How-to: call variants from RNA-seq data

Last updated on May 19, 2023 1 min read

https://software.broadinstitute.org/gatk/best-practices/workflow?id=11164

Install GATK4

Download the precompiled jar binaries. Set paths.

Adding read groups to bam file

java -jar picard AddOrReplaceReadGroups \
    I=sample.filt.bam \
    O=sample.rg.bam \
    SO=coordinate RGID=id RGLB=library RGPL=platform RGPU=machine RGSM=sample

Marking duplicates

java -jar picard MarkDuplicates \
    I=sample.rg.bam  \
    O=sample.dedupped.bam \
    CREATE_INDEX=true VALIDATION_STRINGENCY=SILENT M=output.metrics

Splitting and trimming

gatk SplitNCigarReads \
   -R genome.fa \
   -I sample.dedupped.bam \
   -O sample.split.filtered.bam

Haplotype calling

gatk HaplotypeCaller \
   -R genome.fa \
   -L 1 2 3 4 5 6 X \
   -I  sample.split.filtered.bam \
   -O  sample.filtered.vcf \
   --dont-use-soft-clipped-bases \
   --standard-min-confidence-threshold-for-calling 20.0