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

Md Fahmid Islam,Atsushi Watanabe,Franco J Vizeacoumar,Maruti Uppalapati,Frederick S Vizeacoumar,C Ronald Geyer,Conor Lazarou,Wayne Hill,Lai Wong,Omar Abuhussein

doi:10.1038/s41598-017-01170-z

Md Fahmid Islam, Atsushi Watanabe + Show 8 more

Open Access

https://doi.org/10.1038/s41598-017-01170-z

Copy DOI

Journal: Scientific Reports	Publication Date: Apr 21, 2017
Citations: 5	License type: open-access

Affiliation: University of Saskatchewan, Akita University

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.

Highlights

Recent advancements in sequencing technologies and their applications in functional genomics have significantly broadened our understanding of cellular functions and our ability to perform translational science
3 million transduced cells were used in the screening process to maintain ~200 fold representation for the shRNA library
Heteroduplex formation results in Ion Sphere Particle (ISP) being surrounded by two different templates, which increases the rate of polyclonality and generates many polyclonal and low quality reads

Summary

Introduction

Recent advancements in sequencing technologies and their applications in functional genomics have significantly broadened our understanding of cellular functions and our ability to perform translational science. Large-scale, genome-wide screens using pooled shRNA or CRISPR libraries query the genome and subsequent sequencing identifies the unique shRNA or sgRNA sequences that affect cell viability[1,2,3,4,5,6] Such methods are increasingly applied to identify therapeutically relevant synthetic lethal targets[4,5,6,7,8,9,10,11] or cancer-specific essential genes[2, 3, 12,13,14,15,16,17,18,19,20]. A common theme in all of these sequencing reactions is that they depend on mixed-oligo PCR reactions wherein unique reads are binned by molecular barcodes distinctively associated with each sequence, allowing multiplexing While these sequencing methods are increasingly used in large core facilities, there are a number of challenges that impede their widespread usage in standard labs where cost-effective bench-top sequencers could be routinely employed. Hairpin structures result from palindromic sequences, and can lead to incomplete, low quality, and polyclonal reads[26, 34,35,36,37]

Methods

Results

Conclusion