FastQC report: Per base N content is a pass but in the overrepresented sequences I have Ns. Why could that be?

Question

[eva]

Hello,

I would appreciate any thoughts on this. In the FastQC report on my Illumina PE reads, the Per base N content is 0 while in the overrepresented sequences I have Ns. I haven't come across any discussion in Biostars or any where else where the overrepresented sequences are Ns. Can anybody explain why this could be and what I need to do about it. Thank you.

Edit: here are the links to the Per base N content and overrepresented sequence report from FastQc:

• https://www.dropbox.com/s/jh5r17szcr6e0yn/per_base_n_content.png?dl=0
• https://www.dropbox.com/s/iblj6zutjf6d2ph/overrepresented.png?dl=0

[Devon Ryan]

Can you post the relevant output somewhere so we can have a look?

Answer 1

[Istvan Albert]

The documentation states that you get a warning if the number of Ns is above 5%

Per Base N

The overrepresented sequences will raise a warning if they are over 0.1%

Overrepresented Sequences

So you can see how the situation you observe could easily happen.

[eva]

Yeah, ok. Any ideas what I should do about those Ns? Thank you.

[Devon Ryan]

If they're near the end, trim them. If they're in the middle, the read won't align anyway so don't worry about it. If you're really bothered by it, just filter out reads with Ns.

[eva]

Alright Devon. Thank you.

Answer 2

[BioApps]

The 'NNNNNNNNNNNNNN.......' sequences (when present in a file) have a high chance to appear in the 'Overrepresented sequences' report because they... well... are all similar.

To prevent their appearance in the report, you can filter out all sequences that have N bases in proportion higher than 3-5% (your choice).

You will have to clean bad sequences from your file anyway, before putting it in your analysis pipeline.

Efficiently process (view, analize, clip ends, convert, demultiplex, dereplicate) SFF/FastQ files