Question: problem determining read directions
0
gravatar for blooria
4.0 years ago by
blooria0
blooria0 wrote:

Hi,

I am new at this, and I am trying to clean CLIP data. I have a SE read results, that are supposed to have adaptors, and I am trying to figure out where these adaptors should be, on 5-prime end or 3-prime (or both?) RNA that had been PCRed using a specific illimina adaptor that adheres to 5', should they have left over adaptor sequences on the 5' end or the 3'? I read lots of posts but still can't wrap my head around the directions... Thanks!

rna-seq • 1.0k views
ADD COMMENTlink modified 4.0 years ago by i.sudbery9.4k • written 4.0 years ago by blooria0
1

Sequencing always happens in 5'-->3' direction and that is what is in the reads. Since you are using illumina technology, adapter contamination, if present should be at the 3'-end of the reads in case the insert is shorter than cycles sequenced.

ADD REPLYlink written 4.0 years ago by genomax91k

does that mean that if the experimental guys give me the adaptor sequence, I should look for its reverse complement? Thanks!

ADD REPLYlink written 4.0 years ago by blooria0
0
gravatar for i.sudbery
4.0 years ago by
i.sudbery9.4k
Sheffield, UK
i.sudbery9.4k wrote:

CLIP or iCLIP?

In both CLIP and iCLIP you will get 3' adaptor contamination. Whether you need to look for the revcomp of the sequence depends on the trimmer, some will take care of that for you.

The way the sequence is processed at the 5' end should take care of the 5' PCR adaptor. If in doubt run your trimmed reads through fastqc which will tell you if you have adaptor contamination.

In iCLIP there will also be a 5' adaptor that consists of the library barcode and the random UMI sequence, in the pattern NNNXXXXNN, where N is a random case and X is a barcode base. You'll need to take that off the 5' end, but retain it for further use.

ADD COMMENTlink modified 4.0 years ago • written 4.0 years ago by i.sudbery9.4k
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