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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2018
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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. |
format | Online Article Text |
id | pubmed-6303091 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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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