Nextra on LCM-material -> strange peak

[marnei]

Hi,

We are using Nextera for making libraries from LCM-material (DNA) and ends up with two peaks in the Bioanalyzer curve (see image). Has anyone seen any sequencing results from such a two-peak-library and/or know the reason?

thanks!
/ Mårten

[pmiguel]

Common library artifact. Called "Bird nesting" by Epicentre and variously "double peaking" and "bubble products". Illumina libraries have long enough adapters that, in the absence of high enough concentrations of PCR primer, products just anneal to each other at the adapters. The middle part will be different, in all likelihood, so you get a "bubble" of single strands in the middle that are the respective inserts of the two library molecules. These seem to run at about 2x the apparent size of fully double-stranded molecules. Hence the extra peak.

Caused by too many cycles of PCR. You should still be able to titre the library (via qPCR) and sequence it.

More info here.

--
Phillip