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is always necessary to do an intermediate index generations and 2nd pass alignment with STAR?

Hi everybody, I've just wondering if is totally necessary to do an intermediate index generations and 2nd pass alignment with STAR. Does anybody has information about when to skip it or not? I would like to know a bit more about this issue and to decide when is the moment to apply them or not.

Thank you very much in advance.

Best,

rnaseq alignment index star
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updated by arta arta • written by Lila M Lila M

1 answer

Well according to documentation, in order to detect novel junction discovery, you need to apply 2-pass mode.

But, fortunately, if you use -- twopassmode, 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 (directly taken from documentation).

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written by arta arta

thank you for this! But in that case, is still necessary to create the intermediate index? or Can I apply the --twopassMode in the 1st pass?

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written by Lila M Lila M

You can apply the --twopassMode in the first pass, at least i do.

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written by arta arta

The 2-pass mode isn't required to detect novel junctions. The 2nd step, however, does increase the mapping of detected novel junctions. Star 2-pass is most commonly used by combining all the sj files from the first pass. You can either use --sjdbFileChrStartEnd to provide a list of sj files (on-the-fly method), or reindex. If you choose to insert junctions to your genome using the on-the-fly method, you might need --limitSjdbInsertNsj when the total number of junctions are greater than 100k. Refer to the documentation quoted above for more details.

Edit: If you only have one sample or intent to perform 2-pass in a sample-by-sample basis, then --twopassMode will do.

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written by Eric Lim Eric Lim

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