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RNA seq TopHat output to variant calls

mpirompiro Member
edited January 2013 in Ask the GATK team

Hello all,

We are planning to use GATK for snp calling on our RNA-Seq. We mapped our RNA-Seq data using TopHat. The pipeline we are planning to use is the same as the DNA sequencing pipeline:

.bam > filtering (fixmate, remove duplicates, ..) > realignment > recalibration > calling variants

I was wondering if anyone has any suggestions/tips for RNA-Seq SNP calling that we need to consider.
Greatly appreciate your input.

Best Regards,

Post edited by Geraldine_VdAuwera on

Best Answer

Answers

  • GermanLGermanL Member
    edited February 2013

    I am also looking into variant calling in RNA-seq data. I would note two things about your approach.

    1) Reassign the TopHat mapping qualities. 255 is used as a "uniquely mapping" read in TopHat, but in the SAM specifications, 255 represents no mapping quality and thus GATK will skip these reads. So you will want to offset these MQ values by -1 or some other arbitrary offset.

    2) You will want to make an exon intervals file, particularly for the realignment step, since you don't want the realigner to try to realign intron spanning reads. With the intervals file you can restrict the realigner to stay within exon boundaries.

    Post edited by Geraldine_VdAuwera on
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