title: Single-step capture and sequencing of natural DNA for detection of BRCA1 mutations.
abstract: Genetic testing for disease risk is an increasingly important component of medical care. However, testing can be expensive, which can lead to patients and physicians having limited access to the genetic information needed for medical decisions. To simplify DNA sample preparation and lower costs, we have developed a system in which any gene can be captured and sequenced directly from human genomic DNA without amplification, using no proteins or enzymes prior to sequencing. Extracted whole-genome DNA is acoustically sheared and loaded in a flow cell channel for single-molecule sequencing. Gene isolation, amplification, or ligation is not necessary. Accurate and low-cost detection of DNA sequence variants is demonstrated for the BRCA1 gene. Disease-causing mutations as well as common variants from well-characterized samples are identified. Single-molecule sequencing generates very reproducible coverage patterns, and these can be used to detect any size insertion or deletion directly, unlike PCR-based methods, which require additional assays. Because no gene isolation or amplification is required for sequencing, the exceptionally low costs of sample preparation and analysis could make genetic tests more accessible to those who wish to know their own disease susceptibility. Additionally, this approach has applications for sequencing integration sites for gene therapy vectors, transposons, retroviruses, and other mobile DNA elements in a more facile manner than possible with other methods.
authors.affiliation: Helicos BioSciences Corporation, Cambridge, Massachusetts 02139, USA. jthompson@helicosbio.com
authors.forename: John F
authors.initials: JF
authors.lastname: Thompson
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Reifenberger
authors.forename: Eldar
authors.initials: E
authors.lastname: Giladi
authors.forename: Kristen
authors.initials: K
authors.lastname: Kerouac
authors.forename: Jaime
authors.initials: J
authors.lastname: Gill
authors.forename: Erik
authors.initials: E
authors.lastname: Hansen
authors.forename: Avak
authors.initials: A
authors.lastname: Kahvejian
authors.forename: Philipp
authors.initials: P
authors.lastname: Kapranov
authors.forename: Travis
authors.initials: T
authors.lastname: Knope
authors.forename: Doron
authors.initials: D
authors.lastname: Lipson
authors.forename: Kathleen E
authors.initials: KE
authors.lastname: Steinmann
authors.forename: Patrice M
authors.initials: PM
authors.lastname: Milos
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons; D019398:Genes, BRCA1; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D016415:Sequence Alignment; D017384:Sequence Deletion