Hi,

I'm trying to figure out how to run a second mapping pass, as described in the paper

"It is also possible to run a second mapping pass, supplying it with splice junction loci found in the first mapping pass. In this case, STAR will not discover any new junctions but will align spliced reads with short overhangs across the previously detected junctions." (Dobin et al., 2012)

I didt found any examples so I don't understand how to run it...

thanks in advance

M.

Hi Mauro,

for a quicker response, please post your questions at the STAR google group (https://groups.google.com/forum/?fromgroups=#!forum/rna-star).

The 2-pass scheme is not automated at the moment. I have attached a simple script with an example of a 2-pass run. You would need to run the 1st pass with usual parameters, than use the SJ.out.tab as a splice junction database file to generate a new genome index for STAR, and then run the 2nd pass of STAR with the new genome index. You can also add annotated junctions to the 1st and 2nd passes.

If you have many samples, you can collect all the novel junctions from all the samples (SJ.out.tab files), possibly filter them for reliability, and create one common set of novel junctions for all samples by merging them. Then you generate a new genome using annotated junctions and the common set of novel junctions, and re-run all the samples with this new genome - this would be the 2-nd pass.