How To Handle Ns In The Middle Of Reads
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8.4 years ago
kautilya ▴ 430

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 has 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?

Following is the image for per base n content from fastqc

qc fastqc • 2.9k views
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Shouldn't you first investigate why you got those weird Ns at these positions?

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These are possibly due to machine read errors during sequencing. These are particular to only 1 of 3 runs. Looking for a way of handling these without losing a lot of sequence data.

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7.1 years ago
Gabriel R. ★ 2.8k

If you want to do a BWA followed by GATK, I would use your reads as is. They likely have a base quality of 0 and GATK overlooks them. BWA will substitute them for random bases but fallacious alignments induced by those bases will be rare. 

The cause ? If this is Illumina, sometimes the reagents do not make it to the flowcell for a cycle or two due to pump problems or air bubbles. 


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