Titration-free 454 sequencing using Y adapters
2011 (English)In: Nature Protocols, ISSN 1754-2189, Vol. 6, no 9, 1367-1376 p.Article in journal (Refereed) Published
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 similar to 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.
Place, publisher, year, edition, pages
2011. Vol. 6, no 9, 1367-1376 p.
IdentifiersURN: urn:nbn:se:kth:diva-46193DOI: 10.1038/nprot.2011.369ISI: 000295362900008ScopusID: 2-s2.0-80052425126OAI: oai:DiVA.org:kth-46193DiVA: diva2:453566
FunderSwedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
QC 201111032011-11-032011-11-022011-12-06Bibliographically approved