Generating an isoform level mtx file

[krtitus]

Hello,

I am currently following the kallisto | bustools tutorial on processing nuclear RNA-seq, which runs bustools count on spliced and unspliced transcripts. I know that you can specify --genecounts, which gives you the gene level counts, but what I want are the isoform level counts. When I try to run the command without the --genecounts flag, it outputs the counts for the equivalence classes, which I don't want either. Given that I'm working with bulk, nuclear RNA-seq data, is there a way I can achieve what I want?

[Yenaled]

You can use kallisto quant-tcc to go from EC matrix to isoform matrix. That said, it has never been benchmarked on nucleus data so I'm not sure how well it will work or if the numbers will even be biologically meaningful. The nucleus is tricky because it's mostly nascent, unspliced transcripts that are in the process of being processed. Even 10x single cell data is tricky to get isoform quantification because nearly everything is sequenced at the 3' end so different isoforms share the same 3' end.