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RNAseq_Process_auto.sh
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RNAseq_Process_auto.sh
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#!/bin/bash
help(){
cat << EOF
Description:
This mini tool can be used to process clean RNA-seq data and produce bam, bigwig, bedgraph files.
CAUTION: (1) THE INPUT DATA MUST BE CLEANED!!!
(2) Until now, this tool can only deal with strand specific RNA-seq data (ssRNA-seq).
Required tools:
- STAR
- bedtools
- samtools
- featureCounts
Usage:
RNAseq_Process_auto.sh -1 <r1.fq> -2 <r2.fq> -r <ref> -g <gtf> -b <bed> -n <processers> -p <prefix> -o <output>
Options:
-1 the path of read1 fastq, can't be gzip. [PATH]
-2 the path of read2 fastq, can't be gzip. [PATH]
-r the ref genome for STAR to do reads alignments. [PATH]
-g the GTF annotation file for reads summary. [PATH]
-b the genome annotation bed file used for determine library type. [PATH]
-n the number of processers to perform this task, at least 2. [NUM]
-p the prefix of output file. [CHAR]
-o the output dir for this task, can be created automatically. [CHAR]
Owner:
Kunming Shui, [email protected], Nanjing University.
Last Modified:
2022-07-20
Modified Log:
2022-07-18 RNAseq_Process_auto.sh first released.[v 0.1.0]
2022-07-20 Modified a bug about the strand information determination.[v 0.1.1]
EOF
}
#version information
version="v 0.1.1"
if [ $# -eq 0 ] || [ $1 = "-h" ] || [ $1 = "--help" ]
then
help
exit 1
fi
if [ $1 = "-v" ] || [ $1 = "--version" ]
then
echo $version
exit 1
fi
echo "=================== Begin Analysis at $(date) ==================="
while getopts "1:2:r:g:b:n:p:o:" option
do
case $option in
1) read1=$OPTARG;;
2) read2=$OPTARG;;
r) ref=$OPTARG;;
g) gtf=$OPTARG;;
b) bed=$OPTARG;;
n) num=$OPTARG;;
p) prefix=$OPTARG;;
o) out=$OPTARG;;
esac
done
echo "=================== Reads Alignment with STAR ==================="
if [ -d $out ]
then
echo "$out is exiting."
else
mkdir -p $out
fi
#data check
echo "The read1 file is $read1"
echo "The read2 file is $read2"
STAR --runThreadN $num \
--genomeDir $ref \
--readFilesIn $read1 $read2 \
--outSAMtype BAM SortedByCoordinate \
--outBAMsortingThreadN $[ $num/2 ] \
--outFileNamePrefix $out/$prefix
echo "=================== Filter out low quality alignments ==================="
samtools view -q 255 -b -@ $num -o $out/$prefix.highquality.bam $out/${prefix}Ali*.bam
echo "Finished..."
echo "=================== Determine the library type ==================="
class=`infer_experiment.py -i $out/$prefix.highquality.bam -r $bed`
num1=`echo "$class" | grep 'Fraction of reads explained by "1++,1--,2+-,2-+":' | cut -d " " -f7`
num2=`echo "$class" | grep 'Fraction of reads explained by "1+-,1-+,2++,2--":' | cut -d " " -f7`
if [ `echo "$num1 > $num2" | bc` -eq 1 ]
then
library="S"
else
library="R"
fi
if [ $library = "S" ]
then
echo "Your RNA-seq library is stranded..."
else
echo "Your RNA-seq library is reverse stranded..."
fi
cd $out
samtools index $prefix.highquality.bam
samtools view -f 99 -b -@ $num -o $prefix.r1.fwd.bam $prefix.highquality.bam
samtools view -f 163 -b -@ $num -o $prefix.r2.fwd.bam $prefix.highquality.bam
samtools view -f 147 -b -@ $num -o $prefix.r2.rev.bam $prefix.highquality.bam
samtools view -f 83 -b -@ $num -o $prefix.r1.rev.bam $prefix.highquality.bam
if [ $library = "S" ]
then
samtools merge -@ $num tmp.forward.bam $prefix.r1.fwd.bam $prefix.r2.rev.bam
samtools merge -@ $num tmp.reverse.bam $prefix.r1.rev.bam $prefix.r2.fwd.bam
else
samtools merge -@ $num tmp.forward.bam $prefix.r1.rev.bam $prefix.r2.fwd.bam
samtools merge -@ $num tmp.reverse.bam $prefix.r1.fwd.bam $prefix.r2.rev.bam
fi
echo "=================== Perform reads summary with featureCounts ==================="
if [ $library = "S" ]
then
type=1
else
type=2
fi
mkdir featureCounts
featureCounts -a $gtf \
-o featureCounts/${prefix}.txt \
-t exon \
-g gene_id \
-s $type \
-p \
--countReadPairs \
-B \
-C \
-T $num $prefix.highquality.bam
echo "=================== Produce bedgraph, bigwig files ==================="
samtools sort -@ $num -o $prefix.fwd.bam tmp.forward.bam
samtools sort -@ $num -o $prefix.rev.bam tmp.reverse.bam
rm tmp.forward.bam tmp.reverse.bam
samtools index $prefix.fwd.bam
samtools index $prefix.rev.bam
#bedgraph file
genomeCoverageBed -bga -ibam $prefix.fwd.bam -split > $prefix.fwd.bedgraph
genomeCoverageBed -bga -ibam $prefix.rev.bam -split > $prefix.rev.bedgraph
#bigwig file
bamCoverage -b $prefix.fwd.bam -o $prefix.fwd.bigwig -bs 10 --normalizeUsing CPM -p $num
bamCoverage -b $prefix.rev.bam -o $prefix.rev.bigwig -bs 10 --normalizeUsing CPM -p $num
echo "$class" >> Process.log
echo $library >> Process.log
cd -
echo "=================== Finished at $(date) ==================="