Titration-free 454 sequencing using y adapters

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalNot applicablepeer-review

20 Scopus Citations
View graph of relations

Author(s)

  • Abdolreza Advani
  • Öjar Melefors
  • Steve Glavas
  • Henrik Nordström
  • Weimin Ye
  • Lars Engstrand
  • Anders F. Andersson

Detail(s)

Original languageEnglish
Pages (from-to)1367-1376
Journal / PublicationNature Protocols
Volume6
Issue number9
Publication statusPublished - Sep 2011
Externally publishedYes

Abstract

We describe a protocol for construction and quantification of libraries for emulsion PCR (emPCR)-based sequencing platforms such as Roche 454 or Ion Torrent PGM. The protocol involves library construction using customized Y adapters, quantification using TaqMan-MGB (minor groove binder) probe-based quantitative PCR (qPCR) and calculation of an optimal template-to-bead ratio based on Poisson statistics, thereby avoiding the need for a laborious titration assay. Unlike other qPCR methods, the TaqMan-MGB probe specifically quantifies effective libraries in molar concentration and does not require specialized equipment. A single quality control step prior to emulsion PCR ensures that libraries contain no adapter dimers and have an optimal length distribution. The presented protocol takes ∼7 h to prepare eight barcoded libraries from genomic DNA into libraries that are ready to use for full-scale emPCR. It will be useful, for example, to allow analyses of precious clinical samples and amplification-free metatranscriptomics. © 2011 Nature America, Inc. All rights reserved.

Citation Format(s)

Titration-free 454 sequencing using y adapters. / Zheng, Zongli; Advani, Abdolreza; Melefors, Öjar; Glavas, Steve; Nordström, Henrik; Ye, Weimin; Engstrand, Lars; Andersson, Anders F.

In: Nature Protocols, Vol. 6, No. 9, 09.2011, p. 1367-1376.

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalNot applicablepeer-review