Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens

Next generation sequencingis becoming the method of choice for functional genomicstudies that use pooled shRNA or CRISPRlibraries. A key challenge in sequencingthese mixed-oligo libraries is that they are highly susceptible to hairpin and/or heteroduplexformation. This results in polyclonal, low quality, and incomplete reads and reduces sequencingthroughput. Unfortunately, this challenge is significantly magnified in low-to-medium throughput bench-top sequencersas failed reads significantly perturb the maximization of sequencecoverage and multiplexing capabilities. Here, we report a methodology that can be adapted to maximize the coverage on a bench-top, Ion PGM System for smaller shRNA libraries with high efficiency. This ligation-based, half-shRNA sequencingstrategy minimizes failed sequencesand is also equally amenable to high-throughput sequencersfor increased multiplexing. Towards this, we also demonstrate that our strategy to reduce heteroduplexformation improves multiplexing capabilities of pooled CRISPRscreens using Illumina NextSeq 500. Overall, our method will facilitate sequencingof pooled shRNA or CRISPRlibraries from genomic DNA and maximize sequencecoverage.