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Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens
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Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens
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Journal Article
Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens
2017
Overview
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.
Publisher
Springer Science and Business Media LLC,Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 45/77
/ 631/61
/ Clustered Regularly Interspaced Short Palindromic Repeats
/ CRISPR
/ DNA
/ Genomes
/ High-Throughput Nucleotide Sequencing
/ High-Throughput Nucleotide Sequencing - instrumentation
/ High-Throughput Nucleotide Sequencing - methods
/ Humanities and Social Sciences
/ Humans
/ Medicine
/ Methods
/ Q
/ R
/ Science
