Question: Strandness setting for HISAT2 : BGI sequencing platform
1
gravatar for venura
6 weeks ago by
venura60
University of Peradeniya
venura60 wrote:

Hi,

I have this question (prob a silly question). We have a set of RNASEq data that came from BGI sequencing platform. The dataset is PE 150bp (and stranded).

I am going to use HISAT2 for alignment and wondering about the strandness setting to be used. This is the exp procedure they used:

enter image description here

Any thoughts on what setting I should use R/RF or F/FR . By the Image I feel the latter should be used. Just trying to make sure. Thanks in advance.

hisat2 rna-seq • 168 views
ADD COMMENTlink modified 6 weeks ago by GenoMax92k • written 6 weeks ago by venura60
1

Run GUESSmyLT and you should have your answer.

ADD REPLYlink written 6 weeks ago by Juke344.9k

Thank you for the link, Jacques!

ADD REPLYlink written 6 weeks ago by venura60

Is this even a stranded library? I would ask the facility that made the libraries for advise.

ADD REPLYlink written 6 weeks ago by ATpoint42k

I did. They said to follow the Illumina pipeline. :| When we placed the order we asked for stranded sequencing and PE 150

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by venura60
1

Following Illumina pipeline is one thing but as ATPoint said earlier this library does not appear to be stranded (see: HISAT2 rna-strandness option )

ADD REPLYlink written 6 weeks ago by GenoMax92k
1

That is not an answer I would accept if I was you. You paid for it, so I personally would insist on details which means a protocol and details on the library prep. As of the picture you provide it does not seem that the library is stranded (at least based on the image) because (for me) it is not obvious whether the the first strand synthesis preserves the strand information. Ask for details, don't let them waive you off until you get the details you need for your analysis, you are the paying customer ;-)

ADD REPLYlink written 6 weeks ago by ATpoint42k
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Writing back to them now! You are correct, I am going to give them a hard time! :)

ADD REPLYlink written 6 weeks ago by venura60

They replied and said it was a mistake :) and sent their original logs as proof. and asked me to replace the old image with this.

New Image

ADD REPLYlink modified 6 weeks ago by Istvan Albert ♦♦ 85k • written 6 weeks ago by venura60
1

Please edit the original post and replace the image in it.

Looks like this is a standard dUTP library.

ADD REPLYlink written 6 weeks ago by GenoMax92k

Just updated the original post.

ADD REPLYlink written 6 weeks ago by venura60
1

So standard dUTP I guess: https://bioinformatics.stackexchange.com/questions/4074/hisat2-which-option-should-mention-for-strand-specific-library-read

ADD REPLYlink modified 6 weeks ago • written 6 weeks ago by ATpoint42k
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