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 polyc...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2017-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-017-01170-z |
| _version_ | 1818428202340581376 |
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| author | Md. Fahmid Islam Atsushi Watanabe Lai Wong Conor Lazarou Frederick S. Vizeacoumar Omar Abuhussein Wayne Hill Maruti Uppalapati C. Ronald Geyer Franco J. Vizeacoumar |
| author_facet | Md. Fahmid Islam Atsushi Watanabe Lai Wong Conor Lazarou Frederick S. Vizeacoumar Omar Abuhussein Wayne Hill Maruti Uppalapati C. Ronald Geyer Franco J. Vizeacoumar |
| author_sort | Md. Fahmid Islam |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T14:57:52Z |
| format | Article |
| id | doaj.art-89b483b604f54dc19de4121b5e8ea367 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-14T14:57:52Z |
| publishDate | 2017-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-89b483b604f54dc19de4121b5e8ea3672022-12-21T22:56:57ZengNature PortfolioScientific Reports2045-23222017-04-017111210.1038/s41598-017-01170-zEnhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screensMd. Fahmid Islam0Atsushi Watanabe1Lai Wong2Conor Lazarou3Frederick S. Vizeacoumar4Omar Abuhussein5Wayne Hill6Maruti Uppalapati7C. Ronald Geyer8Franco J. Vizeacoumar9Department of Biochemistry, University of SaskatchewanDepartment of Pathology, University of SaskatchewanDepartment of Biochemistry, University of SaskatchewanDepartment of Pathology, University of SaskatchewanDepartment of Pathology, University of SaskatchewanCollege of Pharmacy and Nutrition, University of SaskatchewanDepartment of Pathology, University of SaskatchewanDepartment of Pathology, University of SaskatchewanDepartment of Pathology, University of SaskatchewanDepartment of Pathology, University of SaskatchewanAbstract 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.https://doi.org/10.1038/s41598-017-01170-z |
| spellingShingle | Md. Fahmid Islam Atsushi Watanabe Lai Wong Conor Lazarou Frederick S. Vizeacoumar Omar Abuhussein Wayne Hill Maruti Uppalapati C. Ronald Geyer Franco J. Vizeacoumar Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens Scientific Reports |
| title | Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens |
| title_full | Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens |
| title_fullStr | Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens |
| title_full_unstemmed | Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens |
| title_short | Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens |
| title_sort | enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shrna and crispr screens |
| url | https://doi.org/10.1038/s41598-017-01170-z |
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