title: Rapid flow-sorting to simultaneously resolve multiplex massively parallel sequencing products.
abstract: Sample preparation for Roche/454, ABI/SOLiD and Life Technologies/Ion Torrent sequencing are based on amplification of library fragments on the surface of beads prior to sequencing. Commonly, libraries are barcoded and pooled, to maximise the sequence output of each sequence run. Here, we describe a novel approach for normalization of multiplex next generation sequencing libraries after emulsion PCR. Briefly, amplified libraries carrying unique barcodes are prepared by fluorescent tagging of complementary sequences and then resolved by high-speed flow cytometric sorting of labeled emulsion PCR beads. The protocol is simple and provides an even sequence distribution of multiplex libraries when sequencing the flow-sorted beads. Moreover, since many empty and mixed emulsion PCR beads are removed, the approach gives rise to a substantial increase in sequence quality and mean read length, as compared to that obtained by standard enrichment protocols.
authors.affiliation: Science for Life Laboratory, Department of Gene Technology, Royal Institute of Technology (KTH), Box 1031, SE-17121, Solna, Sweden.
authors.forename: Julia
authors.initials: J
authors.lastname: Sandberg
authors.forename: Beata
authors.initials: B
authors.lastname: Werne
authors.forename: Mark
authors.initials: M
authors.lastname: Dessing
authors.forename: Joakim
authors.initials: J
authors.lastname: Lundeberg
mesh_terms: D004655:Emulsions; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004655:Emulsions; D005456:Fluorescent Dyes