<p>So, I am analysing 18S amplicon data (fastq files) to be able to eventually investigate the taxa composition.</p>
<p>After removing primers (using <code>cutadapt</code>) from both R1 and R2 (assuming primers are found at the beginning of my sequences), I am still able to detect, apparently at random, a few reads that contain my primers (i.e. a part of the sequence matching the primer) in, again apparently random, positions.</p>
<p>I am able to detect almost all my primers (16 out of 24, both forward and reverse) in at least one sequence and I am wondering how common (or how weird) this is. I guess it is important to consider that my primers contains a few, i.e. from 1 to 3, wild-card bases that may match multiple other bases (apparently called <a href="https://www.biosearchtech.com/support/faqs/custom-oligonucleotides-modifications/what-are-wobbles" rel="nofollow noreferrer">whobble bases</a>), which makes it easier to match a random sequence (there are multiple possibilities for an actual match).</p>
<p>Problem is that the presence of primers in the sequences may affect downstream analyses (e.g. with DADA2 <a href="https://benjjneb.github.io/dada2/tutorial_1_8.html" rel="nofollow noreferrer">they are very clear</a> about the fact that primers have to be removed) in several ways and I was wondering if these sequences I am finding may be interfering somehow of if I can live with it.</p>

CC BY-SA 4.0
2021-08-23T09:43:23.280Z
The primers will be at the 3'-end of the reads. Make sure you check for them there.
Devon Ryan
CC BY-SA 4.0
2021-08-23T10:54:53.607Z
@DevonRyan yes, that I know and that I did so no primers at 3'. I guess it just happens to find them inside the reads, as random matches, but I wasn't expecting that many actual matches. I guess it's statistics, then.
gbt
CC BY-SA 4.0
2021-08-30T14:25:23.100Z
Interesting, have you tried to map a read with a sequence matching a primer to the reference, or even simply blasting it? Does the reference contain the "primer sequence"? ("" as we don't know if it's a homologous sequence or not)
Kamil S Jaron
CC BY-SA 4.0
2021-09-01T08:03:57.933Z
@KamilSJaron apparently this is something that should not be happening and I'm trying to understand why it does.
gbt
<phylogenetics><sequence-analysis><fastq><primer>
How likely is it to find primers sequences in already-trimmed reads?
# How likely is it to find primers sequences in already-trimmed reads?

(Asked by: username_0 on 2021-08-23)

So, I am analysing 18S amplicon data (fastq files) to be able to eventually investigate the taxa composition.

After removing primers (using `cutadapt`) from both R1 and R2 (assuming primers are found at the beginning of my sequences), I am still able to detect, apparently at random, a few reads that contain my primers (i.e. a part of the sequence matching the primer) in, again apparently random, positions.

I am able to detect almost all my primers (16 out of 24, both forward and reverse) in at least one sequence and I am wondering how common (or how weird) this is. I guess it is important to consider that my primers contains a few, i.e. from 1 to 3, wild-card bases that may match multiple other bases (apparently called [whobble bases](https://www.biosearchtech.com/support/faqs/custom-oligonucleotides-modifications/what-are-wobbles)), which makes it easier to match a random sequence (there are multiple possibilities for an actual match).

Problem is that the presence of primers in the sequences may affect downstream analyses (e.g. with DADA2 [they are very clear](https://benjjneb.github.io/dada2/tutorial_1_8.html) about the fact that primers have to be removed) in several ways and I was wondering if these sequences I am finding may be interfering somehow of if I can live with it.

**Tags:** phylogenetics, sequence-analysis, fastq, primer
> **Comments:**
> **username_1**: The primers will be at the 3'-end of the reads. Make sure you check for them there.  
> **username_0**: @DevonRyan yes, that I know and that I did so no primers at 3'. I guess it just happens to find them inside the reads, as random matches, but I wasn't expecting that many actual matches. I guess it's statistics, then.  
> **username_2**: Interesting, have you tried to map a read with a sequence matching a primer to the reference, or even simply blasting it? Does the reference contain the "primer sequence"? ("" as we don't know if it's a homologous sequence or not)  
> **username_0**: @KamilSJaron apparently this is something that should not be happening and I'm trying to understand why it does.  

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## 0 answers

