2024-03-29T06:28:19Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1122052021-12-28T15:49:52Zcom_10261_9676com_10261_8col_10261_9677
2015-03-11T13:25:31Z
urn:hdl:10261/112205
Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate
Džunková, Mária
Garcia-Garcerà, Marc
Martínez-Priego, Llucia
D’Auria, Giuseppe
Calafell, Francesc
Moya-Simarro, Andrés
Instituto de Salud Carlos III
Ministerio de Economía y Competitividad (España)
Generalitat Valenciana
Ministerio de Educación, Cultura y Deporte (España)
Notice 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.
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 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.
2015-03-11T13:25:31Z
2015-03-11T13:25:31Z
2014-06-02
2015-03-11T13:25:31Z
artículo
PLoS ONE 9(6): e97379 (2014)
http://hdl.handle.net/10261/112205
10.1371/journal.pone.0097379
http://dx.doi.org/10.13039/501100004587
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.13039/501100003359
http://dx.doi.org/10.13039/501100003176
24887077
eng
Publisher's version
Džunková, 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
http://dx.doi.org/10.1371/journal.pone.0097379
Sí
http://creativecommons.org/licenses/by/4.0/
openAccess
Public Library of Science