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Old 01-07-2014, 11:07 PM   #1
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Question How to handle Ns in the middle of reads

For my illumina data fastqc shows presence of N's at positions 13,14,15 in 101 bp longs reads. If i go for cropping first 15 bases by using trimmomatic, it solves the problem but i lose a lot of data. I wanted to know that if i retain the N's what sort of problems would they cause during alignment(bwa+stampy)/variant calling(unified genotyper) and how can i handle these problems?

If any body faced a similar problem how did you handle it? Similar questions asked on different forums but none answered. Could not find a resourse on how variant calling programs handle N's. Do they ignore them? Or consider them as a variation with low confidence scores?

Last edited by themysticgeek; 01-08-2014 at 01:55 AM.
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Old 01-08-2014, 03:04 AM   #2
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Are there N's at those positions in *all* reads? That would almost certainly indicate a technical problem of some kind with this run. In general your sequence provider should not have released this data if that is the case.
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Old 01-08-2014, 04:42 AM   #3
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The N's are in ~50% of the reads. I have attached the Fasqc image for per base n content. This is particular to this sequening run. Did not observe this problem in the other runs

Last edited by themysticgeek; 01-08-2014 at 04:45 AM.
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Old 01-20-2014, 04:25 AM   #4
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Default Similar problem

I've just run FastQC on a published RNAseq dataset (SRX294957) and I see a very similar pattern:

where on position 21-23, 80% of reads are N's. As I'm only interested in expression, I could accept the low read quality as long as the aligner accepts it. I'm using RSEM+bowtie for the purpose, so I'm wondering if bowtie will match NNN against anything in the reference?
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Old 01-20-2014, 04:27 AM   #5
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It will. You can alter the mismatch score due to an N and the maximum number of allowed Ns if you need to (you'll likely need to tweak the minimum allowable score if you do so).
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ambiguous bases, fastqc

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