We're currently testing some new methods in the lab to find an optimal exome library prep, not the capture just the prep. The ideal would be a PCR-free exome, however we want to work with limited material and so maximising library prep efficiency is key, and we'll still use some PCR. The two main factors we're considering are ligation temperature/time, and DNA:adapter molar ratio. The major impact of increasing ligation efficiency is to maximise library diversity, and this applies whatever your DNA input. Even if you're not working with low-input samples, high-diversity libraries minimise the sequencing required for almost all applications.
During discussions with some users it became evident that not everyone knows what the critical bits of a DNA ligation reaction are and since adpater ligation is key to the success of many NGS library preps I thought it would be worthwhile summarising some key points here.
|Image from taken from Bob Lehman's 1974 Science paper|