title: Titration-free 454 sequencing using Y adapters.
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.
authors: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. zhengzongli@gmail.com
authors: Zongli
authors: Z
authors: Zheng
authors: Abdolreza
authors: A
authors: Advani
authors: Öjar
authors: Ö
authors: Melefors
authors: Steve
authors: S
authors: Glavas
authors: Henrik
authors: H
authors: Nordström
authors: Weimin
authors: W
authors: Ye
authors: Lars
authors: L
authors: Engstrand
authors: Anders F
authors: AF
authors: Andersson
mesh_terms: D015723:Gene Library; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D009841:Oligonucleotides