Department of Neurology, University of Tokyo, Graduate School of Medicine, Tokyo, Japan.
J Hum Genet. 2010 Jul;55(7):448-55. doi: 10.1038/jhg.2010.46. Epub 2010 May 20.
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.
我们最近发现，多个葡萄糖脑苷脂酶基因 (GBA) 的罕见变异赋予了帕金森病的强大风险，支持“常见疾病-多个罕见变异”假说。为了开发一种在大量样本中识别罕见变异的有效方法，我们应用了下一代测序仪的多重重测序来鉴定 GBA 的罕见变异。从六个混合 DNA 样本中制备了十六组混合 DNA。每组混合 DNA 都经过聚合酶链反应 (PCR) 扩增目标基因 (GBA)，覆盖 6.5kb，汇集到一个带有条形码索引的管中，然后使用 SOLiD 系统进行广泛的序列分析。个别样本也进行了直接核苷酸序列分析。通过数据处理的优化，我们能够从 96 个样本中提取出所有的变异，假阳性单核苷酸变异的比率可接受。
