Extra peak after TruSeq DNA library prep

[anna_m]

Hello,
We made library preps following the standard TruSeq DNA library prep method, beginning with 3ug gDNA and took this through the steps shearing-end repair-A tailing-ligation-size selection-PCR.

We use the blue pippin for size selection and 6 cycles of amplification.

Now, we are seeing an extra peak over double the size of the intended library peak (see attached). Is this a primer-dimer peak? Or a single-stranded artefact? Do you think it will affect the sequencing of the library?

Any advice you could give me would be much appreciated.

Thank you.

[microgirl123]

It's not primer dimer (that would be ~128 bp). It may just be a bubble peak from too many cycles of PCR. Sometimes, when the strands reanneal only the adapters anneal with a mismatched fragment in the middle. This creates a "bubble" and the DNA runs strangely on the BioAnalyzer.

[Genohub]

Typically bubble products won't affect your sequencing results because the double stranded product is denatured prior to cluster generation. To verify, heat a small aliquot of the sample to 95°C for 5 minutes and place on ice. The denatured product should appear as a single band on a Bioanalyzer RNA Pico 6000 Chip Kit.

- Genohub