For those readers who are making Illumina libraries for NGS, which I assume is many of you, I’d like to direct you to this new paper by Sheila Fisher’s group at the Broad Institute. In this paper Fisher describes the Broad’s method for targeted re-sequencing. However, of broader (pun intended) interest is her description of a ‘tweak’ the the general Illumina Library prep protocol.
This tweak is a simplification of the reaction clean-ups. I’m assuming most labs have moved away from Qiagen PCR purifications to magnetic bead cleanups. If you haven’t I highly recommend doing so. I also recommend the Agencourt SPRIPlate Super Magnet Plate. Although it’s a bit more expensive than an entry level 96 well plate magnet, the extra strength of the rare-earth magnets really keeps the beads stuck to the sides of the wells. Anyway, I digress. The tweak Fisher describes involves keeping the beads in the wells during the End-Repair, Plus-A, and Ligation steps. This reduces the number of beads used by two-thirds, but more importantly it helps keep your DNA in the tubes and thus increases yields. Honestly, I can’t wait to try this out.
Anyway, this paper is definitely worth a look-see. There’s a lot more in it that I glossed over.