Question: Request to validate my RNAseq workflow
gravatar for umn_bist
2.4 years ago by
umn_bist310 wrote:

The samples are tumor/match_normal PE RNA-seq samples from TCGA. I need to call/annotate somatic mutation

1. preprocessing QC: trim adapter using cutadapt (may use BBMap), remove low quality (<10) reads

2. align: TopHat2, add RG with bowtie2 (is this common practice?)

  • I am interested in using STAR instead of TopHat2 but my advisor warned that the STAR/Tophat does not share similar format for removing multimapped/unmapped reads (Tophat is part of the workflow right now)
for f in *_nodupe.bam
do fname=`basename $f .bam`

#Remove Create Unique and Multi/Unmapped Bams
    samtools view -h -F 1024 $f | awk 'substr($0,1,1)=="@"||match($0, /NH:i:1\t/) {print $0}' | samtools view -hSb - > ${fname}_unique.bam
    samtools view -F 1024 -h $f | awk 'substr($0,1,1)=="@"||!/NH:i:1\t/  {print $0}' | samtools view -hSb - > ${fname}_multimapped.bam

    #Unique Reads Aligning to hg19 Repeatitive Sequence

        samtools view ${fname}_unique.bam | wc -l > ${fname}.count;
        bamToBed -i ${fname}_unique.bam -bed12 |
        intersectBed -a packages/tracks/hg19/hg19_rmsk_FLL1.bed -b stdin -split -sorted -c > ${fname}_MappedRE.bed;
        awk -F '\t' '{print $4"\t"$7}' ${fname}_MappedRE.bed > ${fname}.names;

3. postprocessing QC: dedupe (mark duplicates)

4. variant calling/annotation: muTect2, snpSift (select against low MQ, AF, QUAL variants)

5. annotate: wAnnovar (may use snpEff)

rna-seq ngs • 789 views
ADD COMMENTlink modified 2.4 years ago • written 2.4 years ago by umn_bist310
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