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Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens.
Islam, Md Fahmid; Watanabe, Atsushi; Wong, Lai; Lazarou, Conor; Vizeacoumar, Frederick S; Abuhussein, Omar; Hill, Wayne; Uppalapati, Maruti; Geyer, C Ronald; Vizeacoumar, Franco J.
Afiliação
  • Islam MF; Department of Biochemistry, University of Saskatchewan, Saskatoon, S7N 5E5, Canada.
  • Watanabe A; Department of Pathology, University of Saskatchewan, Saskatoon, S7N 0W8, Canada.
  • Wong L; Department of Hematology, Nephrology and Rheumatology, Graduate School of Medicine, Akita University, Akita, Japan.
  • Lazarou C; Department of Biochemistry, University of Saskatchewan, Saskatoon, S7N 5E5, Canada.
  • Vizeacoumar FS; Department of Pathology, University of Saskatchewan, Saskatoon, S7N 0W8, Canada.
  • Abuhussein O; Department of Pathology, University of Saskatchewan, Saskatoon, S7N 0W8, Canada.
  • Hill W; College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, S7N 5C9, Canada.
  • Uppalapati M; Department of Pathology, University of Saskatchewan, Saskatoon, S7N 0W8, Canada.
  • Geyer CR; Department of Pathology, University of Saskatchewan, Saskatoon, S7N 0W8, Canada.
  • Vizeacoumar FJ; Department of Pathology, University of Saskatchewan, Saskatoon, S7N 0W8, Canada. ron.geyer@usask.ca.
Sci Rep ; 7(1): 1040, 2017 04 21.
Article em En | MEDLINE | ID: mdl-28432350
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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Análise de Sequência de RNA / Sequenciamento de Nucleotídeos em Larga Escala Limite: Humans Idioma: En Revista: Sci Rep Ano de publicação: 2017 Tipo de documento: Article País de afiliação: Canadá

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Análise de Sequência de RNA / Sequenciamento de Nucleotídeos em Larga Escala Limite: Humans Idioma: En Revista: Sci Rep Ano de publicação: 2017 Tipo de documento: Article País de afiliação: Canadá