Skip to content

Another reason not to overamplify your NGS libraries

February 5, 2012

I’m taking a long weekend off to read about plants, do some surfing and contemplate what life would be like in Western Australia. Anyway, somehow I came across this paper (DNA rehybridization during PCR: the ‘Cot effect’ and its consequences) last night which has nothing to do with either but a lot to do with what I think is a mistake most people are making in their NGS library preps. The process of sequencing on all the NGS platforms creates systemic biases in the samples sequenced, which of course not only confuses the analysis but can also lead to false conclusions being made. The major contributor to bias for epigenetic and gene expression assays comes from PCR amplification of the library, for example biases against regions with very low and very high GC content. The Kapa HF polymerase causes less of this than some other polymerases which is why I prefer it. Obviously, the more cycles you do the worse it gets. The paper linked above documents another bias created by PCR that I had not thought about. Fortunately this type of bias can be minimalized by simply not over-amplifying your libraries and keeping primers in excess. Thus we have another reason (there are many more) to quantitate the minimum number of PCR cycles you need to amplify your library with using the method I describe in my protocols. It may not be standard practice and I have never seen it recommended in any other protocols but that doesn’t mean you should skip it. Over-amplification is exactly what it is and should be avoided if for no other reason than it is easy to avoid.

Advertisements

From → Posts

2 Comments
  1. Dear Ethan,
    This is good explanation for reducing the bias! It would be great if you can create a hyperlink to your protocol from this page!

  2. ethanomics permalink

    Good idea and done. Edit: Thinking about it, this is a step where a student in the lab made a mistake and I always have to explain in better detail which means it is not very clear. So I have given it its own protocol page. https://ethanomics.wordpress.com/ngs-pcr-cycle-quantitation-protocol/

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: