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LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms

Molecular characterization of genetically modified organisms (GMOs) yields basic information on exogenous DNA integration, including integration sites, entire inserted sequences and structures, flanking sequences and copy number, providing key data for biosafety assessment. However, there are few ef...

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Autores principales: Zhang, Hanwen, Li, Rong, Guo, Yongkun, Zhang, Yuchen, Zhang, Dabing, Yang, Litao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9055813/
https://www.ncbi.nlm.nih.gov/pubmed/34990051
http://dx.doi.org/10.1111/pbi.13776
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author Zhang, Hanwen
Li, Rong
Guo, Yongkun
Zhang, Yuchen
Zhang, Dabing
Yang, Litao
author_facet Zhang, Hanwen
Li, Rong
Guo, Yongkun
Zhang, Yuchen
Zhang, Dabing
Yang, Litao
author_sort Zhang, Hanwen
collection PubMed
description Molecular characterization of genetically modified organisms (GMOs) yields basic information on exogenous DNA integration, including integration sites, entire inserted sequences and structures, flanking sequences and copy number, providing key data for biosafety assessment. However, there are few effective methods for deciphering transgene integration, especially for large DNA fragment integration with complex rearrangement, inversion and tandem repeats. Herein, we developed a universal Large Integrated DNA Fragments Enrichment strategy combined with PacBio Sequencing (LIFE‐Seq) for deciphering transgene integration in GMOs. Universal tilling DNA probes targeting transgenic elements and exogenous genes facilitate specific enrichment of large inserted DNA fragments associated with transgenes from plant genomes, followed by PacBio sequencing. LIFE‐Seq were evaluated using six GM events and four crop species. Target DNA fragments averaging ~6275 bp were enriched and sequenced, generating ~26 352 high fidelity reads for each sample. Transgene integration structures were determined with high repeatability and sensitivity. Compared with next‐generation whole‐genome sequencing, LIFE‐Seq achieved better data integrity and accuracy, greater universality and lower cost, especially for transgenic crops with complex inserted DNA structures. LIFE‐Seq could be applied in molecular characterization of transgenic crops and animals, and complex DNA structure analysis in genetics research.
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spelling pubmed-90558132022-05-03 LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms Zhang, Hanwen Li, Rong Guo, Yongkun Zhang, Yuchen Zhang, Dabing Yang, Litao Plant Biotechnol J Research Articles Molecular characterization of genetically modified organisms (GMOs) yields basic information on exogenous DNA integration, including integration sites, entire inserted sequences and structures, flanking sequences and copy number, providing key data for biosafety assessment. However, there are few effective methods for deciphering transgene integration, especially for large DNA fragment integration with complex rearrangement, inversion and tandem repeats. Herein, we developed a universal Large Integrated DNA Fragments Enrichment strategy combined with PacBio Sequencing (LIFE‐Seq) for deciphering transgene integration in GMOs. Universal tilling DNA probes targeting transgenic elements and exogenous genes facilitate specific enrichment of large inserted DNA fragments associated with transgenes from plant genomes, followed by PacBio sequencing. LIFE‐Seq were evaluated using six GM events and four crop species. Target DNA fragments averaging ~6275 bp were enriched and sequenced, generating ~26 352 high fidelity reads for each sample. Transgene integration structures were determined with high repeatability and sensitivity. Compared with next‐generation whole‐genome sequencing, LIFE‐Seq achieved better data integrity and accuracy, greater universality and lower cost, especially for transgenic crops with complex inserted DNA structures. LIFE‐Seq could be applied in molecular characterization of transgenic crops and animals, and complex DNA structure analysis in genetics research. John Wiley and Sons Inc. 2022-02-01 2022-05 /pmc/articles/PMC9055813/ /pubmed/34990051 http://dx.doi.org/10.1111/pbi.13776 Text en © 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
spellingShingle Research Articles
Zhang, Hanwen
Li, Rong
Guo, Yongkun
Zhang, Yuchen
Zhang, Dabing
Yang, Litao
LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms
title LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms
title_full LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms
title_fullStr LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms
title_full_unstemmed LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms
title_short LIFE‐Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms
title_sort life‐seq: a universal large integrated dna fragment enrichment sequencing strategy for deciphering the transgene integration of genetically modified organisms
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9055813/
https://www.ncbi.nlm.nih.gov/pubmed/34990051
http://dx.doi.org/10.1111/pbi.13776
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