fr-firststrand

Library Types:

• fr-unstranded: Standard Illumina Reads from the left-most end of the fragment (in transcript coordinates) map to the transcript strand, and the right-most end maps to the opposite strand.
• fr-firststrand:dUTP, NSR, NNSR Same as above except we enforce the rule that the right-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during first strand synthesis is sequenced.
• fr-secondstrand: Ligation, Standard SOLiD Same as above except we enforce the rule that the left-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during second strand synthesis is sequenced.

If you use Truseq stranded protocol, the library type should be fr-firststrand

check this analysis guide from Illumina, in the pdf file, they clearly suggest using fr-firststrand http://res.illumina.com/documents/products/technotes/rnaseqanalysistophat.pdf

This could illustrate how to select your library-type if you really want to know why so. http://onetipperday.blogspot.com/2012/07/how-to-tell-which-library-type-to-use.html

So I've to do :

tophat -library-type fr-firststrand index R2.fastq R1.fastq and not tophat -library-type fr-firststrand index R1.fastq R2.fastq because R2 reads are aligning on the sens of the RNA fragment that is sequenced ? Is it correct ?

Thanks