strand-specificity in RSEM

shurjo · 10-29-2015, 10:25 AM

Hello,

I'm trying to figure out the settings for analysing strand-specific RNA-Seq data from Illumina TruSeq chemistry with rsem-calculate-expression

Specifically, regarding the "--forward-prob" parameter, the manual has this to say:

"....Set to 1 for a strand-specific protocol where all (upstream) reads are derived from the forward strand, 0 for a strand-specific protocol where all (upstream) read are derived from the reverse strand, or 0.5 for a non-strand-specific protocol. (Default: 0.5)"

Question: based upon my understanding of the molecular biology of the TruSeq strand-specific kit, the strand actually getting sequenced in read 1 of a pair is the cDNA sequence, (or in RSEM manual terminology, the reverse strand).

As such, my hunch is that I should be using --forward-prob=0, but I'd like to check with the community to see if I'm getting things right here.

Any help much appreciated,

Thanks,

Shurjo

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10-29-2015, 10:25 AM	#1
shurjo Senior Member Location: Rockville, MD Join Date: Jan 2009 Posts: 126	strand-specificity in RSEM Hello, I'm trying to figure out the settings for analysing strand-specific RNA-Seq data from Illumina TruSeq chemistry with rsem-calculate-expression Specifically, regarding the "--forward-prob" parameter, the manual has this to say: "....Set to 1 for a strand-specific protocol where all (upstream) reads are derived from the forward strand, 0 for a strand-specific protocol where all (upstream) read are derived from the reverse strand, or 0.5 for a non-strand-specific protocol. (Default: 0.5)" Question: based upon my understanding of the molecular biology of the TruSeq strand-specific kit, the strand actually getting sequenced in read 1 of a pair is the cDNA sequence, (or in RSEM manual terminology, the reverse strand). As such, my hunch is that I should be using --forward-prob=0, but I'd like to check with the community to see if I'm getting things right here. Any help much appreciated, Thanks, Shurjo