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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.:
- Align RNAseq fastq with STAR;
- Pre-process the
Aligned.toTranscriptome.out.bamusing the transcriptomic sequences (such as gencode.v26.transcripts.fa.gz) as the reference sequence;
- Call variants with HC using the pre-processed sequence and references mentioned in (2);
- 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!