Question: Are these reads all PCR replicates ?
0
gravatar for Nicolas Rosewick
4.6 years ago by
Belgium, Brussels
Nicolas Rosewick9.1k wrote:

Hi,

I did an sequencing run where I enriched specific DNA regions. Thus I expect to have a lot of PCR duplicates. In the figure below you can see a IGV print screen of a specific region. You can see that pretty all reads are the same. But several of them has some mismatches (see arrows) (less than 1% of the reads have some mismatches). Can I consider that they are PCR duplicates ? or are they real different DNA fragments ?

enter image description here

Thanks

dna-seq • 1.1k views
ADD COMMENTlink modified 4.6 years ago by stolarek.ir650 • written 4.6 years ago by Nicolas Rosewick9.1k

I think that checking the Phred score for those bases that are different can give you some insights. But in general I don't think there's an easy way to know if two reads are PCR duplicates.

ADD REPLYlink written 4.6 years ago by Martombo2.6k

they seems to have phred score between 15 and 20. But several of them have good phred score (>30)

ADD REPLYlink written 4.6 years ago by Nicolas Rosewick9.1k

What sequencer was used? Do the differences occur in a homopolymer region?

ADD REPLYlink written 4.6 years ago by 5heikki9.0k

We used a miSeq and it's not a homopolymer region

ADD REPLYlink written 4.6 years ago by Nicolas Rosewick9.1k

Which polymerase enzyme was used in PCR?

ADD REPLYlink written 4.6 years ago by 5heikki9.0k
1
gravatar for surendra
4.6 years ago by
surendra30
South Africa
surendra30 wrote:

Hi,

You can use Picard tools to identify the PCR duplicates (with option MarkDuplicates)

http://broadinstitute.github.io/picard/command-line-overview.html#Overview

ADD COMMENTlink written 4.6 years ago by surendra30

It looks like Haloplex data - the last thing you want to do is run MarkDuplicates on it. This is a terrible idea.

ADD REPLYlink written 4.6 years ago by Daniel Swan13k
0
gravatar for Jenez
4.6 years ago by
Jenez520
Sweden
Jenez520 wrote:

Those differences could have easily arisen during the sequencing of the fragments, as no sequencing machine is flawless and will produce erroneous sequencing reads.

ADD COMMENTlink written 4.6 years ago by Jenez520
0
gravatar for stolarek.ir
4.6 years ago by
stolarek.ir650
Poland
stolarek.ir650 wrote:

Looking at this picture it looks like sequencing error <- more or less random between sequences, however

In aDNA we observe lots of fixed errors in some portion of the reads, the possible explanation for that is that those mismatches if present in the exactly same place in probable duplicate read come not from sequencing error, but either from polymerase error during PCR or from sample contamination. To test if it really was a polymerase you can do analysis cycle by cycle.

ADD COMMENTlink written 4.6 years ago by stolarek.ir650
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