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.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Islam MF,Watanabe A,Wong L,Lazarou C,Vizeacoumar FS,Abuhussein O,Hill W,Uppalapati M,Geyer CR,Vizeacoumar FJdoi
10.1038/s41598-017-01170-zsubject
Has Abstractpub_date
2017-04-21 00:00:00pages
1040issue
1issn
2045-2322pii
10.1038/s41598-017-01170-zjournal_volume
7pub_type
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