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
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