Library peak-let contamination at high end of TruSeq BioAnalyzer trace

[Acacianpunk]

Hi everyone,
We are constructing ddRad-seq libraries using a homebrew variant of the TruSeq system. The library prep was pooled and run on a BioAnalyzer before Kapa quantification and running. The resulting BioAnalyzer profile is below.

The majority of our library is centered at the selected size of 300bp with minor adapter dimer contamination in the top sample. Both samples show some variant sized products at very low concentrations ranging from 800-6000bp. Does anyone know what these could be?

Based on reading threads my assumptions are either beads or bubbled molecules but any suggestions would be appreciated before we sequence.

Thank you
Matt

[nucacidhunter]

They do not look like bubbles that you are reffering. They could be remaining non-target digested DNA themselves or their amplification products or both. If size-selection was done on Pippin instrument, my previous statement would not be true. Maybe the bioanalyser reagents and run (specially ladder) should be inspected more closely for further explanation in first step. The answer might be in the modified protocol that have been used to prep libraries.

[Acacianpunk]

Great point. Size selection was gel based so any DNA coming through without adapters possibly could be amplified from at the last step.

This is becoming problematic in quantifying the amount of material to load. We might try a weighted average and see how the cluster generation goes.

[nucacidhunter]

I would first rerun the sample on Bioanlyser to see if the trace is consistent or is due to anomaly in that particular run. If the trace is repeated the next step would be to see if those off target fragments are amplifiable or not by running a PCR with diluted library. If they are not amplified, their presence can be ignored and a qPCR quantification method should result in consistent cluster numbers. If you are using Qubit for quantification, those fragments would interfere with quantification and likely source of them should be explored. In a gel run in optimum condition and extraction you should not get those large fragments after PCR (assuming size selection is before PCR on digested-ligated DNA), although smaller fragments can be expected.

Size selection was gel based so any DNA coming through without adapters possibly could be amplified from at the last step.

This is less likely as the fragments without adapters will lack priming site for amplification.