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A user-friendly platform for yeast two-hybrid library screening using next generation sequencing

Yeast two-hybrid (Y2H) is a well-established genetics-based system that uses yeast to selectively display binary protein-protein interactions (PPIs). To meet the current need to unravel complex PPI networks, several adaptations have been made to establish medium- to high-throughput Y2H screening pla...

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Autores principales: Erffelinck, Marie-Laure, Ribeiro, Bianca, Perassolo, Maria, Pauwels, Laurens, Pollier, Jacob, Storme, Veronique, Goossens, Alain
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303091/
https://www.ncbi.nlm.nih.gov/pubmed/30576311
http://dx.doi.org/10.1371/journal.pone.0201270
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author Erffelinck, Marie-Laure
Ribeiro, Bianca
Perassolo, Maria
Pauwels, Laurens
Pollier, Jacob
Storme, Veronique
Goossens, Alain
author_facet Erffelinck, Marie-Laure
Ribeiro, Bianca
Perassolo, Maria
Pauwels, Laurens
Pollier, Jacob
Storme, Veronique
Goossens, Alain
author_sort Erffelinck, Marie-Laure
collection PubMed
description Yeast two-hybrid (Y2H) is a well-established genetics-based system that uses yeast to selectively display binary protein-protein interactions (PPIs). To meet the current need to unravel complex PPI networks, several adaptations have been made to establish medium- to high-throughput Y2H screening platforms, with several having successfully incorporated the use of the next-generation sequencing (NGS) technology to increase the scale and sensitivity of the method. However, these have been to date mainly restricted to the use of fully annotated custom-made open reading frame (ORF) libraries and subject to complex downstream data processing. Here, a streamlined Y2H library screening strategy, based on integration of Y2H with NGS, called Y2H-seq, was developed, which allows efficient and reliable screening of Y2H cDNA libraries. To generate proof of concept, the method was applied to screen for interaction partners of two key components of the jasmonate signaling machinery in the model plant Arabidopsis thaliana, resulting in the identification of several previously reported as well as hitherto unknown interactors. Our Y2H-seq method offers a user-friendly, specific and sensitive screening method that allows identification of PPIs without prior knowledge of the organism’s ORFs, thereby extending the method to organisms of which the genome has not entirely been annotated yet. The quantitative NGS readout allows to increase genome coverage, thereby overcoming some of the bottlenecks of current Y2H technologies, which will further strengthen the value of the Y2H technology as a discovery platform.
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spelling pubmed-63030912019-01-08 A user-friendly platform for yeast two-hybrid library screening using next generation sequencing Erffelinck, Marie-Laure Ribeiro, Bianca Perassolo, Maria Pauwels, Laurens Pollier, Jacob Storme, Veronique Goossens, Alain PLoS One Research Article Yeast two-hybrid (Y2H) is a well-established genetics-based system that uses yeast to selectively display binary protein-protein interactions (PPIs). To meet the current need to unravel complex PPI networks, several adaptations have been made to establish medium- to high-throughput Y2H screening platforms, with several having successfully incorporated the use of the next-generation sequencing (NGS) technology to increase the scale and sensitivity of the method. However, these have been to date mainly restricted to the use of fully annotated custom-made open reading frame (ORF) libraries and subject to complex downstream data processing. Here, a streamlined Y2H library screening strategy, based on integration of Y2H with NGS, called Y2H-seq, was developed, which allows efficient and reliable screening of Y2H cDNA libraries. To generate proof of concept, the method was applied to screen for interaction partners of two key components of the jasmonate signaling machinery in the model plant Arabidopsis thaliana, resulting in the identification of several previously reported as well as hitherto unknown interactors. Our Y2H-seq method offers a user-friendly, specific and sensitive screening method that allows identification of PPIs without prior knowledge of the organism’s ORFs, thereby extending the method to organisms of which the genome has not entirely been annotated yet. The quantitative NGS readout allows to increase genome coverage, thereby overcoming some of the bottlenecks of current Y2H technologies, which will further strengthen the value of the Y2H technology as a discovery platform. Public Library of Science 2018-12-21 /pmc/articles/PMC6303091/ /pubmed/30576311 http://dx.doi.org/10.1371/journal.pone.0201270 Text en © 2018 Erffelinck et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Erffelinck, Marie-Laure
Ribeiro, Bianca
Perassolo, Maria
Pauwels, Laurens
Pollier, Jacob
Storme, Veronique
Goossens, Alain
A user-friendly platform for yeast two-hybrid library screening using next generation sequencing
title A user-friendly platform for yeast two-hybrid library screening using next generation sequencing
title_full A user-friendly platform for yeast two-hybrid library screening using next generation sequencing
title_fullStr A user-friendly platform for yeast two-hybrid library screening using next generation sequencing
title_full_unstemmed A user-friendly platform for yeast two-hybrid library screening using next generation sequencing
title_short A user-friendly platform for yeast two-hybrid library screening using next generation sequencing
title_sort user-friendly platform for yeast two-hybrid library screening using next generation sequencing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303091/
https://www.ncbi.nlm.nih.gov/pubmed/30576311
http://dx.doi.org/10.1371/journal.pone.0201270
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