+
+
+ bins = gnomad['MAF_bin'].cat.categories.tolist()
+ bins = ['singleton']+bins
+ gnomad['MAF_bin']=pd.Categorical(gnomad['MAF_bin'], categories = bins, ordered = True,
+ )
+ gnomad.loc[gnomad['INFO/AC']==1, 'MAF_bin']='singleton'
+ gnomad['MAF_bin_rank']=gnomad['MAF_bin'].rank(method = 'dense')
+
+ return gnomad
+if __name__ == '__main__':
+ roulette = pd.read_csv(sys.argv[1],
+ sep = '\t',
+ names = ['CHROM','POS', 'ID','REF','ALT', 'INFO/RBPSITE','INFO/MR', 'INFO/AC','INFO/AN'],
+ na_values = '.',
+ )
+ vep = pd.read_csv(sys.argv[2],
+ sep = '\t',
+ names = ['Uploaded_variation','Location','Allele',
+ 'Gene','Feature',
+ 'Feature_type','Consequence','cDNA_position','CDS_position',
+ 'Protein_position','Amino_acids','Codons','Existing_variation',
+ 'Extra'])
+ model_out = Path(sys.argv[3])
+
+ vep = vep.loc[vep['Consequence']=='intergenic_variant']
+ vep['POS']=vep['Location'].str.split(':', expand = True)[1].astype(int)
+ merged_vep = vep.merge(roulette, right_on = ['POS', 'ALT'],
+ left_on = ['POS', 'Allele'], how = 'left'
+ )
+ merged_vep = make_MAF_bins(merged_vep)
+ merged_vep['mutation rate bin'] = pd.cut(merged_vep['INFO/MR'], bins = 100,
+ duplicates = 'drop')
+ count_per_mr_maf = pd.pivot_table(merged_vep, index = 'mutation rate bin',
+ columns = 'MAF_bin',
+ aggfunc = 'size').dropna()
+ fraction_per_mr_maf = count_per_mr_maf.div(count_per_mr_maf.sum(axis =1),
+ axis = 0)
+ types = fraction_per_mr_maf.columns
+ fraction_per_mr_maf['MR']=[(i.left+i.right)/2 for i in fraction_per_mr_maf.index]
+ fraction_per_mr_maf['counts']=merged_vep['mutation rate bin'].value_counts()
+
+ fraction_per_mr_maf['log_counts'] = np.log10(fraction_per_mr_maf['counts'])
+ count_per_mr_maf['MR']=[(i.left+i.right)/2 for i in count_per_mr_maf.index]
+ for t in types:
+ count_per_mr_maf[f'non_{t}']=count_per_mr_maf.sum(axis = 1)-count_per_mr_maf[t]
+
+ models = {}
+
+ for t in types:
+ binom = sm.families.Binomial()
+
+ lrTest = smf.glm(f'{t} + non_{t} ~ MR',
+ count_per_mr_maf.loc[count_per_mr_maf[t]+count_per_mr_maf[f'non_{t}']>0],
+ family=binom).fit()
+ models[t]=lrTest
+ fraction_per_mr_maf[f'binom_predicted_fraction_{t}'] = lrTest.predict(count_per_mr_maf[['MR']])
+ models[t].save(model_out/"{t}.pickle")
+
+ f, ax = plt.subplots(figsize = (3,3))
+ for t, color in zip(types[:1], ['tomato', 'gold', 'seagreen', 'royalblue']):
+
+ fraction_per_mr_maf.plot.scatter(x = 'MR',
+ y = t,
+ s = 'log_counts',
+ ax = ax,
+ color = color
+ )
+
+ # sub.plot(x = 'MR',
+ # y = 'predicted_fraction_singleton', ax = ax)
+ fraction_per_mr_maf.plot(x = 'MR',
+ y = f'binom_predicted_fraction_{t}', ax = ax,
+ color = color,
+ label = f'{t} model')
+
+ plt.ylabel('Fraction')
+ plt.xlabel('Mutation Rate')
+ plt.legend(bbox_to_anchor=(1,0.5))
+ plt.savefig(model_out/ 'fit.pdf')
\ No newline at end of file
diff --git a/utils/fix_output.sh b/utils/fix_output.sh
new file mode 100644
index 0000000..ac76774
--- /dev/null
+++ b/utils/fix_output.sh
@@ -0,0 +1,21 @@
+find . -type f -empty -print -delete
+for f in *.score.txt;
+do
+LINECOUNT=`wc -l $f | cut -f1 -d ' '`;
+
+
+if [[ $LINECOUNT -eq 1 ]]; then
+rm $f
+echo $f $LINECOUNT;
+fi
+
+done
+
+
+for f in ~/scratch/ENCO*_*/output/variants/clinvar;
+do
+cd $f
+echo $f
+pwd
+find . -name '*.vep.tsv' -type f -empty -print
+done
\ No newline at end of file
diff --git a/utils/installing_eugene.sh b/utils/installing_eugene.sh
new file mode 100644
index 0000000..919729e
--- /dev/null
+++ b/utils/installing_eugene.sh
@@ -0,0 +1,6 @@
+conda create -n eugene2
+
+conda activate eugene2
+mamba install python==3.9.2
+python -m pip install eugene-tools pandas seaborn seqdata statsmodels seqpro torch scipy numpy tqdm pybedtools xarray tensorboard tabulate
+python -m pip install eugene-tools==0.1.2 pandas seaborn seqdata==0.1.3 statsmodels seqpro==0.1.11 torch scipy numpy tqdm pybedtools xarray tensorboard tabulate modisco-lite==2.2.1
\ No newline at end of file
diff --git a/utils/non_RBP_intron_rates.sh b/utils/non_RBP_intron_rates.sh
new file mode 100644
index 0000000..c10fe46
--- /dev/null
+++ b/utils/non_RBP_intron_rates.sh
@@ -0,0 +1,42 @@
+# non-RBP site introns
+intron=/tscc/nfs/home/hsher/gencode_coords/gencode.v35.basic.intron.gff3
+
+# bedtools intersect multiple rbps # rename stupid shit
+cat ~/scratch/ENCODE3_HepG2/output/finemapping/mapped_sites/*finemapped_windows.bed.gz > ~/scratch/allrbpsite.bed.gz
+
+bedtools intersect -a $intron -b ~/scratch/allrbpsite.bed.gz \
+-s -v | awk '{ gsub(/chr/,"", $1); print }' OFS="\t" | \
+awk '{ print $1,$4,$5,$6,$3,$7}' OFS="\t" > ~/scratch/non_rbp_intron.bed
+
+# https://bedtools.readthedocs.io/en/latest/content/tools/intersect.html
+
+bcftools query -R ~/scratch/non_rbp_intron.bed \
+ -f '%CHROM\t%POS\t%ID\t%REF\t%ALT\t%FILTER\t%INFO/MR\t%INFO/AR\t%INFO/MG\t%INFO/MC\n' \
+ /tscc/nfs/home/hsher/ps-yeolab5/roulette/22_rate_v5.2_TFBS_correction_all.vcf.bgz \
+ | awk '$1="chr"$1' OFS="\t" > ~/scratch/non_rbp_intron.roulette.vcf
+
+# rename
+awk '$1="chr"$1' OFS="\t" ~/scratch/non_rbp_intron.bed > ~/scratch/non_rbp_intron.chr.bed
+
+
+# query genes
+gencode=/tscc/nfs/home/hsher/gencode_coords/gencode.v40.primary_assembly.annotation.gff3
+genes_bed=~/scratch/genes.bed
+gene_vcf=~/scratch/genes.vcf
+
+awk '$3=="gene" {print $1,$4,$5,$6,$8,$7}' OFS="\t" $gencode > $genes_bed
+
+bcftools query -R $genes_bed \
+ -f '%CHROM\t%POS\t%ID\t%REF\t%ALT\t%INFO/AC\t%INFO/AN\n' \
+ /tscc/nfs/home/hsher/ps-yeolab5/gnomAD/v3/gnomad.genomes.v3.1.2.sites.chr22.vcf.bgz \
+ > $gene_vcf
+
+
+bcftools query -f '%CHROM\t%POS\t%ID\t%REF\t%ALT\t%INFO/AC\t%INFO/AN\n' \
+ /tscc/nfs/home/hsher/ps-yeolab5/gnomAD/v3/gnomad.genomes.v3.1.2.sites.chr22.vcf.bgz \
+ > ~/scratch/gnomad.chr22.tsv
+
+bcftools query \
+ -f '%CHROM\t%POS\t%ID\t%REF\t%ALT\t%FILTER\t%INFO/MR\t%INFO/AR\t%INFO/MG\t%INFO/MC\n' \
+ /tscc/nfs/home/hsher/ps-yeolab5/roulette/22_rate_v5.2_TFBS_correction_all.header.filtered.vcf \
+ > ~/scratch/roulette.chr22.vcf
diff --git a/utils/score_any_variants.smk b/utils/score_any_variants.smk
new file mode 100644
index 0000000..bc3135d
--- /dev/null
+++ b/utils/score_any_variants.smk
@@ -0,0 +1,178 @@
+VARIANT='/tscc/nfs/home/hsher/ps-yeolab5/ENCODE_paper_tables/CHD_variants.tsv'
+#'CHROM','POS','ID','REF','ALT' first 5 columns needs to be this, no index, no header
+
+
+TABLE='/tscc/projects/ps-yeolab4/software/skipper/bb63a25/bin/skipper/annotations/GTEx_Analysis_2017-06-05_v8_RNASeQCv1.1.9_gene_median_tpm.Heart_Left_Ventricle.tiled_partition.bed.gz'
+VEP_CACHEDIR='/tscc/nfs/home/hsher/scratch/vep_cache/'
+import pandas as pd
+locals().update(config)
+
+tabledir = Path('/tscc/nfs/home/hsher/ps-yeolab5/ENCODE_paper_tables/')
+metrics=pd.read_csv(tabledir/'model_performance.csv')
+selected_models = metrics.loc[metrics['selected'], 'Experiment'].sort_values().tolist()
+
+workdir: "/tscc/nfs/home/hsher/scratch/CHD_analysis"
+from pathlib import Path
+model_dir = Path('/tscc/nfs/home/hsher/scratch/')
+models = model_dir.glob('ENCO*/output/ml/rbpnet_model/*')
+model_dict = {}
+for f in models:
+ model_dict[f.name] = f
+
+
+print(model_dict)
+"""
+snakemake -s utils/score_any_variants.smk \
+ --configfile /tscc/nfs/home/hsher/projects/skipper/encode_configs/encode_pe_rules_config_K562_20230929.yaml \
+ --profile profiles/tscc2 \
+ -n
+"""
+
+rule all:
+ input:
+ expand("output/variants/{model_name}.score.csv",
+ model_name = selected_models
+ ),
+ #"output/variants/vep.tsv"
+
+
+rule slop_finemap:
+ input:
+ finemapped_windows = TABLE
+ output:
+ "output/table.slop.bed.gz"
+ threads: 2
+ params:
+ error_file = "stderr/slop_finemap",
+ out_file = "stdout/slop_finemap",
+ run_time = "06:20:00",
+ cores = 1,
+ memory = 40000,
+ container:
+ "docker://howardxu520/skipper:bigwig_1.0"
+ shell:
+ """
+ bedtools slop -i {input.finemapped_windows} -g {CHROM_SIZES} -b 100 | gzip -c > {output}
+ """
+
+rule fetch_peak_sequence:
+ input:
+ finemapped_windows = rules.slop_finemap.output,
+ output:
+ finemapped_fa = "output/sequence/table.slop.fa",
+ params:
+ error_file = "stderr/fetch_sequence.err",
+ out_file = "stdout/fetch_sequence.out",
+ run_time = "40:00",
+ memory = "2000",
+ job_name = "run_homer",
+ fa = config['GENOME']
+ container:
+ "docker://howardxu520/skipper:bedtools_2.31.0"
+ shell:
+ '''
+ bedtools getfasta -fo {output.finemapped_fa} -fi {params.fa} -bed {input.finemapped_windows} -s
+ '''
+
+rule fetch_variant_sequence:
+ input:
+ subset_vcf=VARIANT,
+ seq_fa = rules.fetch_peak_sequence.output.finemapped_fa,
+ finemapped_windows = rules.slop_finemap.output
+ output:
+ ref_fa = temp("output/variants/annotation.ref.fa"),
+ alt_fa = temp("output/variants/annotation.alt.fa"),
+ all_fa = "output/variants/annotation.all.fa",
+ csv = "output/variants/annotation.csv"
+ threads: 2
+ params:
+ error_file = "stderr/fetch_variant",
+ out_file = "stdout/fetch_variant",
+ run_time = "01:20:00",
+ cores = 1,
+ out_prefix = lambda wildcards, output: output.csv.replace('.csv', ''),
+ memory = 80000,
+ conda:
+ "/tscc/nfs/home/hsher/projects/skipper/rules/envs/metadensity.yaml"
+ shell:
+ """
+ if [ -s {input.subset_vcf} ]; then
+ python {TOOL_DIR}/generate_variant_sequence.py \
+ {input.subset_vcf} \
+ {input.seq_fa} \
+ {input.finemapped_windows} \
+ {params.out_prefix}
+ else
+ touch {output.csv}
+ touch {output.ref_fa}
+ touch {output.alt_fa}
+ fi
+ """
+
+rule score_fa:
+ input:
+ fa = "output/variants/annotation.all.fa"
+ output:
+ score=temp("output/variants/{model_name}.score.csv"),
+ threads: 1
+ params:
+ error_file = "stderr/score_fa.{model_name}",
+ out_file = "stdout/score_fa.{model_name}",
+ run_time = "00:20:00",
+ cores = 1,
+ memory = 80000,
+ model_path = lambda wildcards: model_dict[wildcards.model_name]
+ conda:
+ "/tscc/nfs/home/hsher/projects/skipper/rules/envs/eugene.yaml"
+ shell:
+ """
+ if [ -s {input.fa} ]; then
+ python {RBPNET_PATH}/score_fa.py \
+ {params.model_path} \
+ {input.fa} \
+ {output.score}
+ else
+ touch {output.score}
+ fi
+ """
+
+rule to_vcf:
+ input:
+ TABLE
+ output:
+ "output/variants/variants.vcf"
+ container:
+ "docker://brianyee/bcftools:1.17"
+ params:
+ error_file = "stderr/tovcf",
+ out_file = "stdout/tovcf",
+ run_time = "00:20:00",
+ cores = 1,
+ memory = 80000,
+ shell:
+ """
+ bcftools convert -c CHROM,POS,REF,ALT,-,-,filter,- -f {GENOME} --tsv2vcf {input} -o {output}
+ """
+rule vep:
+ input:
+ "output/variants/variants.vcf"
+ output:
+ "output/variants/vep.tsv"
+ threads: 2
+ params:
+ error_file = "stderr/vep",
+ out_file = "stdout/vep",
+ run_time = "1:20:00",
+ cores = 1,
+ memory = 40000,
+ cache= VEP_CACHEDIR
+ container:
+ "docker://ensemblorg/ensembl-vep:latest"
+ shell:
+ """
+ vep \
+ -i {input} \
+ --force_overwrite \
+ -o {output} -offline --cache {params.cache}
+ """
+
diff --git a/utils/vep_annotate_gnomADref.smk b/utils/vep_annotate_gnomADref.smk
new file mode 100644
index 0000000..b1fe4cc
--- /dev/null
+++ b/utils/vep_annotate_gnomADref.smk
@@ -0,0 +1,171 @@
+VEP_CACHEDIR='/tscc/nfs/home/hsher/scratch/vep_cache/'
+ROULETTE = '/tscc/nfs/home/hsher/ps-yeolab5/roulette/22_rate_v5.2_TFBS_correction_all.vcf.bgz'
+HEADER = '/tscc/nfs/home/hsher/bin/Roulette/header.hr'
+N_SPLIT=1000
+ROULETTE_DIR=Path('/tscc/nfs/home/hsher/ps-yeolab5/roulette/')
+JVARKIT_DIR='/tscc/nfs/home/hsher/bin/jvarkit/JVARKIT'
+GENCODE='/tscc/nfs/home/hsher/gencode_coords/gencode.v40.primary_assembly.annotation.gff3'
+MODEL_DIR='/tscc/nfs/home/hsher/ps-yeolab5/roulette/model'
+
+locals().update(config)
+
+workdir: '/tscc/nfs/home/hsher/scratch/annotate_roulette/'
+"""
+snakemake -s utils/vep_annotate_gnomADref.smk \
+ --profile /tscc/nfs/home/hsher/projects/skipper/profiles/tscc2 \
+ -n
+"""
+
+rule all:
+ input:
+ "22_rate_v5.2_TFBS_correction_all.vep.tsv",
+ '22_rate_v5.2_TFBS_correction_all.header.filtered.rename.annotated.filtered.tsv.gz',
+ MODEL_DIR+'/singleton.pickle'
+
+rule split_vcf:
+ input:
+ ROULETTE_DIR/'22_rate_v5.2_TFBS_correction_all.header.filtered.rename.annotated.vcf.gz'
+ output:
+ temp(expand("output/{prefix}.{chunk}.vcf.gz",
+ chunk = [f"{i:05d}" for i in range(1, N_SPLIT+1)],
+ prefix="{prefix}")),
+ temp(expand("{prefix}.{chunk}.vcf.gz.tbi",
+ chunk = [f"{i:05d}" for i in range(1, N_SPLIT+1)],
+ prefix="{prefix}"))
+ threads: 1
+ params:
+ error_file = "stderr/split_vcf",
+ out_file = "stdout/split_vcf",
+ run_time = "6:20:00",
+ cores = 1,
+ memory = 80000,
+ conda:
+ "envs/java.yaml"
+ shell:
+ """
+ java -jar {JVARKIT_DIR}/jvarkit.jar vcfsplitnvariants \
+ --vcf-count {N_SPLIT} \
+ --prefix output/{wildcards.prefix} \
+ {input}
+ """
+
+rule vep:
+ input:
+ "output/{prefix}.{chunk}.vcf.gz"
+ output:
+ temp("output/{prefix}.{chunk}.tsv"),
+ temp("output/{prefix}.{chunk}.tsv_summary.html")
+ threads: 2
+ params:
+ error_file = "stderr/vep",
+ out_file = "stdout/vep",
+ run_time = "1:20:00",
+ cores = 1,
+ memory = 40000,
+ container:
+ "docker://ensemblorg/ensembl-vep:latest"
+ shell:
+ """
+ vep \
+ -i {input} \
+ --force_overwrite \
+ -o {output} -offline --cache {VEP_CACHEDIR}
+ """
+
+rule combine_vep:
+ input:
+ expand("output/{prefix}.{chunk}.tsv",
+ chunk = [f"{i:05d}" for i in range(1, N_SPLIT+1)],
+ prefix="{prefix}")
+ output:
+ "{prefix}_all.vep.tsv"
+ threads: 2
+ params:
+ error_file = "stderr/combine_vep",
+ out_file = "stdout/combine_vep",
+ run_time = "1:20:00",
+ cores = 1,
+ memory = 40000,
+ shell:
+ """
+ cat {input} | grep -v '#' > {output}
+ """
+
+rule annotate_RBP_site:
+ input:
+ vcf=ROULETTE_DIR/'22_rate_v5.2_TFBS_correction_all.header.filtered.rename.annotated.vcf.gz'
+ output:
+ allrbp="allrbpsite.bed.gz",
+ allrbp_rename="allrbpsite.bed.rename.gz",
+ vcf=ROULETTE_DIR/'22_rate_v5.2_TFBS_correction_all.header.filtered.rename.annotated.filtered.vcf.gz'
+ threads: 1
+ params:
+ error_file = "stderr/annotate_RBP_site",
+ out_file = "stdout/annotate_RBP_site",
+ run_time = "1:20:00",
+ cores = 1,
+ memory = 40000,
+ container:
+ "docker://brianyee/bcftools:1.17"
+ shell:
+ """
+ cat /tscc/nfs/home/hsher/scratch/ENCO*_*/output/finemapping/mapped_sites/*finemapped_windows.bed.gz > {output.allrbp}
+ zcat {output.allrbp} | \
+ sort -k1,1 -k2,2n -k3,3n | \
+ awk -v OFS=\"\t\" '{{print $1, $2, $3, "RBP_site", $5, $6}}' | \
+ bgzip > {output.allrbp_rename}
+ tabix -p bed {output.allrbp_rename}
+
+ bcftools annotate \
+ -a {output.allrbp_rename} \
+ -c CHROM,FROM,TO,RBPSITE \
+ -h <(echo '##INFO=') \
+ -Oz -o {output.vcf} \
+ {input.vcf}
+
+ bcftools index {output.vcf}
+ """
+
+rule make_tsv:
+ input:
+ vcf=ROULETTE_DIR/'22_rate_v5.2_TFBS_correction_all.header.filtered.rename.annotated.filtered.vcf.gz'
+ output:
+ tsv='22_rate_v5.2_TFBS_correction_all.header.filtered.rename.annotated.filtered.tsv.gz'
+ threads: 1
+ params:
+ error_file = "stderr/make_tsv",
+ out_file = "stdout/make_tsv",
+ run_time = "1:20:00",
+ cores = 1,
+ memory = 40000,
+ container:
+ "docker://brianyee/bcftools:1.17"
+ shell:
+ """
+ bcftools query -f '%CHROM\t%POS\t%ID\t%REF\t%ALT\t%INFO/RBPSITE\t%INFO/MR\t%INFO/AC\t%INFO/AN\n' \
+ {input.vcf} \
+ | gzip > {output.tsv}
+ """
+
+rule fit_expected_singleton_rate:
+ input:
+ vcf=ROULETTE_DIR/'22_rate_v5.2_TFBS_correction_all.header.filtered.rename.annotated.filtered.vcf.gz',
+ vep='/tscc/nfs/home/hsher/scratch/annotate_roulette/22_rate_v5.2_TFBS_correction_all.vep.tsv'
+ output:
+ expected=MODEL_DIR+'/singleton.pickle'
+ threads: 1
+ params:
+ error_file = "stderr/fit_expected_singleton_rate",
+ out_file = "stdout/fit_expected_singleton_rate",
+ run_time = "1:20:00",
+ cores = 1,
+ memory = 40000,
+ conda:
+ "/tscc/nfs/home/hsher/projects/skipper/rules/envs/metadensity.yaml"
+ shell:
+ """
+ python /tscc/nfs/home/hsher/projects/skipper/utils/expected_singleton_rate_regression.py \
+ {input.vcf} \
+ {input.vep} \
+ {MODEL_DIR}
+ """
\ No newline at end of file