Question: Doubt on removing duplicates on amplicon sequencing data
2
gravatar for Picasa
21 months ago by
Picasa490
Picasa490 wrote:

Hi,

I am looking to make a simple SNP analysis.

I have different individuals from which we have targeted specific markers. Then the reads I have come from amplicon sequencing. My questions are:

1) Do I have to remove duplicates ? From what I understand, tools like Picard look for the same 5', but by definition, amplicon sequencing reads start by the same position?

2) If no: how can I treat these data, because if if an error is propagate during the PCR, it will be a bad call at the end ?

Edit: 3) There are 2 type of duplicates: optical and pcr, in that case do I have to remove only optical duplicates ? if yes, do you know how ? seems that Picard doest not separate optical and pcr.

Thanks for your help.

amplicon duplicates • 1.1k views
ADD COMMENTlink modified 21 months ago by Devon Ryan94k • written 21 months ago by Picasa490
2
gravatar for Devon Ryan
21 months ago by
Devon Ryan94k
Freiburg, Germany
Devon Ryan94k wrote:
  1. No for the reasons you listed.
  2. Correct, that's the down-side to amplicons (unless you put UMIs on your PCR primers).
  3. Removing optical duplicates can be done with clumpify from BBTools. However, this doesn't end up working that well for amplicons unless you spiked in a lot of PhiX or had a very large number of amplicons on the same lane. Otherwise you end up overly removing sequence (not that this ends up being a huge problem).
ADD COMMENTlink modified 21 months ago • written 21 months ago by Devon Ryan94k

Thanks Devon, I have edited my post with another question. Maybe you have not seen it:

3) There are 2 type of duplicates: optical and pcr, in that case do I have to remove only optical duplicates ? if yes, do you know how ? seems that Picard doest not separate optical and pcr.

ADD REPLYlink modified 21 months ago • written 21 months ago by Picasa490
1

I just edited my response accordingly.

ADD REPLYlink written 21 months ago by Devon Ryan94k
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