Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples.
Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples.
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DOI：
10.1128/mbio.02257-16
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发表时间：
2017-02-07
期刊：
影响因子：
6.4
通讯作者：
Winzeler EA
中科院分区：
文献类型：
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作者：
Cowell AN;Loy DE;Sundararaman SA;Valdivia H;Fisch K;Lescano AG;Baldeviano GC;Durand S;Gerbasi V;Sutherland CJ;Nolder D;Vinetz JM;Hahn BH;Winzeler EA
来自临床样本的微生物病原体的全基因组测序（WGS）是一种高度敏感的工具，用于更深入地了解许多感染的生物学、流行病学和耐药性机制。然而，在其宿主中表现出低密度的生物体的WGS是具有挑战性的，这是由于高水平的宿主基因组DNA（gDNA），这导致微生物基因组的覆盖率非常低。间日疟原虫是分布最广泛的疟疾形式，由于寄生虫密度低和缺乏离体培养系统，因此WGS特别困难。目前用于从临床样品中富集间日疟原虫DNA的技术需要大量的资源或不是一贯有效的。在这里，我们证明了选择性全基因组扩增（SWGA）可以从未处理的人类血液样本和干血斑中富集间日疟原虫gDNA，以获得高质量的WGS，从而对分离株进行遗传表征，否则这些分离株将非常昂贵或无法测序。我们实现了24×的平均基因组覆盖率，其中高达95%的间日疟原虫核心基因组被≥5个读段覆盖。所观察到的单核苷酸多态性（SNP）特征和耐药性突变与来自秘鲁相似区域的其他间日疟原虫序列的特征和耐药性突变一致，表明SWGA产生用于下游分析的高质量序列。SWGA是一种强大的工具，将使来自临床样本的间日疟原虫分离株的高效、具有成本效益的WGS能够应用于其他被忽视的微生物病原体。疟疾是一种由疟原虫寄生虫引起的疾病，2015年造成2.14亿例症状病例和43.8万例死亡。间日疟原虫是分布最广的物种，造成撒哈拉以南非洲以外的大多数疟疾感染。疟原虫全基因组测序为疟疾的流行病学和耐药机制研究提供了重要线索。然而，间日疟原虫的WGS是具有挑战性的，因为人体内的寄生虫水平低，并且缺乏培养寄生虫的常规系统。选择性全基因组扩增（SWGA）优先从靶标和宿主gDNA的混合物扩增病原体的基因组。在这里，我们证明了SWGA是一种简单、稳健的方法，可用于从未经处理的人血液样本和干血斑中富集间日疟原虫基因组DNA（gDNA），以获得具有成本效益的高质量WGS。
Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.