Alternate Cluster Generation for Picomolar NGS Libraries.

While advancements in library preparation techniques now enable NGS library preparation from single cells and nano- to picograms of DNA or RNA, it can still be difficult to reach Illumina's recommended final library concentration of 2 nM for DNA template preparation prior to cluster generation. This can be an acute issue for ChIP-seq, RIP-seq, and LCM-based samples. We have developed a simple and robust alternative method that maximizes number of reads from an Illumina library without having to sacrifice precious sample or perform excessive amplification. This new method requires between 10- and 100-fold less library material than the standard Illumina protocol. By direct comparison to the Illumina standard DNA template preparation method, we demonstrate that cluster density, Q-scores, raw reads and PF reads, and data quality are not affected by this new protocol. We have successfully used this method on over 115 HiSeq runs and 100 MiSeq runs of both single- and paired-end varieties.