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

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.

tophat illumina strand • 38k views
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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

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14
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8.7 years ago
Irsan ★ 7.4k

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

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7
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7.6 years ago
dbrg77 ▴ 70

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

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

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

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

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8.1 years ago
Puriney ▴ 330

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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

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

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