A flexible approach for highly multiplexed candidate gene targeted resequencing.
A flexible approach for highly multiplexed candidate gene targeted resequencing.
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DOI：
10.1371/journal.pone.0021088
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Ji HP
中科院分区：
文献类型：
--
作者：
Natsoulis G;Bell JM;Xu H;Buenrostro JD;Ordonez H;Grimes S;Newburger D;Jensen M;Zahn JM;Zhang N;Ji HP
我们已经开发了一种整合策略，用于对来自人类外显子组的基因子集进行靶向重测序和变异分析。我们的捕获技术是面向重测序基因子集大大大于可以有效地完成与单一或多重PCR，但规模小于外显子组测序。我们描述了从最初的捕获检测到单核苷酸变异（SNV）发现的所有步骤。捕获方法使用溶液中80聚体寡核苷酸。为了在选择人类基因靶点时提供最佳的灵活性，我们设计了一组计算机模拟的寡核苷酸，即人类寡外显子组，其覆盖了由共识编码测序项目（CCDS）注释的基因外显子。该资源作为互联网可访问的数据库公开提供，其中可以下载任何CCDS基因的捕获寡核苷酸序列并设计定制捕获测定。使用该资源，我们通过定制设计捕获测定来证明该测定的灵活性，所述捕获测定范围从10个到超过100个基因靶标，总捕获大小从超过100千碱基到近1兆碱基。我们建立了一种方法，以减少捕获变异性和纳入索引计划，以增加样品的吞吐量。我们的方法具有多种应用，包括但不限于特定基因子集的群体靶向重测序研究，全基因组测序调查中发现的变体的验证以及疾病基因子集的可能诊断分析。我们还提出了一个成本分析，证明其成本效益的大人口研究。
We have developed an integrated strategy for targeted resequencing and analysis of gene subsets from the human exome for variants. Our capture technology is geared towards resequencing gene subsets substantially larger than can be done efficiently with simplex or multiplex PCR but smaller in scale than exome sequencing. We describe all the steps from the initial capture assay to single nucleotide variant (SNV) discovery. The capture methodology uses in-solution 80-mer oligonucleotides. To provide optimal flexibility in choosing human gene targets, we designed an in silico set of oligonucleotides, the Human OligoExome, that covers the gene exons annotated by the Consensus Coding Sequencing Project (CCDS). This resource is openly available as an Internet accessible database where one can download capture oligonucleotides sequences for any CCDS gene and design custom capture assays. Using this resource, we demonstrated the flexibility of this assay by custom designing capture assays ranging from 10 to over 100 gene targets with total capture sizes from over 100 Kilobases to nearly one Megabase. We established a method to reduce capture variability and incorporated indexing schemes to increase sample throughput. Our approach has multiple applications that include but are not limited to population targeted resequencing studies of specific gene subsets, validation of variants discovered in whole genome sequencing surveys and possible diagnostic analysis of disease gene subsets. We also present a cost analysis demonstrating its cost-effectiveness for large population studies.
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影响因子：
1.7
作者：
Jurka, J;Kapitonov, VV;Walichiewicz, J
通讯作者：
Walichiewicz, J
DOI：
10.1073/pnas.0702165104
发表时间：
2007-05-29
影响因子：
11.1
作者：
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通讯作者：
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影响因子：
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作者：
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作者：
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通讯作者：
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影响因子：
14.9
作者：
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通讯作者：
CLARK, JM