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

Abstract

Next generation sequencing is becoming the method of choice for functional genomic studies that use pooled shRNA or CRISPR libraries. A key challenge in sequencing these mixed-oligo libraries is that they are highly susceptible to hairpin and/or heteroduplex formation. This results in polyclonal, low quality, and incomplete reads and reduces sequencing throughput. Unfortunately, this challenge is significantly magnified in low-to-medium throughput bench-top sequencers as failed reads significantly perturb the maximization of sequence coverage 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 sequencing strategy minimizes failed sequences and is also equally amenable to high-throughput sequencers for increased multiplexing. Towards this, we also demonstrate that our strategy to reduce heteroduplex formation improves multiplexing capabilities of pooled CRISPR screens using Illumina NextSeq 500. Overall, our method will facilitate sequencing of pooled shRNA or CRISPR libraries from genomic DNA and maximize sequence coverage.

Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture

“…Currently, the majority of industrial strains with optimized performance have been generated by mutagenesis screens, resulting in elevated product titres, the utilization of a greater variety of cheap nutrient sources, development of optimized morphologies for improved rheological performance in submerged fermentation, or enhanced resistance to toxic metabolic intermediates, amongst many other desired phenotypes [2, 4]. However, a significant limitation to mutagenesis approaches is that the molecular basis of strain optimisation is extremely difficult to reverse engineer [5], and thus favourable attributes of production strains cannot be easily applied to different isolates or fungal species [4].…”

Downregulation of Blimp1 inhibits the maturation of bone marrow-derived dendritic cells

“…DNA oligos (shRNA sequences) were dissolved in TE (pH 8.0) solution at a concentration of 100 µM. PCR amplification was used to complete the annealing process and the products were preserved at 4˚C (30). The shRNA samples were obtained following the annealing process.…”