Tophat Library-Type : Illumina Truseq Stranded Total Rna Sample Prep Kit

Question

[Nicolas Rosewick]

Hi,

Anybody know which type of tophat library-type do I have to use with IlluminaTruSeq Stranded Total RNA Sample Prep Kit (paired-end 2x50bp) ?

Thanks ,

N.

[Nicolas Rosewick]

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

Answer 1

[Irsan]

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.

[Biomonika (Noolean)]

The funny thing is that what is fr-firststrand for Tophat is RF for Trinity (transcriptome assembler).

Answer 2

[dbrg77]

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

[Christopher Bottoms]

As of 2016-09-07, this is the correct URL for that document: http://www.illumina.com/documents/products/technotes/RNASeqAnalysisTopHat.pdf

[camelbbs]

I like the direct answer like this. So for Illumina Truseq Stranded Total Rna Sample Prep Kit, we can use tophat -library-type fr-firststrand, is that correct?

[shweta.jajoriya]

what would be the library type if data from Illumina HiSeq 2000

Answer 3

[Puriney]

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

[germelcar]

Could you please share the Zhao Zhang's title paper? Thanks in advance.