Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer
Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer
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DOI：
10.1038/jhg.2010.46
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发表时间：
2010-07-01
影响因子：
3.5
通讯作者：
Tsuji, Shoji
中科院分区：
文献类型：
--
作者：
Mitsui, Jun;Fukuda, Yoko;Tsuji, Shoji
我们最近发现，葡萄糖脑苷脂酶基因（GBA）的多种罕见变体赋予帕金森病的强大风险，支持“常见病-多种罕见变体”假说。为了开发一种在大量样品中鉴定罕见变体的有效方法，我们使用下一代测序仪应用多重重测序来鉴定GBA的罕见变体。从6个合并的DNA样品制备16组合并的DNA。对每组合并的DNA进行聚合酶链反应以扩增覆盖6.5 kb的靶基因（GBA），合并到一个带有条形码索引的试管中，然后使用SOLiD系统进行广泛的序列分析。还对单个样品进行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率可接受。人类遗传学杂志（2010）55，448-455; doi：10.1038/jhg.2010.46;在线发表于2010年5月20日
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. Journal of Human Genetics (2010) 55, 448-455; doi:10.1038/jhg.2010.46; published online 20 May 2010