Question: Adapter Clipping
3
gravatar for Steffi
7.5 years ago by
Steffi560
Germany
Steffi560 wrote:

I have a question regarding paired-end sequencing and adapter clipping (Solexa, HiSeq, TruSeq Protocol). I have the sequence of the "PE Read 1 Sequencing Primer" and the "PE Read 2 Sequencing Primer". Are these the sequences to clip away? And if so, at which end? 5' and 3' end?

Best, Stefanie

paired adaptor • 3.0k views
ADD COMMENTlink modified 5.8 years ago by Dan D6.8k • written 7.5 years ago by Steffi560

As sequence data are generated and read from 5' to 3', you want to clip at the 3' side of the primers.

ADD REPLYlink written 7.5 years ago by Larry_Parnell16k

I suppose you mean to clip at the 3' side of the reads. And it never may happen that the sequencing reaction starts already within the 5' adapter?

ADD REPLYlink written 7.5 years ago by Steffi560

It's not where the reaction starts, but where the data start that should be a concern. Sometimes, only a portion of the adapter is seen. You need to clip where adapter joins novel seq data.

ADD REPLYlink written 7.5 years ago by Larry_Parnell16k
1
gravatar for Dan D
5.8 years ago by
Dan D6.8k
Tennessee
Dan D6.8k wrote:

In short, yes you do want to clip away the sequencing primer sequences. The question about which end to clip depends on you sequencer, but it will most often be the 3' end.

Before I embark on trimming a collection of samples, I usually run run a few FASTQ files through FastQC. It's easy to use and relatively quick. Before you run it, check the included contaminants file and see if your sequencing primers are located in there. If not, add your sequencing primers.

When you run FastQC, the report will have an "Overrepresented Sequence" section. Check that section and see if your sequence is shown there. An overrepresented sequence that matches with an entry in the contaminants file will be labeled accordingly. Based on this information you can tell your favorite trimming application which sequences to look for and remove.

ADD COMMENTlink modified 5.8 years ago • written 5.8 years ago by Dan D6.8k
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