title: Abridged adapter primers increase the target scope of Hi-Plex.
abstract: Previously, we reported Hi-Plex, an amplicon-based method for targeted massively parallel sequencing capable of generating 60 amplicons simultaneously. In further experiments, however, we found our approach did not scale to higher amplicon numbers. Here, we report a modification to the original Hi-Plex protocol that includes the use of abridged adapter oligonucleotides as universal primers (bridge primers) in the initial PCR mixture. Full-length adapter primers (indexing primers) are included only during latter stages of thermal cycling with concomitant application of elevated annealing temperatures. Using this approach, we demonstrate the application of Hi-Plex across a broad range of amplicon numbers (16-plex, 62-plex, 250-plex, and 1003-plex) while preserving the low amount (25 ng) of input DNA required.
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Melbourne, Australia.
authors.forename: Tú
authors.initials: T
authors.lastname: Nguyen-Dumont
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Melbourne, Australia.
authors.forename: Fleur
authors.initials: F
authors.lastname: Hammet
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Melbourne, Australia.
authors.forename: Maryam
authors.initials: M
authors.lastname: Mahmoodi
authors.affiliation: Victorian Life Sciences Computation Initiative, Melbourne, Australia; Department of Computing and Information Systems, The University of Melbourne, Melbourne, Australia.
authors.forename: Bernard J
authors.initials: BJ
authors.lastname: Pope
authors.affiliation: Cancer Epidemiology Centre, The Cancer Council Victoria, 615 St. Kilda Road, Melbourne, Australia.
authors.forename: Graham G
authors.initials: GG
authors.lastname: Giles
authors.affiliation: Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, Parkville, Australia.
authors.forename: John L
authors.initials: JL
authors.lastname: Hopper
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Melbourne, Australia.
authors.forename: Melissa C
authors.initials: MC
authors.lastname: Southey
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, The University of Melbourne, Melbourne, Australia.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Park
mesh_terms: D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012984:Software
chemical_list: D017931:DNA Primers; D004247:DNA
keywords: Hi-Plex; genetic screening; massively parallel sequencing; mutation screening; next-generation sequencing; targeted sequencing