Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate

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  • dc.contributor.author Dzunková, Máriaca
  • dc.contributor.author García Garcerà, Marcca
  • dc.contributor.author Martínez Priego, Llúciaca
  • dc.contributor.author D'Auria, Giussepeca
  • dc.contributor.author Calafell i Majó, Francescca
  • dc.contributor.author Moya, Andrésca
  • dc.date.accessioned 2015-05-25T09:06:37Z
  • dc.date.available 2015-05-25T09:06:37Z
  • dc.date.issued 2014ca
  • dc.description.abstract The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA.en
  • dc.description.sponsorship This work was funded by grant CP09/00049 Miguel Servet, Instituto de Salud Carlos III, Spain to GD; by projects SAF2009-13032-C02-01 and SAF 2012-31187 (AM), BFU2009-12895-CO2-01 and SAF2010-16240 (FC) from the Spanish Ministry for Science and Innovation (MCINN), FU2008-04501-E from Spanish Ministry for Science and Innovation(MCINN) in the frame of ERA-Net PathoGenoMics and Prometeo/2009/092 from Conselleria D’Educació Generalitat Valenciana,Spain, to AM. MD is recipient of a fellowship from Spanish Ministry of Education FPU2010. MGG was supported by a predoctoral fellowship from the Spanish Ministry of Science and Innovation (Grant number BES-2008-006029)en
  • dc.format.mimetype application/pdfca
  • dc.identifier.citation Dzunkova M, García-Garcerà M, Martínez-Priego L, D'Auria G, Calafell F, Moya A. Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate. PLoS ONE. 2014;9(6):e97379. DOI: 10.1371/journal.pone.0097379ca
  • dc.identifier.doi http://dx.doi.org/10.1371/journal.pone.0097379
  • dc.identifier.issn 1932-6203ca
  • dc.identifier.uri http://hdl.handle.net/10230/23641
  • dc.language.iso engca
  • dc.publisher Public Library of Science (PLoS)ca
  • dc.relation.ispartof PLoS ONE. 2014;9(6):e97379
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/SAF2009-13032
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/SAF2012-31187
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/BFU2009-12895
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/SAF2010-16240
  • dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/BES2008-006029
  • dc.rights © 2014 Dzunkova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are crediteden
  • dc.rights.accessRights info:eu-repo/semantics/openAccessca
  • dc.rights.uri https://creativecommons.org/licenses/by/4.0/
  • dc.subject.other Gens humansca
  • dc.subject.other Genètica bacterianaca
  • dc.subject.other Mapatge
  • dc.title Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplateen
  • dc.type info:eu-repo/semantics/articleca
  • dc.type.version info:eu-repo/semantics/publishedVersionca

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