Question

problem determining read directions

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7.5 years ago

blooria • 0

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 • 1.6k views

ADD COMMENT • link updated 7.5 years ago by i.sudbery 19k • written 7.5 years ago by blooria • 0

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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 REPLY • link 7.5 years ago by GenoMax 141k

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

ADD REPLY • link 7.5 years ago by blooria • 0

score 0 · Answer 1 · 2016-10-20

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.