STAR two step mode

[Mp]

What is the best way to align for  transcription profile studies using two step mode. It is wrong  align using two step procedure without concatenate all junctions file?

[Alexander Dobin]

Hi Mp,

the best way is to concatenate the junctions from all samples, and then re-generate the genome. This yields the best and uniform sensitivity for novel junctions across all samples.

Using the 2-pass approach on a per sample strategy is OK (not really wrong) - it just yields poorer sensitivity.

For instance, if a novel junction is well expressed in only one sample, and weakly (only a few reads with short overhang) in other samples, the per-sample 2-pass approach may only detect this junction in the former sample. On the other hand, the all sample 2-pass will allow to detect this junction in all samples.

Cheers

Alex