title: Identification of genetic variants using bar-coded multiplexed sequencing.
abstract: We developed a generalized framework for multiplexed resequencing of targeted human genome regions on the Illumina Genome Analyzer using degenerate indexed DNA bar codes ligated to fragmented DNA before sequencing. Using this method, we simultaneously sequenced the DNA of multiple HapMap individuals at several Encyclopedia of DNA Elements (ENCODE) regions. We then evaluated the use of Bayes factors for discovering and genotyping polymorphisms. For polymorphisms that were either previously identified within the Single Nucleotide Polymorphism database (dbSNP) or visually evident upon re-inspection of archived ENCODE traces, we observed a false positive rate of 11.3% using strict thresholds for predicting variants and 69.6% for lax thresholds. Conversely, false negative rates were 10.8-90.8%, with false negatives at stricter cut-offs occurring at lower coverage (<10 aligned reads). These results suggest that >90% of genetic variants are discoverable using multiplexed sequencing provided sufficient coverage at the polymorphic base.
authors.affiliation: The Translational Genomics Research Institute, Phoenix, Arizona 85004, USA. dcraig@tgen.org
authors.forename: David W
authors.initials: DW
authors.lastname: Craig
authors.forename: John V
authors.initials: JV
authors.lastname: Pearson
authors.forename: Szabolcs
authors.initials: S
authors.lastname: Szelinger
authors.forename: Aswin
authors.initials: A
authors.lastname: Sekar
authors.forename: Margot
authors.initials: M
authors.lastname: Redman
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Corneveaux
authors.forename: Traci L
authors.initials: TL
authors.lastname: Pawlowski
authors.forename: Trisha
authors.initials: T
authors.lastname: Laub
authors.forename: Gary
authors.initials: G
authors.lastname: Nunn
authors.forename: Dietrich A
authors.initials: DA
authors.lastname: Stephan
authors.forename: Nils
authors.initials: N
authors.lastname: Homer
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Huentelman
mesh_terms: D001330:Electronic Data Processing; D014644:Genetic Variation; D015894:Genome, Human; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D016415:Sequence Alignment
references.citation: Bioinformatics. 2007 Nov 1;23(21):2942-4
references.pmid: 17893086
references.citation: Nat Protoc. 2007;2(10):2492-501
references.pmid: 17947991
references.citation: Genome Res. 2005 Feb;15(2):292-301
references.pmid: 15687293
references.citation: Nat Methods. 2008 Mar;5(3):235-7
references.pmid: 18264105
references.citation: Nucleic Acids Res. 2007;35(19):e130
references.pmid: 17932070
references.citation: Nat Methods. 2007 Nov;4(11):931-6
references.pmid: 17934468
references.citation: Nucleic Acids Res. 2007;35(15):e97
references.pmid: 17670798
references.citation: Nat Genet. 2007 Dec;39(12):1522-7
references.pmid: 17982454
references.citation: Nat Methods. 2007 Nov;4(11):903-5
references.pmid: 17934467
references.citation: Nat Methods. 2007 Nov;4(11):907-9
references.pmid: 17934469
references.citation: Nature. 2007 Jun 14;447(7146):799-816
references.pmid: 17571346
references.citation: Nature. 2007 Jun 7;447(7145):661-78
references.pmid: 17554300
references.citation: Nature. 2007 Oct 18;449(7164):851-61
references.pmid: 17943122