Titration-free 454 sequencing using Y adapters.

Zheng Z, Advani A, Melefors Ö, Glavas S, Nordström H, Ye W, Engstrand L, Andersson AF

Nat Protoc 6 (9) 1367-1376 [2011-08-18; online 2011-08-18]

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.

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PubMed 21886102

DOI 10.1038/nprot.2011.369

Crossref 10.1038/nprot.2011.369

nprot.2011.369