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

AutorDžunková, Mária; Garcia-Garcerà, Marc ; Martínez-Priego, Llucia ; D’Auria, Giussepe; Calafell, Francesc ; Moya-Simarro, Andrés
Fecha de publicación2-jun-2014
EditorPublic Library of Science
CitaciónPLoS ONE 9(6): e97379 (2014)
ResumenThe 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 fs 24 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. © 2014 Džunková et al.
DescripciónNotice of Republication: This article was republished on June 17, 2014, to correct an error in the title. The publisher apologizes for the error. In addition, a typographical error was corrected in the Abstract. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected article are provided here for reference.
Versión del editorhttp://dx.doi.org/10.1371/journal.pone.0097379
Identificadoresdoi: 10.1371/journal.pone.0097379
issn: 1932-6203
ReferenciasDžunková, Mária; García-Garcerà, Marc; Martínez-Priego, Llúcia; D’Auria, Giussepe; Calafell, Francesc; Moya, Andrés. Correction: Direct Sequencing from the Minimal Number of DNA Molecules Needed to Fill a 454 Picotiterplate. http://dx.doi.org/10.1371/journal.pone.0102719 . http://hdl.handle.net/10261/112211
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