title: Multiplex amplification of large sets of human exons.
abstract: A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify approximately 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.
authors.affiliation: Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Porreca
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.forename: Jin Billy
authors.initials: JB
authors.lastname: Li
authors.forename: Bin
authors.initials: B
authors.lastname: Xie
authors.forename: Derek
authors.initials: D
authors.lastname: Austin
authors.forename: Sara L
authors.initials: SL
authors.lastname: Vassallo
authors.forename: Emily M
authors.initials: EM
authors.lastname: LeProust
authors.forename: Bill J
authors.initials: BJ
authors.lastname: Peck
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Emig
authors.forename: Fredrik
authors.initials: F
authors.lastname: Dahl
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.forename: George M
authors.initials: GM
authors.lastname: Church
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
mesh_terms: D045744:Cell Line, Tumor; D005091:Exons / Q000235:genetics*; D015723:Gene Library; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D009838:Oligodeoxyribonucleotides