From 163ebec93230f77faf4b84be98aa5a15b823b640 Mon Sep 17 00:00:00 2001
From: lecook <lecook@student.unimelb.edu.au>
Date: Fri, 10 Dec 2021 20:51:19 +1100
Subject: [PATCH] annotated
---
mouse-chipseq/README.md | 87 +---
mouse-chipseq/Snakefile1_H3K4me3 | 717 -------------------------------
mouse-chipseq/Snakefile_H3K4me3 | 56 +--
3 files changed, 32 insertions(+), 828 deletions(-)
delete mode 100755 mouse-chipseq/Snakefile1_H3K4me3
diff --git a/mouse-chipseq/README.md b/mouse-chipseq/README.md
index e71fa9d..842b3b6 100755
--- a/mouse-chipseq/README.md
+++ b/mouse-chipseq/README.md
@@ -54,52 +54,12 @@ snakemake -j 6 --snakefile Snakefile_H3K4me3 --cluster-config configs/cluster.js
```
-
-
Cluster configuration file: configs/cluster.json\
Sample configuration file: configs/config.yaml\
Sample text file: configs/SSR.text\
multiQC configuration file: configs/.multiqc_config.yaml\
-
-# 3. FILTERING
-
-### rule filter:
-
-### rule markDups:
-
-### rule properPairs:
-
-### rule indexBam:
-
-# 4. GENERAL ALIGNMENT QC
-
-### rule mappingStats:
-
-### rule preseq:
-
-### rule get_picard_complexity_metrics:
-
-
-Library Complexity
-
-ChIP-seq Standards:
-
-| PBC1 | PBC2 | Bottlenecking level | NRF | Complexity | Flag colors |
-|:-:|:-:|:-:|:-:|:-:|:-:|
-| < 0.5 | < 1 | Severe | < 0.5 | Concerning | Orange |
-| 0.5 ≤ PBC1 < 0.8 | 1 ≤ PBC2 < 3 | Moderate | 0.5 ≤ NRF < 0.8 | Acceptable | Yellow |
-| 0.8 ≤ PBC1 < 0.9 | 3 ≤ PBC2 < 10 | Mild | 0.8 ≤ NRF < 0.9 | Compliant | None |
-| ≥ 0.9 | ≥ 10 | None | > 0.9 | Ideal | None |
-
-# 5. deepTools
-
-### rule deeptools_summary:
-
-
-### rule deeptools_correlation:
-
-
+# deepTools
### rule deeptools_coverage:
Normalised to the reads per genomic content (normalized to 1x coverage)
Produces a coverage file
@@ -111,17 +71,7 @@ The bigWig format is an indexed binary format useful for dense, continuous data
- `smoothLength`: defines a window, larger than the binSize, to average the number of reads over. This helps produce a more continuous plot.
- `centerReads`: reads are centered with respect to the fragment length as specified by extendReads. This option is useful to get a sharper signal around enriched regions.
-
-
-### rule deeptools_fingerprint:
-
-
-### rule deeptools_plotCoverage:
-
-### rule deeptools_bamPEFragmentSize:
-
-
-# 6. phantomPeakQuals
+# phantomPeakQuals
Information from: https://docs.google.com/document/d/1lG_Rd7fnYgRpSIqrIfuVlAz2dW1VaSQThzk836Db99c/edit
@@ -154,10 +104,8 @@ RSC; RSC>0.8 (0 = no signal; <1 low quality ChIP; >1 high enrichment
Quality tag based on thresholded RSC (codes: -2:veryLow,-1:Low,0:Medium,1:High; 2:veryHigh)
-### rule phantomPeakQuals:
-
-# 7. Call peaks (MACS2)
+# Call peaks (MACS2)
__Input file options__
@@ -196,34 +144,7 @@ I've left all the shifting model and peak calling arguments as default
- `peaks.xls`: a tabular file which contains information about called peaks. Additional information includes pileup and fold enrichment
- `summits.bed`: peak summits locations for every peak. To find the motifs at the binding sites, this file is recommended
-
-## Compare peaks to ENCODE peaks
-With p < 0.01 I only call ~85000 peaks but ENCODE call ~125000
-Look at pvalues, qvalues and peak length between the two lists.
-
-Average Peak Length:
-
-
-Plot pvalues of ENCODE on the x and my calls on the y for each sample
-
-
-
-# 8. Peak QC
-
-
-### rule get_narrow_peak_counts_for_multiqc:
-
-
-
-### rule bamToBed:
-
-Convert BAM to tagAlign file for calculating FRiP QC metric (Fraction of reads in peaks)
-
-
-### rule frip:
-
-
-# 9. Create consensus peaksets for replicates
+# Create consensus peaksets for replicates
Edited version of ENCODE `overlap_peaks.py` - recommended for histone marks.
diff --git a/mouse-chipseq/Snakefile1_H3K4me3 b/mouse-chipseq/Snakefile1_H3K4me3
deleted file mode 100755
index c06f816..0000000
--- a/mouse-chipseq/Snakefile1_H3K4me3
+++ /dev/null
@@ -1,717 +0,0 @@
-## Author: Laura E Cook, University of Melbourne
-## Purpose: ChIP-seq pipeline for histone marks
-## This workflow only works for single-end reads
- # 1. FastQC on raw reads
- # 2. Alignment (downloaded from ENCODE)
- # 3. Filtering
- # 4. Alignment QC & Library Complexity
- # 5. deepTools
- # 7. cross correlation (SPP)
- # 8. Call narrow peaks (MACS2)
- # 9. Create consensus peaksets
- # 10. Present QC for raw read, alignment, peak-calling in MultiQC
-
-
-configfile: "configs/config_H3K4me3.yaml"
-# Contains sample and genome information
-# calls the SRR.txt file that contains the samples as either IP or control
-
-import csv
-import os
-
-IPS = []
-INPUTS = []
-MARK = []
-STAGE = []
-
-with open(config['SAMPLES'], "r") as f:
- reader = csv.reader(f, delimiter = "\t")
- # skip the header
- header = next(reader)
- for row in reader:
- STAGE.append(row[2])
- MARK.append(row[1])
- IPS.append(row[3])
- INPUTS.append(row[4])
-
-## multiple samples may use the same control input files
-unique_inputs = list(set(INPUTS))
-unique_marks = list(set(MARK))
-unique_stages = list(set(STAGE))
-
-## combine all samples
-all_samples = IPS + INPUTS
-
-# ===============================================================================================
-# Output targets
-# ===============================================================================================
-
-rule all:
- input:
- # Alignment and Filtering in results folder
- expand("results/bwa/{sample}_{stage}_{mark}_q30.sorted.bam", zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results/bwa/{sample}_{stage}_{mark}_q30.dupmark.bam", zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam", zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam", zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bai", zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results/bwa/{sample}_{stage}_{mark}_q30.dupmark.tmp.bam", zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results/qc/{sample}_{stage}_{mark}.dedup.flagstat.qc", zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results/qc/{sample}_{stage}_{mark}.dupmark.flagstat.qc", zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results/qc/{sample}_{stage}_{mark}.q30.flagstat.qc", zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results/qc/{sample}_{stage}_{mark}.unfiltered.flagstat.qc", zip, sample=all_samples, stage=STAGE, mark=MARK),
- # Merging the BAM replicates for the subsampled dedup BAM files
- "results_10M/bwa/E10.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- "results_10M/bwa/E11.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- "results_10M/bwa/E12.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- "results_10M/bwa/E13.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- "results_10M/bwa/E14.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- "results_10M/bwa/E15.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- "results_10M/bwa/input_E10.5_H3K4me3_q30.sorted.dedup.bam",
- "results_10M/bwa/input_E11.5_H3K4me3_q30.sorted.dedup.bam",
- "results_10M/bwa/input_E12.5_H3K4me3_q30.sorted.dedup.bam",
- "results_10M/bwa/input_E13.5_H3K4me3_q30.sorted.dedup.bam",
- "results_10M/bwa/input_E14.5_H3K4me3_q30.sorted.dedup.bam",
- "results_10M/bwa/input_E15.5_H3K4me3_q30.sorted.dedup.bam",
- "results_10M/logs/E10.5_H3K4me3.mergeBAM",
- "results_10M/logs/E11.5_H3K4me3.mergeBAM",
- "results_10M/logs/E12.5_H3K4me3.mergeBAM",
- "results_10M/logs/E13.5_H3K4me3.mergeBAM",
- "results_10M/logs/E14.5_H3K4me3.mergeBAM",
- "results_10M/logs/E15.5_H3K4me3.mergeBAM",
- "results_10M/logs/input_E10.5_H3K4me3.mergeBAM",
- "results_10M/logs/input_E11.5_H3K4me3.mergeBAM",
- "results_10M/logs/input_E12.5_H3K4me3.mergeBAM",
- "results_10M/logs/input_E13.5_H3K4me3.mergeBAM",
- "results_10M/logs/input_E14.5_H3K4me3.mergeBAM",
- "results_10M/logs/input_E15.5_H3K4me3.mergeBAM",
- # Quality controls for deepTools
- "results_10M/qc/H3K4me3_multibamsum.npz",
- "results_10M/qc/H3K4me3_multibamsum.tab",
- "results_10M/qc/H3K4me3_pearsoncor_multibamsum.pdf",
- "results_10M/qc/H3K4me3_pearsoncor_multibamsum_matrix.txt",
- expand("results_10M/qc/{sample}_{stage}_{mark}.SeqDepthNorm.bw", zip, sample=all_samples, stage=STAGE, mark=MARK),
- "results_10M/qc/H3K4me3_multiBAM_fingerprint.pdf",
- "results_10M/qc/H3K4me3_multiBAM_fingerprint_metrics.txt",
- "results_10M/qc/H3K4me3_multiBAM_fingerprint_rawcounts.txt",
- "results_10M/qc/H3K4me3_plot_coverage.pdf",
- "results_10M/qc/H3K4me3_plot_coverage_rawcounts.tab",
- # Quality control non-redundant fraction etc.
- expand("results/logs/{sample}_{stage}_{mark}.pbc.sort", zip, sample=INPUTS, stage=STAGE, mark=MARK),
- expand("results/qc/{sample}_{stage}_{mark}.pbc.qc", zip, sample=INPUTS, stage=STAGE, mark=MARK),
- expand("results/qc/{sample}_{stage}_{mark}_filt_15Mreads.SE.cc.qc",zip, sample=all_samples, stage=STAGE, mark=MARK),
- expand("results/qc/{sample}_{stage}_{mark}_filt_15Mreads.SE.cc.plot.pdf", zip, sample=all_samples, stage=STAGE, mark=MARK),
- # MACS2 peak calling
- expand("results_10M/macs2/{case}_vs_{control}_{stage}_{mark}_macs2_peaks.narrowPeak", zip, case=IPS, control=INPUTS, stage=STAGE, mark=MARK),
- expand("results_10M/macs2/{case}_vs_{control}_{stage}_{mark}_macs2_peaks.xls", zip, case=IPS, control=INPUTS, stage=STAGE, mark=MARK),
- expand("results_10M/macs2/{case}_vs_{control}_{stage}_{mark}_macs2_summits.bed", zip, case=IPS, control=INPUTS, stage=STAGE, mark=MARK),
- expand("results_10M/logs/{case}_vs_{control}_{stage}_{mark}_call_peaks_macs2.log", zip, case=IPS, control=INPUTS, stage=STAGE, mark=MARK),
- expand("results_10M/bwa/{case}_{stage}_{mark}_q30.sorted.dedup.bed", zip, case=IPS, stage=STAGE, mark=MARK),
- expand("results_10M/logs/{case}_{stage}_{mark}.bamToBed", zip, case=IPS, stage=STAGE, mark=MARK),
- expand("results_10M/qc/{case}_vs_{control}_{stage}_{mark}.frip.txt", zip, case=IPS, control=INPUTS, stage=STAGE, mark=MARK),
- directory("results_10M/macs2/pooled/"),
- "results_10M/macs2/H3K4me3_E10.5_overlap.narrowPeak",
- "results_10M/macs2/H3K4me3_E11.5_overlap.narrowPeak",
- "results_10M/macs2/H3K4me3_E12.5_overlap.narrowPeak",
- "results_10M/macs2/H3K4me3_E13.5_overlap.narrowPeak",
- "results_10M/macs2/H3K4me3_E14.5_overlap.narrowPeak",
- "results_10M/macs2/H3K4me3_E15.5_overlap.narrowPeak",
- "results_10M/qc/H3K4me3_E10.5_overlap.frip",
- "results_10M/qc/H3K4me3_E11.5_overlap.frip",
- "results_10M/qc/H3K4me3_E12.5_overlap.frip",
- "results_10M/qc/H3K4me3_E13.5_overlap.frip",
- "results_10M/qc/H3K4me3_E14.5_overlap.frip",
- "results_10M/qc/H3K4me3_E15.5_overlap.frip",
- # directory("results_10M/multiqc/multiqc_report_data/"),
- # "results_10M/multiqc/multiqc_report.html"
-
-# ===============================================================================================
-# 1. FASTQC
-# ===============================================================================================
-
-## Done separately because fastq and BAM files don't share file prefixes so it becomes very messy
-# without renaming everything
-
-# ===============================================================================================
-# 2. ALIGNMENT
-# > ENCODE alignment downloaded from ENCODE database
-# ===============================================================================================
-
-## Singled-end 36-50nt reads
-## bwa-aln used for aligning reads to the mouse genome (mm10 assembly)
-## Alignment command: bwa-aln -q 5 -l 32 -k 2 <reference_file> <read_file>
-## bwa version version 0.7.7
-
-## -q = parameter for read trimming
-## -l = read length
-## -k = Maximum edit distance in the seed
-
-# ===============================================================================================
-# 4. FILTERING
-# > remove unmapped, mate unmapped
-# > remove low MAPQ reads (-q 30)
-# > -F 1804
-# -Read unmapped
-# -Mate unmapped
-# -Not primary alignment
-# -Read fails platform/vendor quality checks
-# -Read is PCR or optical duplicate
-# > mark duplicates & remove duplicates (picard)
-# > re-filter, sort and index final BAM
-# ===============================================================================================
-
-rule filter:
- input:
- "results/bwa/{sample}_{stage}_{mark}.bam"
- output:
- "results/bwa/{sample}_{stage}_{mark}_q30.sorted.bam"
- log:
- "results/logs/{sample}_{stage}_{mark}.filter"
- shell:
- "samtools view -b -F 1804 -q 30 {input} | samtools sort -o {output} - 2> {log}"
-
-rule markDups:
- input:
- "results/bwa/{sample}_{stage}_{mark}_q30.sorted.bam"
- output:
- bam="results/bwa/{sample}_{stage}_{mark}_q30.dupmark.bam",
- dupQC="results/bwa/{sample}_{stage}_{mark}.dupmark.qc"
- log:
- "results/logs/{sample}_{stage}_{mark}.dedup"
- shell:
- "picard MarkDuplicates I={input} O={output.bam} \
- METRICS_FILE={output.dupQC} REMOVE_DUPLICATES=false ASSUME_SORTED=true 2> {log}"
-
-rule dedup:
- input:
- "results/bwa/{sample}_{stage}_{mark}_q30.dupmark.bam"
- output:
- "results/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam"
- log:
- "results/logs/{sample}_{stage}_{mark}.dedup"
- shell:
- "samtools view -F 1804 -b {input} | samtools sort -o {output} - 2> {log}"
-
-rule normalise10Mreads:
- input:
- "results/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam"
- output:
- "results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam"
- shell:
- """
- frac=$( samtools idxstats {input} | cut -f3 | awk 'BEGIN {total=0} {total += $1} END {frac=10000000/total; if (frac > 1) {print 1} else {print frac}}' )\
- samtools view -bs $frac {input} > {output}
- """
-
-rule indexBam:
- input:
- "results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam"
- output:
- "results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bai"
- log:
- "results_10M/logs/{sample}_{stage}_{mark}.indexBam"
- shell:
- "samtools index {input} {output} 2> {log}"
-
-rule mergeBAMreplicates:
- input:
- E10 = ["results_10M/bwa/ENCFF124UYX_E10.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF045IPK_E10.5_H3K4me3_q30.sorted.dedup.bam"],
- E11 = ["results_10M/bwa/ENCFF760QYZ_E11.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF717QDV_E11.5_H3K4me3_q30.sorted.dedup.bam"],
- E12 = ["results_10M/bwa/ENCFF182ZPF_E12.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF941QJZ_E12.5_H3K4me3_q30.sorted.dedup.bam"],
- E13 = ["results_10M/bwa/ENCFF485UDC_E13.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF124TAB_E13.5_H3K4me3_q30.sorted.dedup.bam"],
- E14 = ["results_10M/bwa/ENCFF724DMU_E14.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF665QBJ_E14.5_H3K4me3_q30.sorted.dedup.bam"],
- E15 = ["results_10M/bwa/ENCFF258KCR_E15.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF401BKM_E15.5_H3K4me3_q30.sorted.dedup.bam"],
- E10C = ["results_10M/bwa/ENCFF157KEH_E10.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF825AVI_E10.5_H3K4me3_q30.sorted.dedup.bam"],
- E11C = ["results_10M/bwa/ENCFF184CUE_E11.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF376FGM_E11.5_H3K4me3_q30.sorted.dedup.bam"],
- E12C = ["results_10M/bwa/ENCFF203JQV_E12.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF058AUT_E12.5_H3K4me3_q30.sorted.dedup.bam"],
- E13C = ["results_10M/bwa/ENCFF117QRC_E13.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF248PGK_E13.5_H3K4me3_q30.sorted.dedup.bam"],
- E14C = ["results_10M/bwa/ENCFF784ORI_E14.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF002HZV_E14.5_H3K4me3_q30.sorted.dedup.bam"],
- E15C = ["results_10M/bwa/ENCFF727QTS_E15.5_H3K4me3_q30.sorted.dedup.bam", "results_10M/bwa/ENCFF182XFG_E15.5_H3K4me3_q30.sorted.dedup.bam"],
- output:
- E10 = "results_10M/bwa/E10.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E11 = "results_10M/bwa/E11.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E12 = "results_10M/bwa/E12.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E13 = "results_10M/bwa/E13.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E14 = "results_10M/bwa/E14.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E15 = "results_10M/bwa/E15.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E10C = "results_10M/bwa/input_E10.5_H3K4me3_q30.sorted.dedup.bam",
- E11C = "results_10M/bwa/input_E11.5_H3K4me3_q30.sorted.dedup.bam",
- E12C = "results_10M/bwa/input_E12.5_H3K4me3_q30.sorted.dedup.bam",
- E13C = "results_10M/bwa/input_E13.5_H3K4me3_q30.sorted.dedup.bam",
- E14C = "results_10M/bwa/input_E14.5_H3K4me3_q30.sorted.dedup.bam",
- E15C = "results_10M/bwa/input_E15.5_H3K4me3_q30.sorted.dedup.bam"
- log:
- E10 = "results_10M/logs/E10.5_H3K4me3.mergeBAM",
- E11 = "results_10M/logs/E11.5_H3K4me3.mergeBAM",
- E12 = "results_10M/logs/E12.5_H3K4me3.mergeBAM",
- E13 = "results_10M/logs/E13.5_H3K4me3.mergeBAM",
- E14 = "results_10M/logs/E14.5_H3K4me3.mergeBAM",
- E15 = "results_10M/logs/E15.5_H3K4me3.mergeBAM",
- E10C = "results_10M/logs/input_E10.5_H3K4me3.mergeBAM",
- E11C = "results_10M/logs/input_E11.5_H3K4me3.mergeBAM",
- E12C = "results_10M/logs/input_E12.5_H3K4me3.mergeBAM",
- E13C = "results_10M/logs/input_E13.5_H3K4me3.mergeBAM",
- E14C = "results_10M/logs/input_E14.5_H3K4me3.mergeBAM",
- E15C = "results_10M/logs/input_E15.5_H3K4me3.mergeBAM"
- run:
- shell("samtools merge {output.E10} {input.E10} 2> {log.E10}")
- shell("samtools merge {output.E11} {input.E11} 2> {log.E11}")
- shell("samtools merge {output.E12} {input.E12} 2> {log.E12}")
- shell("samtools merge {output.E13} {input.E13} 2> {log.E13}")
- shell("samtools merge {output.E14} {input.E14} 2> {log.E14}")
- shell("samtools merge {output.E15} {input.E15} 2> {log.E15}")
- shell("samtools merge {output.E10C} {input.E10C} 2> {log.E10C}")
- shell("samtools merge {output.E11C} {input.E11C} 2> {log.E11C}")
- shell("samtools merge {output.E12C} {input.E12C} 2> {log.E12C}")
- shell("samtools merge {output.E13C} {input.E13C} 2> {log.E13C}")
- shell("samtools merge {output.E14C} {input.E14C} 2> {log.E14C}")
- shell("samtools merge {output.E15C} {input.E15C} 2> {log.E15C}")
-
-
-# ===============================================================================================
-# 4. ALIGNMENT QC
-# > SAMtools flagstat statistics
-# > CollectMultipleMetrics (picard)
-# > Compute Library Complexity (PreSeq)
-# ===============================================================================================
-
-rule mappingStats:
- input:
- a="results/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam",
- b="results/bwa/{sample}_{stage}_{mark}_q30.dupmark.bam",
- c="results/bwa/{sample}_{stage}_{mark}_q30.sorted.bam",
- d="results/bwa/{sample}_{stage}_{mark}.bam"
- output:
- a="results/qc/{sample}_{stage}_{mark}.dedup.flagstat.qc",
- b="results/qc/{sample}_{stage}_{mark}.dupmark.flagstat.qc",
- c="results/qc/{sample}_{stage}_{mark}.q30.flagstat.qc",
- d="results/qc/{sample}_{stage}_{mark}.unfiltered.flagstat.qc",
- run:
- shell("samtools flagstat {input.a} > {output.a}")
- shell("samtools flagstat {input.b} > {output.b}")
- shell("samtools flagstat {input.c} > {output.c}")
- shell("samtools flagstat {input.d} > {output.d}")
-
-
-rule sort_name:
- input:
- "results/bwa/{sample}_{stage}_{mark}_q30.dupmark.bam"
- output:
- tmp = "results/bwa/{sample}_{stage}_{mark}_q30.dupmark.tmp.bam"
- log:
- "results/logs/{sample}_{stage}_{mark}.pbc.sort"
- shell:
- "samtools sort -n {input} -o {output.tmp} 2> {log}"
-
-rule estimate_lib_complexity:
- input:
- "results/bwa/{sample}_{stage}_{mark}_q30.dupmark.tmp.bam"
- output:
- qc = "results/qc/{sample}_{stage}_{mark}.pbc.qc"
- log:
- "results/logs/{sample}_{stage}_{mark}.pbc"
- shell:
- """
- bedtools bamtobed -i {input} | grep -v 'chrM' \
- | awk 'BEGIN{{OFS="\\t"}}{{print $1,$2,$3,$6}}' \
- | sort | uniq -c \
- | awk 'BEGIN{{mt=0;m0=0;m1=0;m2=0}} ($1==1){{m1=m1+1}} ($1==2){{m2=m2+1}} \
- {{m0=m0+1}} {{mt=mt+$1}} END{{printf "%d\\t%d\\t%d\\t%d\\t%f\\t%f\\t%f\\n" ,mt,m0,m1,m2,m0/mt,m1/m0,m1/m2}}' \
- > {output.qc}
- """
-
-
-## convert to bed
-## print read 1 scaffold, read 1 start coordinate, read 2 scaffold, read 2 end coordinate, strand read 1, strand read 2
-## remove mitochondrial genome
-## sort by position and obtain uniq reads
-## Format of file
-## TotalReadPairs [tab] DistinctReadPairs [tab] OneReadPair [tab] TwoReadPairs [tab] NRF=Distinct/Total [tab] PBC1=OnePair/Distinct [tab] PBC2=OnePair/TwoPair
-
-
-# ===============================================================================================
-# 5. deepTools
-# > multiBAMsummary
-# > plotCorrelation
-# > plotFingerprint
-# > bamCoverage (read depth normalised bigWig files)
-# ===============================================================================================
-
-rule deeptools_summary:
- input:
- expand(["results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam"], zip, sample=all_samples, mark=MARK, stage=STAGE)
- output:
- sum="results_10M/qc/H3K4me3_multibamsum.npz",
- counts="results_10M/qc/H3K4me3_multibamsum.tab"
- threads: 32
- log:
- "results_10M/logs/H3K4me3_multisummary.deepTools"
- shell:
- " multiBamSummary bins \
- -p {threads} \
- -b {input} \
- --centerReads \
- -out {output.sum} \
- --outRawCounts {output.counts} 2> {log}"
-
-rule deeptools_correlation:
- input: "results_10M/qc/H3K4me3_multibamsum.npz"
- output:
- fig="results_10M/qc/H3K4me3_pearsoncor_multibamsum.pdf",
- matrix="results_10M/qc/H3K4me3_pearsoncor_multibamsum_matrix.txt"
- log:
- "results_10M/logs/H3K4me3_correlation.deepTools"
- shell:
- "plotCorrelation \
- --corData {input} \
- --plotFile {output.fig} \
- --outFileCorMatrix {output.matrix} \
- --corMethod pearson \
- --whatToPlot heatmap \
- --skipZeros \
- --plotNumbers \
- --colorMap RdYlBu 2> {log}"
-
-rule deeptools_coverage:
- input:
- bam = "results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam",
- bai = "results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bai"
- output:
- "results_10M/qc/{sample}_{stage}_{mark}.SeqDepthNorm.bw"
- log:
- "results_10M/logs/{sample}_{stage}_{mark}_coverage.deepTools"
- shell:
- "bamCoverage --bam {input.bam} --binSize 10 --normalizeUsing RPGC --effectiveGenomeSize 2308125349 --numberOfProcessors 4 -o {output} 2> {log}"
-
-rule deeptools_fingerprint:
- input:
- bam = expand(["results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam"], zip, sample=all_samples, stage=STAGE, mark = MARK),
- bai = expand(["results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bai"], zip, sample=all_samples, stage=STAGE, mark = MARK)
- output:
- fig="results_10M/qc/H3K4me3_multiBAM_fingerprint.pdf",
- metrics="results_10M/qc/H3K4me3_multiBAM_fingerprint_metrics.txt",
- rawcounts="results_10M/qc/H3K4me3_multiBAM_fingerprint_rawcounts.txt"
- threads: 32
- log:
- "results_10M/logs/fingerprint.deepTools"
- shell:
- "plotFingerprint -p {threads} \
- -b {input.bam} \
- --plotFile {output.fig} \
- --outQualityMetrics {output.metrics} \
- --outRawCounts {output.rawcounts} \
- --minMappingQuality 20 \
- --skipZeros \
- --centerReads 2> {log}"
-
-rule deeptools_plotCoverage:
- input:
- bam = expand(["results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bam"], zip, sample=all_samples, stage=STAGE, mark=MARK),
- bai = expand(["results_10M/bwa/{sample}_{stage}_{mark}_q30.sorted.dedup.bai"], zip, sample=all_samples, stage=STAGE, mark=MARK)
- output:
- fig="results_10M/qc/H3K4me3_plot_coverage.pdf",
- rawcounts="results_10M/qc/H3K4me3_plot_coverage_rawcounts.tab"
- log:
- "results_10M/logs/H3K4me3_plotCoverage.deepTools"
- shell:
- "plotCoverage --bamfiles {input.bam} --plotFile {output.fig} \
- -n 1000000 \
- --outRawCounts {output.rawcounts} \
- --ignoreDuplicates 2> {log}"
-
-
-# ===============================================================================================
-# 6. Cross-correlation scores - following ENCODE code
-# ===============================================================================================
-
-rule bamtobed_crossC:
- input:
- "results/bwa/{sample}_{stage}_{mark}_q30.dupmark.bam"
- output:
- tagAlign = "results/bed/{sample}_{stage}_{mark}_q30_SE.tagAlign.gz"
- shell:
- """
- bedtools bamtobed -i {input} | \
- awk 'BEGIN{{OFS="\\t"}}{{$4='N';$5='1000';print $0}}' |\
- gzip -nc > {output.tagAlign}
- """
-
-## Subsample 15 million reads for cross-correlation analysis
-## Estimate read length from first 100 reads
-rule subsample_aligned_reads:
- input:
- "results/bed/{sample}_{stage}_{mark}_q30_SE.tagAlign.gz"
- output:
- subsample = "results/bed/{sample}_{stage}_{mark}.filt.sample.15Mreads.SE.tagAlign.gz",
- tmp = "results/bed/{sample}_{stage}_{mark}_R1_trimmed_q30_SE.tagAlign.tmp"
- params:
- nreads= 15000000
- run:
- shell("""zcat {input} | shuf -n {params.nreads} --random-source=<(openssl enc -aes-256-ctr -pass pass:$(zcat -f {input} | wc -c) -nosalt </dev/zero 2>/dev/null) | gzip -nc > {output.subsample}""")
- shell("zcat {input} > {output.tmp}")
-
-# Determine exclusion range for fragment length estimation.
-## From bamPEFragmentSize (deepTools) average fragment length is ~220bp
-## See bamPEFragmentSize.histogram.pdf
-# Use a fixed lowerbound at -500.
-# Upperbound E
-#EXCLUSION_RANGE_MAX is
-# Histone ChIP-seq: max(read_len + 10, 100)
-# lowerbound is fixed at 500 for both
-
-
-rule cross_correlation_SSP:
- input:
- "results/bed/{sample}_{stage}_{mark}.filt.sample.15Mreads.SE.tagAlign.gz"
- output:
- CC_SCORES_FILE="results/qc/{sample}_{stage}_{mark}_filt_15Mreads.SE.cc.qc",
- CC_PLOT_FILE="results/qc/{sample}_{stage}_{mark}_filt_15Mreads.SE.cc.plot.pdf"
- log:
- "results/logs/{sample}_{stage}_{mark}_filt_15Mreads.SE.spp.log"
- params:
- EXCLUSION_RANGE_MIN=-500,
- EXCLUSION_RANGE_MAX=85
- shell:
- "Rscript scripts/run_spp.R -c={input} -savp={output.CC_PLOT_FILE} -out={output.CC_SCORES_FILE} -x={params.EXCLUSION_RANGE_MIN}:{params.EXCLUSION_RANGE_MAX} 2> {log}"
-
-
-# CC_SCORE FILE format
-# Filename <tab> numReads <tab> estFragLen <tab> corr_estFragLen <tab> PhantomPeak <tab> corr_phantomPeak <tab> argmin_corr <tab> min_corr <tab> phantomPeakCoef <tab> relPhantomPeakCoef <tab> QualityTag
-
-
-# ===============================================================================================
-# 7. Call peaks (MACS2)
-# ===============================================================================================
-
-rule call_peaks_macs2:
- input:
- control = "results_10M/bwa/{control}_{stage}_{mark}_q30.sorted.dedup.bam",
- case = "results_10M/bwa/{case}_{stage}_{mark}_q30.sorted.dedup.bam"
- output:
- "results_10M/macs2/{case}_vs_{control}_{stage}_{mark}_macs2_peaks.xls",
- "results_10M/macs2/{case}_vs_{control}_{stage}_{mark}_macs2_summits.bed",
- "results_10M/macs2/{case}_vs_{control}_{stage}_{mark}_macs2_peaks.narrowPeak",
- log:
- "results_10M/logs/{case}_vs_{control}_{stage}_{mark}_call_peaks_macs2.log"
- params:
- name = "{case}_vs_{control}_{stage}_{mark}_macs2",
- shell:
- " macs2 callpeak -f BAM -t {input.case} \
- -c {input.control} --keep-dup all \
- --outdir results_10M/macs2/ -p 0.01 \
- -n {params.name} \
- -g mm 2> {log} "
-
-rule call_peaks_macs2_pooled_replicates:
- input:
- E10 = "results_10M/bwa/E10.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E11 = "results_10M/bwa/E11.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E12 = "results_10M/bwa/E12.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E13 = "results_10M/bwa/E13.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E14 = "results_10M/bwa/E14.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E15 = "results_10M/bwa/E15.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E10C = "results_10M/bwa/input_E10.5_H3K4me3_q30.sorted.dedup.bam",
- E11C = "results_10M/bwa/input_E11.5_H3K4me3_q30.sorted.dedup.bam",
- E12C = "results_10M/bwa/input_E12.5_H3K4me3_q30.sorted.dedup.bam",
- E13C = "results_10M/bwa/input_E13.5_H3K4me3_q30.sorted.dedup.bam",
- E14C = "results_10M/bwa/input_E14.5_H3K4me3_q30.sorted.dedup.bam",
- E15C = "results_10M/bwa/input_E15.5_H3K4me3_q30.sorted.dedup.bam"
- output:
- directory("results_10M/macs2/pooled/")
- log:
- E10 = "results_10M/qc/E10.5_H3K4me3.pooled.macs2",
- E11 = "results_10M/qc/E11.5_H3K4me3.pooled.macs2",
- E12 = "results_10M/qc/E12.5_H3K4me3.pooled.macs2",
- E13 = "results_10M/qc/E13.5_H3K4me3.pooled.macs2",
- E14 = "results_10M/qc/E14.5_H3K4me3.pooled.macs2",
- E15 = "results_10M/qc/E15.5_H3K4me3.pooled.macs2"
- params:
- E10 = "E10.5_H3K4me3.pooled.macs2",
- E11 = "E11.5_H3K4me3.pooled.macs2",
- E12 = "E12.5_H3K4me3.pooled.macs2",
- E13 = "E13.5_H3K4me3.pooled.macs2",
- E14 = "E14.5_H3K4me3.pooled.macs2",
- E15 = "E15.5_H3K4me3.pooled.macs2"
- run:
- shell("macs2 callpeak -f BAM -t {input.E10} \
- -c {input.E10C} --keep-dup all \
- --outdir results_10M/macs2/ -p 0.01 \
- -n {params.E10} \
- -g mm 2> {log.E10}" )
- shell("macs2 callpeak -f BAM -t {input.E11} \
- -c {input.E11C} --keep-dup all \
- --outdir results_10M/macs2/ -p 0.01 \
- -n {params.E11} \
- -g mm 2> {log.E11}" )
- shell("macs2 callpeak -f BAM -t {input.E12} \
- -c {input.E12C} --keep-dup all \
- --outdir results_10M/macs2/ -p 0.01 \
- -n {params.E12} \
- -g mm 2> {log.E12}" )
- shell("macs2 callpeak -f BAM -t {input.E13} \
- -c {input.E13C} --keep-dup all \
- --outdir results_10M/macs2/ -p 0.01 \
- -n {params.E13} \
- -g mm 2> {log.E13}" )
- shell("macs2 callpeak -f BAM -t {input.E14} \
- -c {input.E14C} --keep-dup all \
- --outdir results_10M/macs2/ -p 0.01 \
- -n {params.E14} \
- -g mm 2> {log.E14}" )
- shell("macs2 callpeak -f BAM -t {input.E15} \
- -c {input.E15C} --keep-dup all \
- --outdir results_10M/macs2/ -p 0.01 \
- -n {params.E15} \
- -g mm 2> {log.E15}" )
-
-# ===============================================================================================
-# 8. Peak QC
-# > narrow peak counts
-# > fraction of reads in peaks (convert BAM to bed first then do intersect with peaks)
-# ===============================================================================================
-
-## Convert BAM to tagAlign file for calculating FRiP QC metric (Fraction of reads in peaks)
-rule bamToBed:
- input:
- "results_10M/bwa/{case}_{stage}_{mark}_q30.sorted.dedup.bam"
- output:
- "results_10M/bwa/{case}_{stage}_{mark}_q30.sorted.dedup.bed"
- log:
- "results_10M/logs/{case}_{stage}_{mark}.bamToBed"
- shell:
- "samtools sort -n {input} | bedtools bamtobed -i - > {output}"
-
-# ## Fraction of reads in peaks
-rule frip:
- input:
- bed = "results_10M/bwa/{case}_{stage}_{mark}_q30.sorted.dedup.bed",
- peak = "results_10M/macs2/{case}_vs_{control}_{stage}_{mark}_macs2_peaks.narrowPeak"
- output:
- "results_10M/qc/{case}_vs_{control}_{stage}_{mark}.frip.txt"
- shell:
- "python2.7 scripts/encode_frip.py {input.bed} {input.peak} > {output}"
-
-# ===============================================================================================
-# 9. Create consensus peaksets for replicates
-# ===============================================================================================
-
-# Based on ENCODE `overlap_peaks.py` - recommended for histone marks.
-# Need to pool peaks first for each replicate
-
-rule overlap_peaks:
- input:
- E10= ["results_10M/macs2/ENCFF124UYX_vs_ENCFF157KEH_E10.5_H3K4me3_macs2_peaks.narrowPeak", "results_10M/macs2/ENCFF045IPK_vs_ENCFF825AVI_E10.5_H3K4me3_macs2_peaks.narrowPeak"],
- E11= ["results_10M/macs2/ENCFF760QYZ_vs_ENCFF184CUE_E11.5_H3K4me3_macs2_peaks.narrowPeak", "results_10M/macs2/ENCFF717QDV_vs_ENCFF376FGM_E11.5_H3K4me3_macs2_peaks.narrowPeak"],
- E12= ["results_10M/macs2/ENCFF941QJZ_vs_ENCFF058AUT_E12.5_H3K4me3_macs2_peaks.narrowPeak", "results_10M/macs2/ENCFF182ZPF_vs_ENCFF203JQV_E12.5_H3K4me3_macs2_peaks.narrowPeak"],
- E13= ["results_10M/macs2/ENCFF485UDC_vs_ENCFF117QRC_E13.5_H3K4me3_macs2_peaks.narrowPeak", "results_10M/macs2/ENCFF124TAB_vs_ENCFF248PGK_E13.5_H3K4me3_macs2_peaks.narrowPeak"],
- E14= ["results_10M/macs2/ENCFF665QBJ_vs_ENCFF002HZV_E14.5_H3K4me3_macs2_peaks.narrowPeak", "results_10M/macs2/ENCFF724DMU_vs_ENCFF784ORI_E14.5_H3K4me3_macs2_peaks.narrowPeak"],
- E15= ["results_10M/macs2/ENCFF401BKM_vs_ENCFF182XFG_E15.5_H3K4me3_macs2_peaks.narrowPeak", "results_10M/macs2/ENCFF258KCR_vs_ENCFF727QTS_E15.5_H3K4me3_macs2_peaks.narrowPeak"],
- E10_pooled="results_10M/macs2/E10.5_H3K4me3.pooled.macs2_peaks.narrowPeak",
- E11_pooled="results_10M/macs2/E11.5_H3K4me3.pooled.macs2_peaks.narrowPeak",
- E12_pooled="results_10M/macs2/E12.5_H3K4me3.pooled.macs2_peaks.narrowPeak",
- E13_pooled="results_10M/macs2/E13.5_H3K4me3.pooled.macs2_peaks.narrowPeak",
- E14_pooled="results_10M/macs2/E14.5_H3K4me3.pooled.macs2_peaks.narrowPeak",
- E15_pooled="results_10M/macs2/E15.5_H3K4me3.pooled.macs2_peaks.narrowPeak",
- output:
- E10= "results_10M/macs2/H3K4me3_E10.5_overlap.narrowPeak",
- E11= "results_10M/macs2/H3K4me3_E11.5_overlap.narrowPeak",
- E12= "results_10M/macs2/H3K4me3_E12.5_overlap.narrowPeak",
- E13= "results_10M/macs2/H3K4me3_E13.5_overlap.narrowPeak",
- E14= "results_10M/macs2/H3K4me3_E14.5_overlap.narrowPeak",
- E15= "results_10M/macs2/H3K4me3_E15.5_overlap.narrowPeak"
- run:
- shell("python2.7 scripts/overlap_peaks.py {input.E10} {input.E10_pooled} {output.E10}")
- shell("python2.7 scripts/overlap_peaks.py {input.E11} {input.E11_pooled} {output.E11}")
- shell("python2.7 scripts/overlap_peaks.py {input.E12} {input.E12_pooled} {output.E12}")
- shell("python2.7 scripts/overlap_peaks.py {input.E13} {input.E13_pooled} {output.E13}")
- shell("python2.7 scripts/overlap_peaks.py {input.E14} {input.E14_pooled} {output.E14}")
- shell("python2.7 scripts/overlap_peaks.py {input.E15} {input.E15_pooled} {output.E15}")
-
-rule overlap_frip:
- input:
- E10bam = "results_10M/bwa/E10.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E11bam = "results_10M/bwa/E11.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E12bam = "results_10M/bwa/E12.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E13bam = "results_10M/bwa/E13.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E14bam = "results_10M/bwa/E14.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E15bam = "results_10M/bwa/E15.5_H3K4me3_q30.sorted.pooled.dedup.bam",
- E10bed = "results_10M/macs2/H3K4me3_E10.5_overlap.narrowPeak",
- E11bed = "results_10M/macs2/H3K4me3_E11.5_overlap.narrowPeak",
- E12bed = "results_10M/macs2/H3K4me3_E12.5_overlap.narrowPeak",
- E13bed = "results_10M/macs2/H3K4me3_E13.5_overlap.narrowPeak",
- E14bed = "results_10M/macs2/H3K4me3_E14.5_overlap.narrowPeak",
- E15bed = "results_10M/macs2/H3K4me3_E15.5_overlap.narrowPeak"
- output:
- E10bed = "results_10M/qc/H3K4me3_E10.5_overlap.frip",
- E11bed = "results_10M/qc/H3K4me3_E11.5_overlap.frip",
- E12bed = "results_10M/qc/H3K4me3_E12.5_overlap.frip",
- E13bed = "results_10M/qc/H3K4me3_E13.5_overlap.frip",
- E14bed = "results_10M/qc/H3K4me3_E14.5_overlap.frip",
- E15bed = "results_10M/qc/H3K4me3_E15.5_overlap.frip"
- run:
- shell("python2.7 scripts/encode_frip.py {input.E10bam} {input.E10bed} > {output.E10bed}")
- shell("python2.7 scripts/encode_frip.py {input.E11bam} {input.E11bed} > {output.E11bed}")
- shell("python2.7 scripts/encode_frip.py {input.E12bam} {input.E12bed} > {output.E12bed}")
- shell("python2.7 scripts/encode_frip.py {input.E13bam} {input.E13bed} > {output.E13bed}")
- shell("python2.7 scripts/encode_frip.py {input.E14bam} {input.E14bed} > {output.E14bed}")
- shell("python2.7 scripts/encode_frip.py {input.E15bam} {input.E15bed} > {output.E15bed}")
-
-
-
-# ===============================================================================================
-# 10. Combine all QC into multiqc output
-# ===============================================================================================
-#
-# rule multiqc:
-# input:
- # fastqc
- # expand("results_10M/fastqc/{sample}_R1_fastqc.html", sample=all_samples),
- # expand("results_10M/fastqc/{sample}_R2_fastqc.html", sample=all_samples),
- # expand("results_10M/fastqc/{sample}_R1_fastqc.zip", sample=all_samples),
- # expand("results_10M/fastqc/{sample}_R2_fastqc.zip", sample=all_samples),
- # # bwa
- # expand("results_10M/logs/{sample}.align", sample=all_samples),
- # expand("results_10M/logs/{sample}.flagstat.qc", sample=all_samples),
- # # preseq
- # expand("results_10M/preseq/{sample}.ccurve.txt", sample=all_samples),
- # # picard
- # expand("results_10M/picard/{sample}_est_lib_complex_metrics.txt", sample=all_samples),
- # # deepTools
- # "results_10M/deeptools/multibamsum.npz",
- # "results_10M/deeptools/multibamsum.tab",
- # "results_10M/deeptools/pearsoncor_multibamsum.pdf",
- # "results_10M/deeptools/pearsoncor_multibamsum_matrix.txt",
- # expand("results_10M/deeptools/{sample}.SeqDepthNorm.bw", sample=all_samples),
- # "results_10M/deeptools/multiBAM_fingerprint.pdf",
- # "results_10M/deeptools/multiBAM_fingerprint_metrics.txt",
- # "results_10M/deeptools/multiBAM_fingerprint_rawcounts.txt",
- # "results_10M/deeptools/plot_coverage.pdf",
- # "results_10M/deeptools/plot_coverage_rawcounts.tab",
- # "results_10M/deeptools/bamPEFragmentSize_hist.pdf",
- # "results_10M/deeptools/bamPEFragmentSize_rawcounts.tab",
- # # phantomPeaks
- # expand("results_10M/phantomPeaks/{sample}.spp.pdf", sample = IPS),
- # expand("results_10M/phantomPeaks/{sample}.spp.Rdata", sample = IPS),
- # expand("results_10M/phantomPeaks/{sample}.spp.out", sample = IPS),
- # # macs2
- # expand("results_10M/macs2/{case}_vs_{control}_macs2_peaks.narrowPeak", zip, case=IPS, control=INPUTS),
- # expand("results_10M/macs2/{case}_vs_{control}_macs2_peaks.xls", zip, case=IPS, control=INPUTS),
- # expand("results_10M/macs2/{case}_vs_{control}_macs2_summits.bed", zip, case=IPS, control=INPUTS),
- # expand("results_10M/macs2/{case}-vs-{control}-narrowpeak-count_mqc.json", zip, case=IPS, control=INPUTS),
- # expand("results_10M/frip/{case}.frip.txt", case=IPS)
- # params:
- # "results_10M/fastqc/",
- # "results_10M/bwa/",
- # "results_10M/logs/",
- # "results_10M/deeptools/",
- # "results_10M/macs2/",
- # "results_10M/phantomPeaks/",
- # "results_10M/frip/",
- # "results_10M/picard/"
- # output:
- # directory("results_10M/multiqc/multiqc_report_data/"),
- # "results_10M/multiqc/multiqc_report.html",
- # log:
- # "results_10M/logs/multiqc.log"
- # shell:
- # "multiqc -f {params} -m fastqc -m samtools -m picard -m preseq -m deeptools -m phantompeakqualtools -m macs2 -o results_10M/multiqc/ \
- # -n multiqc_report.html 2> {log}"
diff --git a/mouse-chipseq/Snakefile_H3K4me3 b/mouse-chipseq/Snakefile_H3K4me3
index f35f705..bce0f86 100755
--- a/mouse-chipseq/Snakefile_H3K4me3
+++ b/mouse-chipseq/Snakefile_H3K4me3
@@ -9,7 +9,7 @@
# 7. cross correlation (SPP)
# 8. Call narrow peaks (MACS2)
# 9. Create consensus peaksets
- # 10. Present QC for raw read, alignment, peak-calling in MultiQC
+ # 10. Present QC for raw read, alignment, peak-calling in MultiQC (work in progress)
configfile: "configs/config_H3K4me3.yaml"
@@ -274,33 +274,33 @@ rule mappingStats:
#shell("samtools flagstat {input.d} > {output.d}")
-# rule sort_name:
-# input:
-# "results_10M/bwa/{sample}_{stage}_{mark}_q30.dupmark.bam"
-# output:
-# tmp = "results_10M/bwa/{sample}_{stage}_{mark}_q30.dupmark.tmp.bam"
-# log:
-# "results_10M/logs/{sample}_{stage}_{mark}.pbc.sort"
-# run:
-# shell("samtools sort -n {input} -o {output.tmp} 2> {log}")
-#
-# rule estimate_lib_complexity:
-# input:
-# "results_10M/bwa/{sample}_{stage}_{mark}_q30.dupmark.tmp.bam"
-# output:
-# qc = "results_10M/qc/{sample}_{stage}_{mark}.pbc.qc",
-# log:
-# "results_10M/logs/{sample}_{stage}_{mark}.pbc"
-# shell:
-# """
-# bedtools bamtobed -i {input} \
-# | awk 'BEGIN{{OFS="\\t"}}{{print $1,$2,$4,$6}}' \
-# | sort | uniq -c \
-# | awk 'BEGIN{{mt=0;m0=0;m1=0;m2=0}} ($1==1){{m1=m1+1}} ($1==2){{m2=m2+1}} \
-# {{m0=m0+1}} {{mt=mt+$1}} END{{printf "%d\\t%d\\t%d\\t%d\\t%f\\t%f\\t%f\\n" ,mt,m0,m1,m2,m0/mt,m1/m0,m1/m2}}' \
-# > {output.qc}
-# """
-#
+rule sort_name:
+ input:
+ "results_10M/bwa/{sample}_{stage}_{mark}_q30.dupmark.bam"
+ output:
+ tmp = "results_10M/bwa/{sample}_{stage}_{mark}_q30.dupmark.tmp.bam"
+ log:
+ "results_10M/logs/{sample}_{stage}_{mark}.pbc.sort"
+ run:
+ shell("samtools sort -n {input} -o {output.tmp} 2> {log}")
+
+rule estimate_lib_complexity:
+ input:
+ "results_10M/bwa/{sample}_{stage}_{mark}_q30.dupmark.tmp.bam"
+ output:
+ qc = "results_10M/qc/{sample}_{stage}_{mark}.pbc.qc",
+ log:
+ "results_10M/logs/{sample}_{stage}_{mark}.pbc"
+ shell:
+ """
+ bedtools bamtobed -i {input} \
+ | awk 'BEGIN{{OFS="\\t"}}{{print $1,$2,$4,$6}}' \
+ | sort | uniq -c \
+ | awk 'BEGIN{{mt=0;m0=0;m1=0;m2=0}} ($1==1){{m1=m1+1}} ($1==2){{m2=m2+1}} \
+ {{m0=m0+1}} {{mt=mt+$1}} END{{printf "%d\\t%d\\t%d\\t%d\\t%f\\t%f\\t%f\\n" ,mt,m0,m1,m2,m0/mt,m1/m0,m1/m2}}' \
+ > {output.qc}
+ """
+
## convert to bedPE
## print read 1 scaffold, read 1 start coordinate, read 2 scaffold, read 2 end coordinate, strand read 1, strand read 2
## remove mitochondrial genome
--
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