Overamplified library question

[Buckethead84]

Hi all,

We've recently run into an issue where a member of our lab accidentally added an extra cycle to the final amplification step of the library prep protocol. As many of you know, this results in secondary structure bubble products that run roughly 2X the size of the major peak of the perfectly double-stranded library.

After QC, we also found an abundance of adapter-adapter ligation products, which we subsequently size selected out using ampure. The resulting library looked like only the 2X overamplified library, with no presence of adapter-adapter ligation products around 120 bp.

My question is this - in the event of overamplification in the prescence of adapter-adapter ligation products, is it possible that there could be adapter-adapter ligation products hidden in our overamplified library, even after size selection? I don't believe the library molecules denature during the DNA precipitation step in Ampure & these molecules have P5 & P7 sequences on them as well, so it's reasonable to assume they'll be able to hybridize to the library molecules with inserts - which makes them appear artificially larger.

We're trying to justify if it is worth sequencing this sample. Thanks for the input!

[HESmith]

Yes, adapter dimers can anneal to insert-containing molecules via the adapter ends. The simplest solution is to perform one new round of PCR using ~1/2 (or less, depending upon the amount) of the library as template, followed by clean-up and QC. Insert-containing library and adapter dimer will now migrate at their true sizes.