A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms

Reference-free reduced representation bisulfite sequencing uses enzymatic digestion for reducing genome complexity and allows detection of markers to study DNA methylation of a high number of individuals in natural populations of non-model organisms. Current methods like epiGBS enquire the use of a...

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Autores: Werner, Olaf, Prudencio, Ángela S., Cruz Martínez, Elena de la, Nieto-Lugilde, Marta, Martínez-Gómez, Pedro, Ros, Rosa M.
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2020
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/235410
Acceso en línea:http://hdl.handle.net/10261/235410
Access Level:acceso abierto
Palabra clave:DNA methylation
Epi genotyping by sequencing
Population genetics
Reduced representation bisulfite sequencing
Non-model organisms
Prunus dulcis
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spelling A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model OrganismsWerner, OlafPrudencio, Ángela S.Cruz Martínez, Elena de laNieto-Lugilde, MartaMartínez-Gómez, PedroRos, Rosa M.DNA methylationEpi genotyping by sequencingPopulation geneticsReduced representation bisulfite sequencingNon-model organismsPrunus dulcisReference-free reduced representation bisulfite sequencing uses enzymatic digestion for reducing genome complexity and allows detection of markers to study DNA methylation of a high number of individuals in natural populations of non-model organisms. Current methods like epiGBS enquire the use of a higher number of methylated DNA oligos with a significant cost (especially for small labs and first pilot studies). In this paper, we present a modification of this epiGBS protocol that requires the use of only one hemimethylated P2 (common) adapter, which is combined with unmethylated barcoded adapters. The unmethylated cytosines of one chain of the barcoded adapter are replaced by methylated cytosines using nick translation with methylated cytosines in dNTP solution. The basic version of our technique uses only one restriction enzyme, and as a result, genomic fragments are integrated into two orientations with respect to the adapter sequences. Comparing the sequences of two chain orientations makes it possible to reconstruct the original sequence before bisulfite treatment with the help of standard software and newly developed software written in C and described here. We provide a proof of concept via data obtained from almond (Prunus dulcis). Example data and a detailed description of the complete software pipeline starting from the raw reads up until the final differentially methylated cytosines are given in Supplementary Material making this technique accessible to non-expert computer users. The adapter design showed in this paper should allow the use of a two restriction enzyme approach with minor changes in software parameters.This study was funded by the Spanish “Fundación Séneca” of the Region of Murcia (Grants 19308/PI/and 19879/GERM/15) and the “Ministerio de Economía y Competitividad” (Projects CGL2014-52579-R and RTI2018-095556-B-I00), co-financed by ERDF of the European Union.Frontiers MediaFundación SénecaMinisterio de Economía y Competitividad (España)Ministerio de Ciencia, Innovación y Universidades (España)European CommissionConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2021202120202021info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/235410reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/ CGL2014-52579-Rinfo:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/ RTI2018-095556-B-I00http://dx.doi.org/10.3389/fpls.2020.00694Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2354102026-05-22T06:33:51Z
dc.title.none.fl_str_mv A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms
title A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms
spellingShingle A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms
Werner, Olaf
DNA methylation
Epi genotyping by sequencing
Population genetics
Reduced representation bisulfite sequencing
Non-model organisms
Prunus dulcis
title_short A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms
title_full A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms
title_fullStr A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms
title_full_unstemmed A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms
title_sort A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms
dc.creator.none.fl_str_mv Werner, Olaf
Prudencio, Ángela S.
Cruz Martínez, Elena de la
Nieto-Lugilde, Marta
Martínez-Gómez, Pedro
Ros, Rosa M.
author Werner, Olaf
author_facet Werner, Olaf
Prudencio, Ángela S.
Cruz Martínez, Elena de la
Nieto-Lugilde, Marta
Martínez-Gómez, Pedro
Ros, Rosa M.
author_role author
author2 Prudencio, Ángela S.
Cruz Martínez, Elena de la
Nieto-Lugilde, Marta
Martínez-Gómez, Pedro
Ros, Rosa M.
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Fundación Séneca
Ministerio de Economía y Competitividad (España)
Ministerio de Ciencia, Innovación y Universidades (España)
European Commission
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv DNA methylation
Epi genotyping by sequencing
Population genetics
Reduced representation bisulfite sequencing
Non-model organisms
Prunus dulcis
topic DNA methylation
Epi genotyping by sequencing
Population genetics
Reduced representation bisulfite sequencing
Non-model organisms
Prunus dulcis
description Reference-free reduced representation bisulfite sequencing uses enzymatic digestion for reducing genome complexity and allows detection of markers to study DNA methylation of a high number of individuals in natural populations of non-model organisms. Current methods like epiGBS enquire the use of a higher number of methylated DNA oligos with a significant cost (especially for small labs and first pilot studies). In this paper, we present a modification of this epiGBS protocol that requires the use of only one hemimethylated P2 (common) adapter, which is combined with unmethylated barcoded adapters. The unmethylated cytosines of one chain of the barcoded adapter are replaced by methylated cytosines using nick translation with methylated cytosines in dNTP solution. The basic version of our technique uses only one restriction enzyme, and as a result, genomic fragments are integrated into two orientations with respect to the adapter sequences. Comparing the sequences of two chain orientations makes it possible to reconstruct the original sequence before bisulfite treatment with the help of standard software and newly developed software written in C and described here. We provide a proof of concept via data obtained from almond (Prunus dulcis). Example data and a detailed description of the complete software pipeline starting from the raw reads up until the final differentially methylated cytosines are given in Supplementary Material making this technique accessible to non-expert computer users. The adapter design showed in this paper should allow the use of a two restriction enzyme approach with minor changes in software parameters.
publishDate 2020
dc.date.none.fl_str_mv 2020
2021
2021
2021
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/235410
url http://hdl.handle.net/10261/235410
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/ CGL2014-52579-R
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/ RTI2018-095556-B-I00
http://dx.doi.org/10.3389/fpls.2020.00694
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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