Answers.Body: <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>
  
Answers.ContentLicense: CC BY-SA 3.0
Answers.CreationDate: 2017-08-07T11:08:18.387Z
Answers.Text: 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.
Answers.UserDisplayName: jxx_fa
Answers.ContentLicense: CC BY-SA 3.0
Answers.CreationDate: 2017-08-04T17:36:59.603Z
Answers.LastActivityDate: 2017-08-04T17:36:59.603Z
Answers.UserDisplayName: Devon Ryan
Body: <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>
  
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2017-08-04T16:39:10.683Z
Comments.Text: Looks like you trimmed your sequences to nothing.  Do you have empty fastq entries in the file?
Comments.UserDisplayName: Bioathlete
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2017-08-07T11:06:07.277Z
Comments.Text: Yes, thanks for your comment, this was the problem.
Comments.UserDisplayName: jxx_fa
Tags: <ngs><fastq><trimming>
Title: FASTQC overrepresented sequences after trimming
ThreadText: # 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.  
  
  ---
  
  ## 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.  
  
  ---
  
  