diff --git a/dunnart/Snakefile b/dunnart/Snakefile
index 41cccb94e461e1e483474d7db459605b66cf4fd3..0ae59b3a962cfede76ef8e3be3653d35e95cad72 100644
--- a/dunnart/Snakefile
+++ b/dunnart/Snakefile
@@ -57,15 +57,19 @@ rule all:
expand("results/bowtie2/{sample}_PPq30.sorted.dupmark.bam", sample=all_samples),
expand("results/bowtie2/{sample}_PPq30.sorted.dedup.bam", sample=all_samples),
expand("results/bowtie2/{sample}_PPq30.sorted.dedup.bai", sample=all_samples),
- expand("results/qc/{sample}.sorted.flagstat.qc", sample=all_samples),
+ expand("results/qc/{sample}.unfiltered.flagstat.qc", sample=all_samples),
expand("results/qc/{sample}.dedup.flagstat.qc", sample=all_samples),
expand("results/qc/{sample}.dupmark.flagstat.qc", sample=all_samples),
expand("results/qc/{sample}.PPq30.flagstat.qc", sample=all_samples),
expand("results/qc/{sample}.ccurve.txt", sample=all_samples),
+ expand("results/qc/{sample}.extrap.txt", sample=all_samples),
+ expand("logs/{sample}.ccurve.preseq", sample=all_samples),
+ expand("logs/{sample}.extrap.preseq", sample=all_samples),
expand("results/qc/{sample}_est_lib_complex_metrics.txt", sample=all_samples),
expand("logs/{sample}.picardLibComplexity", sample=all_samples),
expand("results/qc/{sample}.pbc.qc", sample=all_samples),
expand("results/bowtie2/{sample}_PPq30.sorted.tmp.bam",sample=all_samples),
+ expand("results/bowtie2/{sample}_PPq30.sorted.dupmark_downSampled.bam",sample=all_samples),
"results/qc/multibamsum.npz",
"results/qc/multibamsum.tab",
"results/qc/pearsoncor_multibamsum.png",
@@ -76,21 +80,22 @@ rule all:
"results/qc/multiBAM_fingerprint_rawcounts.txt",
"results/qc/bamPEFragmentSize_hist.png",
"results/qc/bamPEFragmentSize_rawcounts.tab",
- expand("results/qc/{sample}_R1_trimmed.flagstat.qc", sample=all_samples),
expand("results/bowtie2/{sample}_R1_trimmed_q30.bam", sample=all_samples),
- expand("{sample}_filt_15Mreads.SE.cc.qc", sample=all_samples),
- expand("{sample}_filt_15Mreads.SE.cc.plot.pdf", sample=all_samples)
- # expand("results/macs2/{case}_vs_{control}_macs2_peaks.narrowPeak", zip, case=IPS, control=INPUTS),
- # expand("results/macs2/{case}_vs_{control}_macs2_peaks.xls", zip, case=IPS, control=INPUTS),
- # expand("results/macs2/{case}_vs_{control}_macs2_summits.bed", zip, case=IPS, control=INPUTS),
- # expand("results/qxc{case}-vs-{control}-narrowpeak-count_mqc.json", zip, case=IPS, control=INPUTS),
- # expand("results/bowtie2/{case}.bedpe", case=IPS),
- # expand("logs/{case}.bamToBed", case=IPS),
- # expand("results/qc/{case}_vs_{control}.frip.txt", case=IPS, control=INPUTS)
- # "results/macs2/H3K4me3_pooled_macs2_peaks.narrowPeak",
- # "results/macs2/H3K27ac_pooled_macs2_peaks.narrowPeak",
- # "results/macs2/H3K4me3_overlap.narrowPeak",
- # "results/macs2/H3K27ac_overlap.narrowPeak",
+ expand("logs/{sample}_filt_15Mreads.SE.spp.log", sample=all_samples),
+ # expand("results/qc/{sample}_readlength.txt", sample=all_samples),
+ expand("results/qc/{sample}_filt_15Mreads.SE.cc.qc", sample=all_samples),
+ expand("results/qc/{sample}_filt_15Mreads.SE.cc.plot.pdf", sample=all_samples),
+ expand("results/macs2/{case}_vs_{control}_macs2_peaks.narrowPeak", zip, case=IPS, control=INPUTS),
+ expand("results/macs2/{case}_vs_{control}_macs2_peaks.xls", zip, case=IPS, control=INPUTS),
+ expand("results/macs2/{case}_vs_{control}_macs2_summits.bed", zip, case=IPS, control=INPUTS),
+ expand("results/qc/{case}-vs-{control}-narrowpeak-count_mqc.json", zip, case=IPS, control=INPUTS),
+ expand("results/bowtie2/{case}.bedpe", case=IPS),
+ expand("logs/{case}.bamToBed", case=IPS),
+ expand("results/qc/{case}_vs_{control}.frip.txt", case=IPS, control=INPUTS),
+ "results/macs2/H3K4me3_pooled_macs2_peaks.narrowPeak",
+ "results/macs2/H3K27ac_pooled_macs2_peaks.narrowPeak",
+ "results/macs2/H3K4me3_overlap.narrowPeak",
+ "results/macs2/H3K27ac_overlap.narrowPeak",
# directory("results/multiqc/multiqc_report_data/"),
# "results/multiqc/multiqc_report.html"
# ===============================================================================================
@@ -129,19 +134,18 @@ rule align:
"bowtie2 --threads 8 -q -X 2000 --very-sensitive -x {params.index} -1 {input.R1} -2 {input.R2} \
| samtools view -u -h - | samtools sort -o {output} - 2> {log}"
-
# ===============================================================================================
# 3. FILTERING
# > remove unmapped, mate unmapped
# > remove low MAPQ reads (-q 30 (ENCODE))
# > keep proper pairs (-f)
+# > mark duplicates (picard)
# > -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)
# > index final position sorted BAM
# ===============================================================================================
@@ -153,17 +157,7 @@ rule filter:
log:
"logs/{sample}.dedup"
shell:
- "samtools view -b -q 30 -F 1804 -f 2 {input} | samtools sort -o {output} - 2> {log}"
-
-rule indexUnfiltBam:
- input:
- "results/bowtie2/{sample}.sorted.bam"
- output:
- "results/bowtie2/{sample}.sorted.bai"
- log:
- "logs/{sample}.indexUnfiltBam"
- shell:
- "samtools index -c {input} {output} 2> {log}"
+ "samtools view -b -F 1804 -q 30 -f 2 {input} | samtools sort -o {output} - 2> {log}"
rule markDups:
input:
@@ -175,7 +169,7 @@ rule markDups:
"logs/{sample}.dupmark"
shell:
"picard MarkDuplicates I={input} O={output.bam} \
- METRICS_FILE={output.dupQC} REMOVE_DUPLICATES=false ASSUME_SORTED=true 2> {log}"
+ METRICS_FILE={output.dupQC} REMOVE_DUPLICATES=FALSE ASSUME_SORTED=true 2> {log}"
rule dedup:
input:
@@ -202,6 +196,7 @@ rule indexBam:
# > SAMtools flagstat statistics
# > Compute Library Complexity (PreSeq)
# > picard library complexity
+# > ENCODE library complexity
# ===============================================================================================
rule mappingStats:
@@ -221,31 +216,52 @@ rule mappingStats:
shell("samtools flagstat {input.c} > {output.c}")
shell("samtools flagstat {input.d} > {output.d}")
-rule preseq:
+rule downsample_bam:
input:
- "results/bowtie2/{sample}.sorted.bam"
+ "results/bowtie2/{sample}_PPq30.sorted.dupmark.bam"
output:
- "results/qc/{sample}.ccurve.txt"
+ "results/bowtie2/{sample}_PPq30.sorted.dupmark_downSampled.bam"
log:
- "logs/{sample}.preseq"
+ "logs/{sample}.downsample"
shell:
- "preseq lc_extrap -v -output {output} -pe -bam {input} 2> {log}"
+ "picard DownsampleSam I={input} O={output} P=0.35 \
+ 2> {log}"
+
+# A-1_dupmark = 87165286
+# B-1_dupmark = 72303316
+# A-2_dupmark = 62678360
+# A-3_dupmark = 86503078
+# B-2_dupmark = 67048994
+# B-3_dupmark = 66615704
+
+rule preseq:
+ input:
+ "results/bowtie2/{sample}_PPq30.sorted.dupmark.bam"
+ output:
+ ccurve = "results/qc/{sample}.ccurve.txt",
+ extrap = "results/qc/{sample}.extrap.txt"
+ log:
+ ccurve = "logs/{sample}.ccurve.preseq",
+ extrap = "logs/{sample}.extrap.preseq"
+ run:
+ shell("preseq lc_extrap -v -output {output.extrap} -pe -bam {input} 2> {log.extrap}")
+ shell("preseq c_curve -v -output {output.ccurve} -pe -bam {input} 2> {log.ccurve}")
rule get_picard_complexity_metrics:
input:
- "results/bowtie2/{sample}.sorted.bam"
+ "results/bowtie2/{sample}_PPq30.sorted.dupmark.bam"
output:
"results/qc/{sample}_est_lib_complex_metrics.txt"
log:
- "logs/{sample}.picardLibComplexity"
+ "logs/{sample}.downSampled.picardLibComplexity"
shell:
"picard -Xmx6G EstimateLibraryComplexity INPUT={input} OUTPUT={output} USE_JDK_DEFLATER=TRUE USE_JDK_INFLATER=TRUE VERBOSITY=ERROR"
rule sort_name:
input:
- "results/bowtie2/{sample}_PPq30.sorted.dupmark.bam"
+ "results/bowtie2/{sample}_PPq30.sorted.dupmark_downSampled.bam"
output:
- tmp = "results/bowtie2/{sample}_PPq30.sorted.dupmark.tmp.bam"
+ tmp = "results/bowtie2/{sample}_PPq30.sorted.dupmark_downSampled.tmp.bam"
log:
"logs/{sample}.pbc.sort"
run:
@@ -253,7 +269,7 @@ rule sort_name:
rule estimate_lib_complexity:
input:
- "results/bowtie2/{sample}_PPq30.sorted.dupmark.tmp.bam"
+ "results/bowtie2/{sample}_PPq30.sorted.dupmark_downSampled.tmp.bam"
output:
qc = "results/qc/{sample}.pbc.qc",
log:
@@ -262,7 +278,7 @@ rule estimate_lib_complexity:
"""
bedtools bamtobed -bedpe -i {input} \
| awk 'BEGIN{{OFS="\\t"}}{{print $1,$2,$4,$6,$9,$10}}' \
- | grep -v 'chrM' | sort | uniq -c \
+ | 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+$1}} END{{printf "%d\\t%d\\t%d\\t%d\\t%f\\t%f\\t%f\\n" ,mt,m0,m1,m2,m0/mt,m1/m0,m1/m2}}' \
> {output.qc}
@@ -271,7 +287,7 @@ rule estimate_lib_complexity:
## convert to bedPE
## print read 1 scaffold, read 1 start coordinate, read 2 scaffold, read 2 end coordinate, strand read 1, strand read 2
## remove mitochondrial genome
-## sort by position and obtain uniq counts
+## sort by position and obtain uniq reads
## Format of file
## TotalReadPairs [tab] DistinctReadPairs [tab] OneReadPair [tab] TwoReadPairs [tab] NRF=Distinct/Total [tab] PBC1=OnePair/Distinct [tab] PBC2=OnePair/TwoPair
@@ -323,8 +339,8 @@ rule deeptools_correlation:
rule deeptools_coverage:
input:
- bam ="results/bowtie2/{sample}.sorted.bam",
- bai ="results/bowtie2/{sample}.sorted.bai"
+ bam ="results/bowtie2/{sample}_PPq30.sorted.dedup.bam",
+ bai ="results/bowtie2/{sample}_PPq30.sorted.dedup.bai"
output:
"results/qc/{sample}.SeqDepthNorm.bw"
log:
@@ -411,12 +427,10 @@ rule filter_sort_trimmed_alignment:
input:
"results/bowtie2/{sample}_R1_trimmed.bam"
output:
- flagstat = "results/qc/{sample}_R1_trimmed.flagstat.qc",
bam = "results/bowtie2/{sample}_R1_trimmed_q30.bam"
log:
"logs/{sample}_align_trimmed_read1_filter.log"
run:
- shell("samtools sort -n {input} -O SAM | SAMstats --sorted_sam_file - --outf {output.flagstat}")
shell("samtools view -F 1804 -q 30 -b {input} -o {output.bam}")
rule bamtobed_crossC:
@@ -442,9 +456,8 @@ rule subsample_aligned_reads:
params:
nreads= 15000000
run:
- shell("""zcat {input} | grep -v 'chrM' | shuf -n {params.nreads} --random-source=<(openssl enc -aes-256-ctr -pass pass:$(zcat -f {input} | wc -c) -nosalt </dev/zero 2>/dev/null) | gzip -nc > {output.subsample}""")
+ shell("""zcat {input} | 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}")
- shell("""READ_LEN=\$(head -n 100 {output.tmp} | awk 'function abs(v) {{return v < 0 ? -v : v}} BEGIN{{sum=0}} {{sum+=abs($3-$2)}} END{{print int(sum/NR)}}')""")
# Determine exclusion range for fragment length estimation.
# Use a fixed lowerbound at -500.
@@ -457,20 +470,24 @@ rule cross_correlation_SSP:
input:
"results/bed/{sample}.filt.sample.15Mreads.SE.tagAlign.gz"
output:
- CC_SCORES_FILE="{sample}_filt_15Mreads.SE.cc.qc",
- CC_PLOT_FILE="{sample}_filt_15Mreads.SE.cc.plot.pdf"
+ CC_SCORES_FILE="results/qc/{sample}_filt_15Mreads.SE.cc.qc",
+ CC_PLOT_FILE="results/qc/{sample}_filt_15Mreads.SE.cc.plot.pdf"
+ log:
+ "logs/{sample}_filt_15Mreads.SE.spp.log"
params:
EXCLUSION_RANGE_MIN=-500,
- EXCLUSION_RANGE_MAX=110
- run:
- shell("Rscript scripts/run_spp.R -c={input} -filtchr=chrM -savp={output.CC_PLOT_FILE} -out={output.CC_SCORES_FILE} -x={params.EXCLUSION_RANGE_MIN}:{params.EXCLUSION_RANGE_MAX} 2> {log}")
+ EXCLUSION_RANGE_MAX=230
+ 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)
-# # ===============================================================================================
+# 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)
+# ===============================================================================================
# peak calling for pair-end peaks
# effective genome size for dunnart genome calculated with khmer program (see README.md)
@@ -494,11 +511,11 @@ rule call_peaks_macs2:
-n {params.name} \
-g 2740338543 -B 2> {log} "
-# # # ===============================================================================================
-# # # 8. Peak QC
-# # # > narrow peak counts
-# # # > fraction of reads in peaks (convert BAM to bed first then do intersect with peaks)
-# # # ===============================================================================================
+# ===============================================================================================
+# 8. Peak QC
+# > narrow peak counts
+# > fraction of reads in peaks (convert BAM to bed first then do intersect with peaks)
+# ===============================================================================================
# peak counts in a format that multiqc can handle
rule get_narrow_peak_counts_for_multiqc:
@@ -537,9 +554,9 @@ rule frip:
"python2.7 scripts/encode_frip.py {input.bed} {input.peak} > {output}"
-# # ===============================================================================================
-# # 9. Create consensus peaksets for replicates
-# # ===============================================================================================
+# ===============================================================================================
+# 9. Create consensus peaksets for replicates
+# ===============================================================================================
# Based on ENCODE `overlap_peaks.py` - recommended for histone marks.
# Need to pool peaks first for each replicate
@@ -577,48 +594,23 @@ rule overlap_peaks_H3K27ac:
shell:
"python2.7 scripts/overlap_peaks.py {input.peak1} {input.peak2} {input.pooled} {output}"
-#rule consensus_peaks:
-
-
-#rule enhancer_promoter_peaks:
-# enhancer = peaks with only H3K27ac
-# Promoter = peaks with both H3K27ac & H3K4me3, or just H3K4me3
-# Make a bargraph with the amounts for each
-
-
-# # ===============================================================================================
-# # 10. QC on replicate peaks
-# # ===============================================================================================
-#
-# # FRiP for overlapped peaks
-# rule frip:
-# input:
-# bed = "results/bowtie2/{case}.bedpe",
-# peak = "results/macs2/{}_macs2_peaks.narrowPeak"
-# output:
-# "results/qc/{case}.frip.txt"
-# shell:
-# "python2.7 scripts/encode_frip.py {input.bed} {input.peak} > {output}"
-#
-#
-# # ===============================================================================================
-# # 11. Combine all QC into multiqc output
-# # ===============================================================================================
+# ===============================================================================================
+# 10. Combine all QC into multiqc output
+# ===============================================================================================
#
# rule multiqc:
# input:
# # fastqc
-# expand("results/fastqc/{sample}_R1_fastqc.html", sample=all_samples),
-# expand("results/fastqc/{sample}_R2_fastqc.html", sample=all_samples),
-# expand("results/fastqc/{sample}_R1_fastqc.zip", sample=all_samples),
-# expand("results/fastqc/{sample}_R2_fastqc.zip", sample=all_samples),
+# expand("results/qc/{sample}_R1_fastqc.html", sample=all_samples),
+# expand("results/qc/{sample}_R2_fastqc.html", sample=all_samples),
+# expand("results/qc/{sample}_R1_fastqc.zip", sample=all_samples),
+# expand("results/qc/{sample}_R2_fastqc.zip", sample=all_samples),
# # bowtie2
# expand("logs/{sample}.align", sample=all_samples),
-# expand("logs/{sample}.flagstat.qc", sample=all_samples),
+# expand("results/qc/{sample}.flagstat.qc", sample=all_samples),
# # preseq
-# expand("results/preseq/{sample}.ccurve.txt", sample=all_samples),
-# # picard
-# expand("results/picard/{sample}_est_lib_complex_metrics.txt", sample=all_samples),
+# expand("results/qc/{sample}.ccurve.txt", sample=all_samples),
+# expand("results/qc/{sample}.extrap.txt", sample=all_samples),
# # deepTools
# "results/deeptools/multibamsum.npz",
# "results/deeptools/multibamsum.tab",