title: Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.
abstract: We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.
authors.affiliation: Department of Neurology, University of Tokyo, Graduate School of Medicine, Tokyo, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Mitsui
authors.forename: Yoko
authors.initials: Y
authors.lastname: Fukuda
authors.forename: Kyo
authors.initials: K
authors.lastname: Azuma
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tozaki
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ishiura
authors.forename: Yuji
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Jun
authors.initials: J
authors.lastname: Goto
authors.forename: Shoji
authors.initials: S
authors.lastname: Tsuji
mesh_terms: D000483:Alleles; D001483:Base Sequence; D015982:Bias; D004247:DNA / Q000235:genetics*; D001330:Electronic Data Processing / Q000379:methods*; D015723:Gene Library; D014644:Genetic Variation / Q000235:genetics*; D006801:Humans; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*
chemical_list: D004247:DNA