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Transcript to gene file for Kallisto in a iso-seq transcriptome with no reference genome?

Hi,

I am looking to perform DE between 4 groups (with triplicate replicate for each group) on an organism that has never been sequenced before (i.e. no genome, or even close relative genome). I have gone through the iso-seq pipeline to produce 12 clustered FLNCs, and created a reference, non-redundant transcriptome by merging all of the transcriptomes and running CD-HIT-EST to remove redundant transcripts for diamond/annotation. For Kallisto, I need to provide a transcript2gene (t2g) file for quantification and linking the transcripts across the samples. I have searched literature/documentation and have been unable to find any resources addressing this. My thought was to use the clstr file from CD-HIT-EST as "pseudo-genes" to continue my analysis. Is anyone aware of any published literature using this method or an alternative/better suited method?

Thank you!

iso-seq kallisto

1 answer

Cogent is the tool built for exactly this, reconstructing gene families from Iso-Seq with no reference. Same cDNA_Cupcake ecosystem as the rest of the Iso-Seq tooling, so it's the closest thing to a citable standard for your situation.

The issue with using the cd-hit clstr file as pseudo-genes is that cd-hit clusters on identity plus coverage thresholds, so two isoforms differing by a skipped exon will often fail the coverage cutoff and land in separate clusters. You fragment single genes that way, and recent paralogs merge in the other direction. Corset is the better-shaped alternative, since it clusters transcripts by which reads multi-map across them rather than by sequence identity.

That said, I'd think about whether you need gene level at all here. Isoform resolution is the thing Iso-Seq buys you, and with 4 groups and triplicates you can run transcript-level DE directly and skip t2g entirely, rather than collapsing into pseudo-genes you don't fully trust.

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