<p>As @AaronBerlin mentioned, you didn't remove reads that were completely trimmed. Next time use the <code>--minimum-length</code> option and set it to something reasonable, like 20. Alternatively, use "Trim Galore!", which is a wrapper around cutadapt that has more reasonable defaults.</p>

CC BY-SA 3.0
2017-08-07T11:08:18.387Z
Indeed this was the problem, I set the --minimum-length parameter to 20 and everything is fine. I didn't try Trim Galore! because I am working in a cluster and it is not installed at this moment but in the future I will try it as it seems more intuitive.
jxx_fa
CC BY-SA 3.0
2017-08-04T17:36:59.603Z
2017-08-04T17:36:59.603Z
Devon Ryan
<p>I have a set of RNA-seq samples from different experiments (Single and Paired End, depending on the experiment). I ran <a href="https://www.bioinformatics.babraham.ac.uk/projects/fastqc/" rel="noreferrer">FASTQC</a> in all the samples and found overrepresented adapter sequences:<a href="https://i.sstatic.net/RlZlZ.png" rel="noreferrer"><img src="https://i.sstatic.net/RlZlZ.png" alt="enter image description here"></a></p>

<p>I removed the adaptors (TruSeq adaptors) using <a href="http://cutadapt.readthedocs.io/en/stable/guide.html" rel="noreferrer">Cutadapt</a> (in addition, I removed low quality and N bases from the 3' end of the reads). After that, I ran again FASTQC and the output is the following (representative example) :
<a href="https://i.sstatic.net/tpgVR.png" rel="noreferrer"><img src="https://i.sstatic.net/tpgVR.png" alt="enter image description here"></a></p>

<p>Does anyone know what is happening? Now I have an overrepresented sequence for which no sequence is provided. What does this mean? </p>

CC BY-SA 3.0
2017-08-04T16:39:10.683Z
Looks like you trimmed your sequences to nothing.  Do you have empty fastq entries in the file?
Bioathlete
CC BY-SA 3.0
2017-08-07T11:06:07.277Z
Yes, thanks for your comment, this was the problem.
jxx_fa
<ngs><fastq><trimming>
FASTQC overrepresented sequences after trimming
# FASTQC overrepresented sequences after trimming

(Asked by: username_0 on 2017-08-04)

I have a set of RNA-seq samples from different experiments (Single and Paired End, depending on the experiment). I ran [FASTQC](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) in all the samples and found overrepresented adapter sequences:[![enter image description here](https://i.sstatic.net/RlZlZ.png)](https://i.sstatic.net/RlZlZ.png)

I removed the adaptors (TruSeq adaptors) using [Cutadapt](http://cutadapt.readthedocs.io/en/stable/guide.html) (in addition, I removed low quality and N bases from the 3' end of the reads). After that, I ran again FASTQC and the output is the following (representative example) :
[![enter image description here](https://i.sstatic.net/tpgVR.png)](https://i.sstatic.net/tpgVR.png)

Does anyone know what is happening? Now I have an overrepresented sequence for which no sequence is provided. What does this mean?

**Tags:** ngs, fastq, trimming
> **Comments:**
> **username_1**: Looks like you trimmed your sequences to nothing.  Do you have empty fastq entries in the file?  
> **username_0**: Yes, thanks for your comment, this was the problem.  

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## 1 answers

### Answer by username_2 on 2017-08-04. Score: 6 | Accepted answer

As @AaronBerlin mentioned, you didn't remove reads that were completely trimmed. Next time use the `--minimum-length` option and set it to something reasonable, like 20. Alternatively, use "Trim Galore!", which is a wrapper around cutadapt that has more reasonable defaults.

> **Comments:**
> **username_0**: Indeed this was the problem, I set the --minimum-length parameter to 20 and everything is fine. I didn't try Trim Galore! because I am working in a cluster and it is not installed at this moment but in the future I will try it as it seems more intuitive.  

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