title: Hi-Plex targeted sequencing is effective using DNA derived from archival dried blood spots.
abstract: Many genetic epidemiology resources have collected dried blood spots (predominantly as Guthrie Cards) as an economical and efficient means of archiving sources of DNA, conferring great value to genetic screening methods that are compatible with this medium. We applied Hi-Plex to screen the breast cancer predisposition gene PALB2 in 93 Guthrie Card-derived DNA specimens previously characterized for PALB2 genetic variants via DNA derived from lymphoblastoid cell lines, whole blood, and buffy coat. Of the 93 archival Guthrie Card-derived DNAs, 92 (99%) were processed successfully and sequenced using approximately half of a MiSeq run. From these 92 DNAs, all 59 known variants were detected and no false-positive variant calls were yielded. Fully 98.13% of amplicons (5417/5520) were represented within 15-fold of the median coverage (2786 reads), and 99.98% of amplicons (5519/5520) were represented at a depth of 10 read-pairs or greater. With Hi-Plex, we show for the first time that a High-Plex amplicon-based massively parallel sequencing (MPS) system can be applied effectively to DNA prepared from dried blood spot archival specimens and, as such, can dramatically increase the scopes of both method and resource.
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: T
authors.initials: T
authors.lastname: Nguyen-Dumont
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Mahmoodi
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: F
authors.initials: F
authors.lastname: Hammet
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: T
authors.initials: T
authors.lastname: Tran
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: H
authors.initials: H
authors.lastname: Tsimiklis
authors.affiliation: Cancer Epidemiology Centre, Cancer Council Victoria, Melbourne, Victoria 3004, Australia.
authors.forename: G G
authors.initials: GG
authors.lastname: Giles
authors.affiliation: Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, Parkville, Victoria 3010, Australia.
authors.forename: J L
authors.initials: JL
authors.lastname: Hopper
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: M C
authors.initials: MC
authors.lastname: Southey
authors.affiliation: Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, Victoria 3010, Australia. Electronic address: djp@unimelb.edu.au.
authors.forename: D J
authors.initials: DJ
authors.lastname: Park
mesh_terms: D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000097:blood* / Q000235:genetics*; D004252:DNA Mutational Analysis; D059788:Dried Blood Spot Testing; D005260:Female; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods*; D006801:Humans
chemical_list: D004247:DNA
keywords: Archival DNA; Dried blood spot; Guthrie Card; Hi-Plex; Massively parallel sequencing; Targeted sequencing