Cufflinks Or Cuffdiff (Or Both) For Frag-Bias-Correct?

Question

[buchnerd]

Both Cufflinks and Cuffdiff have a frag-bias-correct option (-b). Does it matter at which step I use this option or if I use it at both steps?

Same question for -u/--multi-read-correct

Thanks!

Answer 1

[Ryan Thompson]

As far as I know, the quantification performed by Cufflinks is not used at all in Cuffdiff. Cuffdiff only uses the assembled transcripts and re-quantifies those transcripts against all the bam files given. Cuffdiff presumably uses the exact same quantificatio procedure as Cufflinks, but it does it once for each sample.

So, if you are assembling transcripts with Cufflinks and then testing for differential expression with Cuffdiff (hopefully after using Cuffmerge), then the quality of quantification at the Cufflinks step only really matters for the purposes of determining whether some minor isoforms meet the various thresholds for discarding.