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

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)...

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Autores principales:	Mária Džunková, Marc Garcia-Garcerà, Llúcia Martínez-Priego, Giussepe D'Auria, Francesc Calafell, Andrés Moya
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/026b9cf2d3ee4033a8f4c252823365a4
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spelling	oai:doaj.org-article:026b9cf2d3ee4033a8f4c252823365a42021-11-18T08:17:39ZDirect squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.1932-620310.1371/journal.pone.0097379https://doaj.org/article/026b9cf2d3ee4033a8f4c252823365a42014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24887077/?tool=EBIhttps://doaj.org/toc/1932-6203The 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.Mária DžunkováMarc Garcia-GarceràLlúcia Martínez-PriegoGiussepe D'AuriaFrancesc CalafellAndrés MoyaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 6, p e97379 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Mária Džunková Marc Garcia-Garcerà Llúcia Martínez-Priego Giussepe D'Auria Francesc Calafell Andrés Moya Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.
description	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.
format	article
author	Mária Džunková Marc Garcia-Garcerà Llúcia Martínez-Priego Giussepe D'Auria Francesc Calafell Andrés Moya
author_facet	Mária Džunková Marc Garcia-Garcerà Llúcia Martínez-Priego Giussepe D'Auria Francesc Calafell Andrés Moya
author_sort	Mária Džunková
title	Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.
title_short	Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.
title_full	Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.
title_fullStr	Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.
title_full_unstemmed	Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.
title_sort	direct squencing from the minimal number of dna molecules needed to fill a 454 picotiterplate.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/026b9cf2d3ee4033a8f4c252823365a4
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Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.

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