STAR two -pass mode - lower alignment rate than one pass mode?

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6.0 years ago

Biogeek ▴ 470

Hi guys,

Quick question. I've performed a two-step alignment with STAR 2.6a. It seems my unique alignment rates are approx 1% (85%) lower when performing two pass, than the original one pass mode (86%). I thought the aim of two-pass mode was to increase the number of reads mapping to novel splice junctions / % of maps aligned?

Am I wrong in my understanding?

star alignment two pass mode • 6.0k views

ADD COMMENT • link updated 3.0 years ago by FantasticAI ▴ 60 • written 6.0 years ago by Biogeek ▴ 470

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If anyone wants to know why... see below:

Some drop in the uniquely mapped reads in the 2nd pass is to be expected, but it's typically ~1-2%. It's caused by the reads with short splice ovehangs, which can be mapped to multiple junctions.

ADD REPLY • link 6.0 years ago by Biogeek ▴ 470

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As in STAR manual, it states:

It does not increase the number of detected novel junctions, but allows to detect more splices reads mapping to novel junctions. The basic idea is to run 1st pass of STAR mapping with the usual parameters, then collect the junctions detected in the first pass, and use them as ”annotated” junctions for the 2nd pass mapping.

ADD REPLY • link 6.0 years ago by Ram ▴ 190

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So is it always recommended to run basic twopass mode for pair-end RNA-seq data?

ADD REPLY • link 3.0 years ago by FantasticAI ▴ 60

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