Nextera Problem: Odd fragment size after gel selection

[Kitty]

Hi guys, I'm constructing a PE library by using the Nextera DNA library Prep Kit (Illumina compatible). However, when I cut down the 300-350bp, 450-500bp, 600-650bp region separately after 2.0% agarose gel electrophoresis, there comes double peaks instead of a single narrow peak (pictures). I'm sure there's no problem during gel selection, but obviously, this cannot meet the requirement of a PE library. Is there anyone who has some ideas on this?

Start with 150ng gDNA, and after 10 cycles of PCR:


gel selection 300-350bp:


gel selection 450-500bp:


gel selection 600-650bp:

[pmiguel]

Hi Kitty,
This is a very common issue with Illumina libraries. The good news is that your libraries are probably fine.
We sometimes just denature the libraries and run them on an RNA chip to determine their size, etc. Info on how to do that here:

http://seqanswers.com/forums/showthread.php?t=12523

As to what causes the issue exactly, I think it is not completely clear. But probably either the fragments are annealing end-to-end (adapter-to-adapter) forming daisy chains of amplicons, or adapters on both sides of otherwise unrelated amplicons are annealing after denaturation ("bubble products"). More discussion of the possibilities here:

http://seqanswers.com/forums/showthread.php?p=45116#post45116

--
Phillip

[Kitty]

Hi Phillip,
I think you're right, and I'll try to check it on an RNA chip.
Well, as far as I know, when we loading the library on the Illumina flow cell, there will be a denature step before bridge PCR. So does that mean the "daisy chains" will be separated and may not disturb the output data (insert size distribution)?

[pmiguel]

Yes. The problem is mainly that the multimers interfere with determining the total amount of your library. Also they might hide adapter dimer contamination. But that should be obvious after denaturation and running on an RNA chip.

--
Phillip