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Calling RNAseq variants using a transcriptome FASTA

I know Best Practices recommends calling RNAseq variants based on the genomic fasta and BAMs with genomic coordinates. With the GENCODE references, it is also possible for us to call variants on a transcriptome basis, i.e.:

  1. Align RNAseq fastq with STAR;
  2. Pre-process the Aligned.toTranscriptome.out.bam using the transcriptomic sequences (such as gencode.v26.transcripts.fa.gz) as the reference sequence;
  3. Call variants with HC using the pre-processed sequence and references mentioned in (2);
  4. Hard filter as appropriate.

Aligned.toTranscriptome.out.bam is the STAR alignment in transcript coordinates, which is usually used for gene expression quantifiers such as RSEM or the "fishes."

While I'm sure Broad won't officially sanction such a pipeline, but can anyone here help me point out whether there are obvious problems with it?

Thanks for your answers in advance!

John

Issue · Github
by Sheila

Issue Number
2035
State
closed
Last Updated
Milestone
Array
Closed By
vdauwera

Best Answer

Answers

  • SheilaSheila Broad InstituteMember, Broadie admin

    @johnma
    Hi John,

    I am checking what the team thinks and will get back to you.

    -Sheila

  • johnmajohnma Member

    Hi @Sheila,

    While right now I'm just thinking it, but I'd answer your questions here:

    1. Yes, this is what I mean. While I do align to the genome plus a GTF/GFF, it can provide a separate BAM in transcriptome coordinates.
    2. By "pre-procesing" I mean MarkDuplicate and BQSR; SplitNCigarReads doesn't apply here.

    The reason that I'm bother thinking about doing this is that I want to have an idea of allele frequencies on a isoform-level, as it's probably unsafe to assume the AF at one locus is the same for every isoforms that overlaps it.

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    Hi @johnma, as you correctly surmised we can't really give you any guidance on this since it's not something we have any direct experience with. We've forwarded your question to a few people we know who might be in a better position to answer you, but I can't guarantee they'll get back to you.

    Good luck!

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