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μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one cont...

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Autores principales: Milon, Nicolas, Chantry-Darmon, Céline, Satge, Carine, Fustier, Margaux-Alison, Cauet, Stephane, Moreau, Sandra, Callot, Caroline, Bellec, Arnaud, Gabrieli, Tslil, Saïas, Laure, Boutonnet, Audrey, Ginot, Frédéric, Bergès, Hélène, Bancaud, Aurélien
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736094/
https://www.ncbi.nlm.nih.gov/pubmed/31505675
http://dx.doi.org/10.1093/nar/gkz632
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author Milon, Nicolas
Chantry-Darmon, Céline
Satge, Carine
Fustier, Margaux-Alison
Cauet, Stephane
Moreau, Sandra
Callot, Caroline
Bellec, Arnaud
Gabrieli, Tslil
Saïas, Laure
Boutonnet, Audrey
Ginot, Frédéric
Bergès, Hélène
Bancaud, Aurélien
author_facet Milon, Nicolas
Chantry-Darmon, Céline
Satge, Carine
Fustier, Margaux-Alison
Cauet, Stephane
Moreau, Sandra
Callot, Caroline
Bellec, Arnaud
Gabrieli, Tslil
Saïas, Laure
Boutonnet, Audrey
Ginot, Frédéric
Bergès, Hélène
Bancaud, Aurélien
author_sort Milon, Nicolas
collection PubMed
description Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.
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spelling pubmed-67360942019-09-16 μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome Milon, Nicolas Chantry-Darmon, Céline Satge, Carine Fustier, Margaux-Alison Cauet, Stephane Moreau, Sandra Callot, Caroline Bellec, Arnaud Gabrieli, Tslil Saïas, Laure Boutonnet, Audrey Ginot, Frédéric Bergès, Hélène Bancaud, Aurélien Nucleic Acids Res Genomics Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence. Oxford University Press 2019-09-05 2019-07-25 /pmc/articles/PMC6736094/ /pubmed/31505675 http://dx.doi.org/10.1093/nar/gkz632 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Genomics
Milon, Nicolas
Chantry-Darmon, Céline
Satge, Carine
Fustier, Margaux-Alison
Cauet, Stephane
Moreau, Sandra
Callot, Caroline
Bellec, Arnaud
Gabrieli, Tslil
Saïas, Laure
Boutonnet, Audrey
Ginot, Frédéric
Bergès, Hélène
Bancaud, Aurélien
μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome
title μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome
title_full μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome
title_fullStr μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome
title_full_unstemmed μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome
title_short μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome
title_sort μlas technology for dna isolation coupled to cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome
topic Genomics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736094/
https://www.ncbi.nlm.nih.gov/pubmed/31505675
http://dx.doi.org/10.1093/nar/gkz632
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