Hello,
I am using Kallisto for transcript expression quantification and I noticed that I am getting a very low percentage of reads that are pseudoaligning. It is my understanding that output below refers to the number of reads that are being associated with transcripts:
[quant] processed 54,011,492 reads, 709,696 reads pseudoaligned
I am using these parameters:
kallisto quant --bias --fr-stranded -i ${index} -o ${opath} ${readline}
Has anyone else seen this problem? Does it have to do with specifying the strandedness of the reads?
Thank you!
Are you using a proper reference transcriptome? Did you build it yourself, or download from somewhere (NCBI, ENSEMBL)? How were you libraries prepared, which kit? Strandedness will depend on library prep kit.
Yes, it could be due to passing an incorrect strand flag, but it could also be an incorrect reference transcriptome, or it could be sample contamination with foreign RNA, or it could be major rRNA contamination, or it could be you did a DNAseq run and are trying to quantify against transcriptome.