三种靶向序列捕获策略的比较[选购宝典]

【字体: 时间:2011年05月04日 来源:生物通

编辑推荐:

  为了评估靶向富集方法在ABI SOLiD 3系统上表现如何,来自迈阿密大学米勒医学院以及Life Technologies的研究人员评估了三种富集技术:罗氏NimbleGen的SeqCap寡核苷酸杂交型芯片捕获,安捷伦的SureSelect寡核苷酸杂交溶液型捕获以及Raindance的多重PCR方法。文章发表在4月29日的《PloS ONE》杂志上。

生物通报道,尽管新一代测序(NGS)的通量越来越高,而费用越来越低,但它仍不是大多数遗传实验室的可行选择。对于复杂疾病的研究更是如此,这类研究至少需要数百个样本,以实现足够的统计能力。然而,这么多样本的全基因组测序,无论从成本考虑,还是从数据分析考虑,都是相对困难的。

为了在大样本量中充分发挥新一代测序技术的潜能,科学家们开发出几种方法,来选择性富集目标区域。与全基因组测序相比,富集后再测序降低了成本,也减轻了后续数据分析的压力,让研究人员能够轻松利用新一代测序的通量。目前有几种靶向富集方法,包括分子倒置探针连接测序(MIPS)、基于寡核苷酸杂交的方法,以及多重PCR。

为了评估这些方法在ABI SOLiD 3系统上表现如何,来自迈阿密大学米勒医学院以及Life Technologies的研究人员评估了三种富集技术:罗氏NimbleGen的SeqCap寡核苷酸杂交型芯片捕获,安捷伦的SureSelect寡核苷酸杂交溶液型捕获以及Raindance的多重PCR方法。文章发表在4月29日的《PloS ONE》杂志上。

罗氏NimbleGen是最早推出商业化序列捕获芯片的公司。它目前拥有多种标准和定制的捕获芯片,包括人外显子组捕获芯片。随后,安捷伦也推出了SureSelect靶向序列富集系统。两者的捕获都是基于寡核苷酸探针,但形式不同。NimbleGen是芯片形式,而安捷伦是溶液(In-Solution)形式。关于这两种技术,生物通之前也有报道,详见《揭开基因组捕获的神秘面纱》。

在本研究中,目标区域选自人类基因组中的外显子和进化保守区域。根据三种方法各自的指示来设计引物和探针。对于三种方法来说,目标区域都是相同的,大约0.8 Mb。富集以及测序之后,利用几个指标来分析SOLiD测序结果,包括各样品间覆盖深度的一致性、击中(on-target)和脱靶(off-target)效率、等位基因偏向以及与芯片数据的基因型一致性。

通过分析,研究人员发现:安捷伦的SureSelect表现出优异的击中效率以及各样品间读取深度的一致性。在读取深度为20倍及以下时,NimbleGen的性能很相似。Raindance和NimbleGen SeqCap的读取深度都更严格分布在平均值左右,但击中效率却不如SureSelect。在分析设计上,Raindance表现出最好的多功能性。它靶定了目标区域的97%,这对于诊断性的重测序可能很有用。

作者还提到,利用三种靶向富集方法所得的测序结果与已知的基因型表现出很好的一致性,表明不存在影响基因型检出的系统偏向。(生物通 薄荷)

原文摘要:

Comparison of Three Targeted Enrichment Strategies on the SOLiD Sequencing Platform

摘要:
Despite the ever-increasing throughput and steadily decreasing cost of next generation sequencing (NGS), whole genome sequencing of humans is still not a viable option for the majority of genetics laboratories. This is particularly true in the case of complex disease studies, where large sample sets are often required to achieve adequate statistical power. To fully leverage the potential of NGS technology on large sample sets, several methods have been developed to selectively enrich for regions of interest. Enrichment reduces both monetary and computational costs compared to whole genome sequencing, while allowing researchers to take advantage of NGS throughput. Several targeted enrichment approaches are currently available, including molecular inversion probe ligation sequencing (MIPS), oligonucleotide hybridization based approaches, and PCR-based strategies. To assess how these methods performed when used in conjunction with the ABI SOLID3+, we investigated three enrichment techniques: Nimblegen oligonucleotide hybridization array-based capture; Agilent SureSelect oligonucleotide hybridization solution-based capture; and Raindance Technologies' multiplexed PCR-based approach. Target regions were selected from exons and evolutionarily conserved areas throughout the human genome. Probe and primer pair design was carried out for all three methods using their respective informatics pipelines. In all, approximately 0.8 Mb of target space was identical for all 3 methods. SOLiD sequencing results were analyzed for several metrics, including consistency of coverage depth across samples, on-target versus off-target efficiency, allelic bias, and genotype concordance with array-based genotyping data. Agilent SureSelect exhibited superior on-target efficiency and correlation of read depths across samples. Nimblegen performance was similar at read depths at 20× and below. Both Raindance and Nimblegen SeqCap exhibited tighter distributions of read depth around the mean, but both suffered from lower on-target efficiency in our experiments. Raindance demonstrated the highest versatility in assay design.

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