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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Md. Fahmid Islam, Atsushi Watanabe, Lai Wong, Conor Lazarou, Frederick S. Vizeacoumar, Omar Abuhussein, Wayne Hill, Maruti Uppalapati, C. Ronald Geyer, Franco J. Vizeacoumar
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/89b483b604f54dc19de4121b5e8ea367
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:89b483b604f54dc19de4121b5e8ea367
record_format	dspace
spelling	oai:doaj.org-article:89b483b604f54dc19de4121b5e8ea3672021-12-02T11:41:12ZEnhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens10.1038/s41598-017-01170-z2045-2322https://doaj.org/article/89b483b604f54dc19de4121b5e8ea3672017-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-01170-zhttps://doaj.org/toc/2045-2322Abstract 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.Md. Fahmid IslamAtsushi WatanabeLai WongConor LazarouFrederick S. VizeacoumarOmar AbuhusseinWayne HillMaruti UppalapatiC. Ronald GeyerFranco J. VizeacoumarNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-12 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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
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.
format	article
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
title	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_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_sort	enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shrna and crispr screens
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/89b483b604f54dc19de4121b5e8ea367
work_keys_str_mv	AT mdfahmidislam enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens AT atsushiwatanabe enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens AT laiwong enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens AT conorlazarou enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens AT fredericksvizeacoumar enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens AT omarabuhussein enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens AT waynehill enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens AT marutiuppalapati enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens AT cronaldgeyer enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens AT francojvizeacoumar enhancingthethroughputandmultiplexingcapabilitiesofnextgenerationsequencingforefficientimplementationofpooledshrnaandcrisprscreens
_version_	1718395471021473792

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

Ejemplares similares