Titration-free massively parallel pyrosequencing using trace amounts of starting material

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
Article numbergkq332
Journal / PublicationNucleic Acids Research
Volume38
Issue number13
Publication statusPublished - 30 Apr 2010
Externally publishedYes

Abstract

Continuous efforts have been made to improve next-generation sequencing methods for increased robustness and for applications on low amounts of starting material. We applied double-stranded library protocols for the Roche 454 platform to avoid the yield-reducing steps associated with single-stranded library preparation, and applied a highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) method. The MGB-probe qPCR, which can detect as low as 100 copies, was used to quantify the amount of effective library, i.e. molecules that form functional clones in emulsion PCR. We also demonstrate that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labour-intensive and costly titration can be eliminated and trace amounts of starting material such as precious clinical samples, transcriptomes of small tissue samples and metagenomics on low biomass environments is applicable. © The Author(s) 2010. Published by Oxford University Press.

Citation Format(s)

Titration-free massively parallel pyrosequencing using trace amounts of starting material. / Zheng, Zongli; Advani, Abdolreza; Melefors, Öjar; Glavas, Steve; Nordström, Henrik; Ye, Weimin; Engstrand, Lars; Andersson, Anders F.

In: Nucleic Acids Research, Vol. 38, No. 13, gkq332, 30.04.2010.

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