how to use 2-pass mapping without genome regeneration?

Question

[wangchengyangghg]

Hi All,

I am a guy studying in alternative splicing from RNA-seq. Now I am using the latest STAR to map the RNA-seq reads.

I want to know much more details about "2-pass mapping without re-generated genome" in the latest document http://labshare.cshl.edu/shares/gingeraslab/www-data/dobin/STAR/STARreleases/2.4.0k/doc/STARmanual.pdf

in which, the author said

2-pass mapping without re-generated genome.

To run STAR 2-pass mapping without re-generating the genome,you need to set --twopass1readsN to the number of reads you want mapped in the 1st pass (the default and best approach is to makeit bigger than the number of reads in the sample, so that all reads are used in the 1st pass). At the moment, this option can only be used with a genome without annotated junctions. STAR will perform the 1st pass mapping, then it will automatically extract junctions, insert them into the genome index, and, finally, re-map all reads in the 2nd mapping pass. In this mode, the --sjdbOverhang parameter has to be specified at the mapping stage.

i have two questions. The first one is the necessity of gene annotation in building genome indices for the 2-pass mapping without re-generate genome. The other one is that whether someone else could share an exemplary codes with regard to it to me? Thank you very much!!! ;)