Titration-free massively parallel pyrosequencing using trace amounts of starting material
2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 13Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2010. Vol. 38, no 13
Gene Library, Oligonucleotide Probes, Poisson Distribution, Polymerase Chain Reaction/*methods, Sequence Analysis, DNA/*methods
IdentifiersURN: urn:nbn:se:kth:diva-80954DOI: 10.1093/nar/gkq332ISI: 000280538600002OAI: oai:DiVA.org:kth-80954DiVA: diva2:496980
QC 201202102012-02-102012-02-102012-02-10Bibliographically approved