Adapter Dimers after Size Selection (?)

[mhapp95]

Hello all!

Recently I ran a library on our lab's new Next Seq 500 and came across an interesting problem. While processing the data from the instrument, I noticed that one of my samples had an excess of 35 bp poly(N) reads...as in 40% (yikes!). From what I can tell this generally means adapter dimer, as the bcl2fastq tool masks adapters with "N" as well as reads below the default of 35 bases. None of my other samples had this problem.

We had previously ran the samples before pooling on an Agilent TapeStation, and noticed this particular sample had a high concentration of adapter dimer. So we ran the library on a Blue Pippin to select for a bp range of 200-500, and rechecked with the TapeStation to confirm the 120 bp peak was gone. Which it was.

So we proceeded with the run. And then I discovered this problem.

I then ran some of what I had left of the size selected library on both a regular agarose gel and a denaturing gel. A ~120 bp band showed up on the denaturing gel only!

My question is, has anyone run into this before or do they know if some secondary structure could be causing it? Where could this band be hiding?

[jdk787]

I've seen this with libraries that had a lot of dimer going into amplification and the library was overamplified.

I think that when the PCR primers have been exhausted, the dimer strands will anneal to library strands at the adapter regions creating a half dimer half library bubble product. When these products are run on a non-denaturing gel they will migrate slower than a 120bp dimer and appear larger, but when you use a denaturing gel the dimer strand will run as expected.

Also, the dimer strands will cluster more efficiently during sequencing which will cause them to be over-represented in the sequencing results.

[pmiguel]

Wow mhapp95, that is awesome that you actually ran the denaturing gel. I always suspected that what jdk787 was describing, was what was going on, but I was too lazy to do what you did.

Can you give details on your procedure for doing the denaturing gel? Seems like if you used the denaturing gel instead of your Blue Pippin, you would be able to completely remove adapter dimers from a library.

--
Phillip

[CoastGen]

I know of a group that saw this years ago when working with miRNA preps. Native gels would show a single band after size selection, but 2 bands on denaturing gels. They actually sequenced it and found that is was indeed an adaptermer. In the native gel, it formed a double-stranded species with actual library fragments, which led to a hairpin structure with aberrant mobility that allowed this structure to migrate with the normal library in a native gel.

[Christine]

We've seen the typical ssDNA-bubble products/daisy chain libraries many times. I used to ignore it until we encountered a library that was hiding enormous amounts of adaptor dimers in there.

Here's an image of the original library we received (a ~600 bp amplicon):



Almost every sequence was clearly a short primer dimer. We reasoned the same as others here: when the primers are limiting in the amplification, dimers could hybridize to the library fragment ends and escape clean-up.

To fix this we re-amplified for a few cycles with fresh PCR reagents to denture these duplexes and make everything properly double-stranded, then we could clean up the dimers.

Here's what the library looked like after that PCR test, prior to clean-up: