Nextera DNA Exome Tagmentation Peaks

[kblake]

I'm new to the library prep side of sequencing and I've been having trouble getting my post-tagmentation, post-amplification libraries to match the profile in the Nextera DNA Exome protocol.

I've repeated the process a few times with increasing care (using low-bind tubes, checking input concentrations with Qubit, testing beads on a DNA ladder, etc.) and I'm finding that I have a flat, right skewed bioanalyzer peak (the peak should be between 200-500bps and mine hangs over 1000bps - see attached trace). Illumina's FAQ claims that a secondary, larger peak is common and should be of no concern since it's often an amplification product but I'm not convinced that's what I'm seeing here.

Has anyone else had a pre-enrichment library prep with a similar profile? Is this cause for concern (over- or under-tagmentation)? If so, do you have any tips on how the protocol can be adjusted to account for it?

[nucacidhunter]

The library profile indicates input DNA overload and it can affect on target capture (enrichment efficiency).

Exon length on average are around 200bp and a 1Kb fragment might have hybridized to target in the middle so sequencing 100 cycles from ends will result in off target reads.

[kblake]

Is it common to have to use less input DNA than the kit recommends? I'm worried about reducing my library complexity and I don't know how much the input concentration influences that.

[nucacidhunter]

You should not use less DNA. Your thermocycler or kit also could be faulty (expired, exposed to high temperatures). You can test lower DNA input and see if you get shorter fragments.