TruSeq PCR-free libraries inefficient ligation of adapters

[kgoglin]

It seems to be well known that the ligation efficiency of the adapters for the TruSeq PCR-free library prep is low. Bioanalyzer concentrations can be 3-30 fold higher than qPCR. I believe the qPCR concentrations are accurate, however, the sequencing runs are under-clustered. I suspect the libraries are not getting denatured properly. Has anyone had this happen?

My theory is that the NaOH is being used up on the DNA fragments with no adapters and therefore less NaOH is available to denature the libraries.

Thanks,
Karrie

[thermophile]

what kind of samples? I'm fighting with difficult really high gc bacteria that I've truseq pcr free prepped. Ive started heat denaturing after NaOH denaturing. it may be helping a bit? I'm still testing the libraries by spiking them in other runs, haven't tried this on its own run yet