removing PCR duplicates from MedipSeq Data

Question

[gskbioinfo143]

Dear all

i have paired end data of medip seq from illumina platform. i have done mapping using bowtie2 and after mapping is it necessary to remove PCR Duplicates from bam file. if it is not removed whether it will effect in DMR or peak calling analysis.

Need help

Thank you

Answer 1

[Medhat]

PCR duplicates are sequence reads that result from sequencing two or more copies of the exact same DNA fragment, which, at worst, may contain erroneous mutations introduced during PCR amplification, or, at the very least, make the occurrence of the allele(s) sequenced in duplicates appear proportionately more often than it should compared to the other allele

It is always recommended to remove PCR duplication and you can use this tool

https://broadinstitute.github.io/picard/command-line-overview.html#MarkDuplicates

you can read more about the topic here:

Evaluating the necessity of PCR duplicate removal from next-generation sequencing data and a comparison of approaches

Answer 2

[Brian Bushnell]

Whether or not you should remove PCR duplicates depends on many factors.

1) Did you PCR-amplify? If not, then there are no PCR duplicates.

2) Are your reads paired? If not, most of your duplicates will be false-positives (coincidental rather than PCR duplicates).

3) Is your coverage locally super-high (RNA-seq, Chip-seq, etc)? If so, most of your duplicates will be false-positives.