Hello,
I am learning how to run the pacbio pipeline for IsoSeq but have found conflicting information. We sent bulk total RNA off to a core to be prepped with the IsoSeq SMRTbell prep kit 3.0 and sequenced on PacBio's Sequel II. I received a subreads.bam file where I then ran ccs from SMRT Tools.
In the SMRT Tools Reference Guide on page 35 it's stated the recommended parameters for running CCS analysis for Iso-Seq are min-pass=1, min accuracy=0.9, and turning Polish to OFF. Then, I would proceed to lima with the ccs.bam file.
ccs --min-passes 1 --min-rq 0.9 --skip-polish subreads.bam ccs.bam
However, in the IsoSeq Docs CLI Workflow it says to generate hifi reads and then proceed to lima.
ccs subreads.bam ccs.bam
extracthifi ccs.bam hifi.bam
What way should it be done? Or does it depend on what question you asking - rare/long transcript (ccs default is 3 passes) or high-fidelity?
