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Experiment: deep sequencing for mutants in 700nt fragment. the fragment of dna was preamplified by primers flanking the fragment followed by hiseq. per base coverage was calculated by coverageBed -d -abam in.bam -b ref.bed > out.cov

Observation: two distinct peaks in coverage at the ends as below plot.. coverage vs positions enter image description here the peaks are made from reads having part of primers..thus also show soft clipping at ends.. there is a huge difference in the calculations if i include such reads And if I exclude them.

Question: is there anyone who knows how to handle such a situation?

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    $\begingroup$ It's absolutely not clear, how you prepared your library for sequencing, where the boundaries of your primers are and what exactly bothers you in this context. $\endgroup$ – har-wradim Apr 9 '14 at 11:08

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