NEBNext® Ultra™ DNA Library Prep QC- extra peak at around 1500bp

[Leo Lee]

I used the NEBNext® Ultra™ DNA Library Prep for illumina to prepare the library and follow the standard protocol with 250ng DNA input, 8 PCR cycle and size selection for target total library size ~400bp.

However, I noted that quite a number of samples in the same preparation showed an extra peak at around 1600bp (slightly >1500bp, refer to the attached Bioanalyzer profile lanes 4, 5, 7 to 11).

From some forum discussion, a extra peak at 1500bp might be due to the carryover of magnetic beads. However, the peak in my libraries is not that strong and board compared with the typical magnetic bead leftover bioanalyzer profile.

Is the extra peak a single-stranded artefact? Do you think it will affect the sequencing of the library? Any another round dual bead size selection is needed to remove that extra peak?

Thanks very much for your comment.

[Leo Lee]

I would like to attached more bioanalyzer profiles here. I have selected 3 samples from my library batch as an example.

All 3 samples are proceed in one batch. However, sample A does not have the extra peak beyond 1500bp while samples B and C have!

Does anyone think that the second peak may represent single-stranded library products that have self-annealed? As I don't think it is due to the magnetic bead carryout as I had already aliquot the library on magnetic stand.

Will it affect the sample pooling and do the downstream TruSeq Exome capture?

What can I do to avoid this problem?

Thanks!
Leo