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polyAmapping.smk
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rule bamToSam:
input: "output/mappings/longReadMapping/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.bam"
output: temp("output/mappings/longReadMapping/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.sam")
conda: "envs/xtools_env.yml"
shell:
'''
uuid=$(uuidgen)
samtools view {input} > {TMPDIR}/$uuid.sam
mv {TMPDIR}/$uuid.sam {output}
'''
rule polyAmapping:
input:
reads = "output/mappings/longReadMapping/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.sam",
genome = lambda wildcards: config["GENOMESDIR"] + CAPDESIGNTOGENOME[wildcards.capDesign] + ".sorted.fa"
params:
minAcontent=0.8
output: "output/mappings/polyAmapping/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAsites.bed.gz"
conda: "envs/perl_env.yml"
shell:
'''
uuidTmpOut=$(uuidgen)
cat {input.reads} | samToPolyA.pl --minClipped=10 --minAcontent={params.minAcontent} --discardInternallyPrimed --minUpMisPrimeAlength=10 --genomeFasta={input.genome} - |sort -T {TMPDIR} -k1,1 -k2,2n -k3,3n |gzip > {TMPDIR}/$uuidTmpOut
mv {TMPDIR}/$uuidTmpOut {output}
'''
rule removePolyAERCCs:
input: "output/mappings/polyAmapping/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAsites.bed.gz"
output: temp("output/mappings/removePolyAERCCs/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAsitesNoErcc.tmp.bed")
shell:
'''
uuidTmpOut=$(uuidgen)
zcat {input} | fgrep -v ERCC > {TMPDIR}/$uuidTmpOut
mv {TMPDIR}/$uuidTmpOut {output}
'''
rule getPolyAreadsList:
input: "output/mappings/removePolyAERCCs/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAsitesNoErcc.bed"
output: "output/mappings/getPolyAreadsList/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAreads.list"
shell:
'''
uuidTmpOut=$(uuidgen)
cat {input} | cut -f4 | sort -T {TMPDIR} |uniq > {TMPDIR}/$uuidTmpOut
mv {TMPDIR}/$uuidTmpOut {output}
'''
rule getPolyAreadsStats:
input:
mappedReads= "output/mappings/longReadMapping/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.bam",
polyAreads = "output/mappings/getPolyAreadsList/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAreads.list"
conda: "envs/xtools_env.yml"
output: "output/statsFiles/" + "tmp/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAreads.stats.tsv"
shell:
'''
uuidTmpOut=$(uuidgen)
mapped=$(samtools view -F4 {input.mappedReads} |cut -f1|sort -T {TMPDIR} |uniq|wc -l)
polyA=$(cat {input.polyAreads} | wc -l)
echo -e "{wildcards.techname}\t{wildcards.capDesign}\t{wildcards.sizeFrac}\t{wildcards.sampleRep}\t$mapped\t$polyA" | awk '{{print $0"\t"$6/$5}}' > {TMPDIR}/$uuidTmpOut
mv {TMPDIR}/$uuidTmpOut {output}
'''
rule aggPolyAreadsStats:
input: expand("output/statsFiles/" + "tmp/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAreads.stats.tsv",filtered_product, techname=TECHNAMES, capDesign=CAPDESIGNS, sizeFrac=SIZEFRACS, sampleRep=SAMPLEREPS)
output: "output/statsFiles/" + "all.polyAreads.stats.tsv"
shell:
'''
uuidTmpOut=$(uuidgen)
echo -e "seqTech\tcapDesign\tsizeFrac\tsampleRep\tcategory\tcount\tpercent" > {TMPDIR}/$uuidTmpOut
cat {input} | awk '{{print $1"\\t"$2"\\t"$3"\\t"$4"\\tnonPolyA\\t"$5-$6"\\t"($5-$6)/$5"\\n"$1"\\t"$2"\\t"$3"\\t"$4"\\tpolyA\\t"$6"\\t"$6/$5}}' | sort -T {TMPDIR} >> {TMPDIR}/$uuidTmpOut
mv {TMPDIR}/$uuidTmpOut {output}
'''
rule plotAllPolyAreadsStats:
input: "output/statsFiles/" + "all.polyAreads.stats.tsv"
output: returnPlotFilenames("output/plots/" + "polyAreads.stats/{techname}/{capDesign}/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAreads.stats")
params:
filterDat=lambda wildcards: multi_figures(wildcards.capDesign, wildcards.sizeFrac, wildcards.sampleRep, wildcards.techname)
conda: "envs/R_env.yml"
shell:
'''
echo "
library(ggplot2)
library(cowplot)
library(plyr)
library(scales)
library(gridExtra)
library(grid)
library(ggplotify)
library(data.table)
dat <- read.table('{input}', header=T, as.is=T, sep='\\t')
{params.filterDat[technameFilterString]}
{params.filterDat[capDesignFilterString]}
{params.filterDat[sizeFracFilterString]}
{params.filterDat[sampleRepFilterString]}
{params.filterDat[substSeqTechString]}
{params.filterDat[substSampleRepString]}
{params.filterDat[graphDimensions]}
plotBase <- \\"p <- ggplot(data=dat, aes(x=1, y=count, fill=category)) +
geom_bar(stat='identity') +
scale_fill_manual(values=c('polyA' = '#c8e09e', 'nonPolyA' = '#e7a198')) +
ylab('# mapped reads') +
xlab('') +
guides(fill = guide_legend(title='Category'))+ scale_y_continuous(labels=scientific)+
geom_text(position = 'stack', size=geom_textSize, aes( y = count, label = paste(sep='',percent(round(percent, digits=2)),'\\n','(',comma(count),')'), hjust = 0.5, vjust = 1))+
{params.filterDat[hideXaxisLabels]}
{GGPLOT_PUB_QUALITY} +
theme(axis.ticks.x = element_blank(), axis.text.x = element_blank()) +
\\"
{params.filterDat[facetPlotSetup]}
save_plot('{output[0]}', legendOnly, base_width=wLegendOnly, base_height=hLegendOnly)
save_plot('{output[1]}', pXy, base_width=wXyPlot, base_height=hXyPlot)
save_plot('{output[2]}', pXyNoLegend, base_width=wXyNoLegendPlot, base_height=hXyNoLegendPlot)
save_plot('{output[3]}', pYx, base_width=wYxPlot, base_height=hYxPlot)
save_plot('{output[4]}', pYxNoLegend, base_width=wYxNoLegendPlot, base_height=hYxNoLegendPlot)
" > $(dirname {output[0]})/$(basename {output[0]} .legendOnly.png).r
cat $(dirname {output[0]})/$(basename {output[0]} .legendOnly.png).r | R --slave
'''
rule clusterPolyAsites:
input: "output/mappings/removePolyAERCCs/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAsitesNoErcc.bed"
output: "output/mappings/clusterPolyAsites/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAsites.clusters.bed"
conda: "envs/xtools_env.yml"
shell:
'''
uuidTmpOut=$(uuidgen)
cat {input} | bedtools merge -s -d 5 -c 4,6 -o distinct -i stdin | awk '{{print $1"\t"$2"\t"$3"\t"$4"\t0\t"$5}}'| perl -F"\\t" -lane 'if($F[5] eq "+"){{$F[1]=$F[2]-1}}elsif($F[5] eq "-"){{$F[2]=$F[1]+1}}else{{die}} print join("\t",@F);'|sort -T {TMPDIR} -k1,1 -k2,2n -k3,3n > {TMPDIR}/$uuidTmpOut
mv {TMPDIR}/$uuidTmpOut {output}
'''
rule makePolyABigWigs:
input:
sites = "output/mappings/removePolyAERCCs/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAsitesNoErcc.bed",
genome = lambda wildcards: config["GENOMESDIR"] + CAPDESIGNTOGENOME[wildcards.capDesign] + ".sorted.genome"
output: "output/mappings/makePolyABigWigs/{techname}_{capDesign}_{sizeFrac}_{sampleRep}.polyAsitesNoErcc.{strand}.bw"
conda: "envs/xtools_env.yml"
shell:
'''
tmpIn=$(uuidgen)
uuidTmpOut=$(uuidgen)
cat {input.sites} | grep -P "^chr" | grep -v "chrIS" > {TMPDIR}/$tmpIn
bedtools genomecov -strand {wildcards.strand} -split -bg -i {TMPDIR}/$tmpIn -g {input.genome} > {TMPDIR}/$uuidTmpOut.bedgraph
bedGraphToBigWig {TMPDIR}/$uuidTmpOut.bedgraph {input.genome} {TMPDIR}/$uuidTmpOut
mv {TMPDIR}/$uuidTmpOut {output}
'''