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Question: Stranded vs unstranded cufflinks
1
gravatar for VY
2.5 years ago by
VY • 120
London
VY • 120 wrote:

Dear Biostar,

Im having issues with my RNAseq libraries.

The library was made with the NexteraXT library which produces unstranded libraries. When I run tophat it only has one option "unstranded" so I set that. But after when it comes to cufflinks and/or cuffdiff there is an fr and and ff unstranded option. Does anyone know which ones I should chose based on their experiance or should I just leave both as "autodetect"?

Thanks Best wishes
nexteraxt strand cufflinks rna-seq • 1.4k views
ADD COMMENT • link •
modified 2.3 years ago by Jennifer Hillman Jackson ♦ 25k • written 2.5 years ago by VY • 120
0
gravatar for Jennifer Hillman Jackson
2.5 years ago by
Jennifer Hillman Jackson ♦ 25k
United States
Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

I couldn't find this in the Illumina protocol documents, oddly, but I believe that the orientation is "fr unstranded". http://www.illumina.com/products/nextera_xt_dna_library_prep_kit.html (If you can find this in the docs, and have time, please comment back about where it is noted/linked - thanks!).

But this can also be autodetected or be doubled checked by you by examining the Tophat/HISAT2 mapped BAM output. Here is post that describes how: https://www.biostars.org/p/45818/. I tend to also visualize data to confirm protocol claims - more information is communicated.

For understanding strandedness and read orientation in general (with many useful references), please see: https://github.com/nekrut/galaxy/wiki/Reference-based-RNA-seq

Best, Jen, Galaxy
ADD COMMENT • link written 2.5 years ago by Jennifer Hillman Jackson ♦ 25k
2

Sorry for the delayed reply to this thread. This isn't actually written in the docs of the protocol for some reason, but after contacting illumuna directly they confirmed that the orientation for nextera-xt is "fr-unstranded" as you said.

ADD REPLY • link written 2.3 years ago by VY • 120

Great! Glad you were able to get the info and thanks for sharing it back, Jen

ADD REPLY • link written 2.3 years ago by Jennifer Hillman Jackson ♦ 25k
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