Combining target enrichment with barcode multiplexing for high throughput SNP discovery
Combining target enrichment with barcode multiplexing for high throughput SNP discovery
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DOI：
10.1186/1471-2164-11-641
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发表时间：
2010-11-18
期刊：
影响因子：
4.4
通讯作者：
Jowett, Jeremy B. M.
中科院分区：
文献类型：
--
作者：
Cummings, Nik;King, Rob;Jowett, Jeremy B. M.
背景：遗传连锁分析的主要目的是确定影响表型性状的基因。在连锁区域定位之后，疾病连锁基因座的有效遗传解剖需要在基因座上鉴定功能变体。由于遗传多样性的程度，这些功能变异很难检测，并且迄今为止，对种群内和种群间存在的大量变异的编目不完整。大规模并行测序平台为变异发现提供了前所未有的能力，但分析的样本数量仍然受到每个样本成本的限制。在使用多重方法或通过利用靶向富集技术降低重测序成本方面已经取得了一些进展，靶向富集技术提供了对感兴趣的基因组区域进行重测序的能力，而不是全基因组测序。我们开发了一种方法，将当前的多重方法与基于溶液的靶富集方法相结合，以进一步降低重测序的成本，其中区域-需要特定的排序。我们的多重/富集策略产生了高质量的数据，测序深度标称降低。我们进行了基因分型研究，并成功地在所有样品中发现新的SNP等位基因在单重，双链和pentaplex levels.Conclusion：我们的工作描述了成功的组合有针对性的富集方法和索引条形码多路复用，以降低成本，时间和劳动力与处理大样本集。此外，我们已经表明，所获得的测序深度足以在单链体、双链体和五链体水平上进行可靠的SNP基因分型分析。
Background: The primary goal of genetic linkage analysis is to identify genes affecting a phenotypic trait. After localisation of the linkage region, efficient genetic dissection of the disease linked loci requires that functional variants are identified across the loci. These functional variations are difficult to detect due to extent of genetic diversity and, to date, incomplete cataloguing of the large number of variants present both within and between populations. Massively parallel sequencing platforms offer unprecedented capacity for variant discovery, however the number of samples analysed are still limited by cost per sample. Some progress has been made in reducing the cost of resequencing using either multiplexing methodologies or through the utilisation of targeted enrichment technologies which provide the ability to resequence genomic areas of interest rather that full genome sequencing.Results: We developed a method that combines current multiplexing methodologies with a solution-based target enrichment method to further reduce the cost of resequencing where region-specific sequencing is required. Our multiplex/enrichment strategy produced high quality data with nominal reduction of sequencing depth. We undertook a genotyping study and were successful in the discovery of novel SNP alleles in all samples at uniplex, duplex and pentaplex levels.Conclusion: Our work describes the successful combination of a targeted enrichment method and index barcode multiplexing to reduce costs, time and labour associated with processing large sample sets. Furthermore, we have shown that the sequencing depth obtained is adequate for credible SNP genotyping analysis at uniplex, duplex and pentaplex levels.