Abnormal Bioanalyzer Read Out after Library Prep

[omy567]

Hey guys. I'm currently preparing some sequencing libraries for the first time from some chip material, and I'm getting some weird results when I run a bioanalyzer trace on them. I prepared my chromatin using a Diagenode Biorupter and ran a size analysis before IPing on my material. I got a nice sizing range between 250-550 bp's on the sizing gel I ran. I used the Illumina Truseq Genomic DNA Prep Kit to prepare libraries from both my inputs and ChIP DNA using their gel-free method. So, I expected some larger fragments in my preps once I ran it on the bioanalyzer. However, I got a huge peak at a high molecular weight (~1300 bp) that just didn't show up in my original sizing gel. It's weird because I IP'd off the same crosslinked material with 2 different antibodies, and one of the antibodies does not show this large molecular weight peak. I could chalk it up to one antibody having a preference for larger fragments, but that is a stretch since there were barely any in that size range in my original sizing gel. If anyone has any input on what this could be or what I could do to fix it, I would appreciate it. I've attached the bioanalyzer traces to this post. The weird trace with the high molecular weight peak is g1 IP2 and the expected trace is H31 IP12. Thanks.

[sara_ahmed]

The large peak could possibly be a PCR artefact. Since the IPs may have had different recovery, the amount of template going into the amplification will be different despite the starting size range being the same. Try denaturing a portion of the library and running it out on the Bioanalyzer to see if it changes things and/or add more primer and do another cycle of PCR on some of the material to see if that will diminish the larger sized peak. Sometimes you get some portion of the library being single stranded (running out of primer) and it runs strange on the Bioanalyzer.

Sara Ahmed, PhD | Director of Sequencing | Cofactor Genomics
3141 Olive St. | St. Louis, MO 63103 | tel. (314) 531-4647

[pmiguel]

The second BA run just looks like a "bubble product". That is the library molecule concentration got high enough in the PCR to allow disparate single-stranded library molecules to anneal at their adapters. (With the single-stranded "bubble" forming from the unrelated center sections of the library molecules.)

As Sara mentions, this partially single-stranded molecule tends to run larger than its actual molecular weight. Possibly the non-annealed part of the molecule causes extra drag during electrophoresis.


BTW, denaturing/renaturing the sample won't work because the kinetics for library molecule strands finding their real complement are poor. Some people have had luck with adding fresh primers and doing another cycle of PCR. However, it is not necessary for sequencing the library. Generally bubble-peak samples work fine for sequencing.
--
Phillip

[sara_ahmed]

We've had mixed results with the denaturing/renaturing so I agree it probably won't work but Illumina tech support seems to think its the way to go so I thought I'd throw it out there.

Adding fresh primers and doing an additional cycle is a good way of diagnosing if the peak is from a bubble product. You're looking for it to diminish. We use it as a diagnostic but as Philip said, it sequences fine and you don't actually have to 'fix' the library.