Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia.
Whole Genome Amplification and Reduced-Representation Genome Sequencing of Schistosoma japonicum Miracidia.
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DOI：
10.1371/journal.pntd.0005292
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发表时间：
2017-01
影响因子：
3.8
通讯作者：
Pollock DD
中科院分区：
文献类型：
--
作者：
Shortt JA;Card DC;Schield DR;Liu Y;Zhong B;Castoe TA;Carlton EJ;Pollock DD
在实施血吸虫病控制项目的地区，发病率和流行率已大大降低。然而，为了维持这些减少并逐步阻断传播，需要新的疾病监测工具。基因组方法有可能帮助追踪新感染的来源，并使我们能够监测耐药性。大规模的基因分型的努力已被成本，有限的目标基因座的数量，并从毛蚴，最容易从人类获得的生命阶段获得的DNA量少的阻碍。在这里，我们提出了一种方法，使用下一代测序提供高分辨率的基因组数据从S。japonicum为基础的研究。我们应用全基因组扩增，然后双消化限制性位点相关DNA测序（ddRADseq）的个人S。保存在Whatman FTA卡上的日本毛蚴。我们发现，我们可以有效地和一致地调查数十万个变异，从10,000到30,000个位点，来自六年前的存档毛蚴。从四川两个村庄的三个主机8毛蚴的变异分析表明，即使在这个有限的样本中，明确的人口结构的村庄和主机。这种高分辨率测序方法产生的信息比过去十年中采用的微卫星基因分型方法多三个数量级，从而有可能回答有关人类感染来源的详细问题并监测耐药性。每个样品的成本范围从$50-$200，这取决于所需的序列信息的量，并且我们预计这些成本可以进一步降低，因为测序成本的持续降低、方案的改进和并行化。这一方法为利用现代基因组规模的采样对S.日本血吸虫的监测，并可应用于其他寄生性物种和其他寄生蠕虫。血吸虫病是一种毁灭性的热带疾病，影响着全世界2亿多人。在过去的几十年里，在中国实施的传播控制策略已经降低了日本血吸虫病在许多地区的流行和发病率。然而，感染仍然持续存在，因此确定复发感染的来源具有很大的意义。基因分析是实现这一目标的一种有希望的手段。为了实现这一目标，我们进行了一项试点研究，以评估使用高通量测序来评估染色体遗传变异的地理分布的可行性。由于毛蚴（最容易获得的生命阶段）的DNA产量不足以进行高通量测序，我们首先采用全基因组扩增来获得足够量的DNA。然后，我们采用了一种技术，可重复测序的基因组在许多样品的相同部分。我们成功地测序了6岁的S。来自中国四川省的日本血吸虫样品，容易和经济地鉴定数以万计的可变基因座，足够的数量来区分精细规模的群体结构。进一步的人口抽样将有助于回答有关感染的持续性、新感染的来源以及寄生虫种群是否经历了抗药性的初期演变等重要问题。
In areas where schistosomiasis control programs have been implemented, morbidity and prevalence have been greatly reduced. However, to sustain these reductions and move towards interruption of transmission, new tools for disease surveillance are needed. Genomic methods have the potential to help trace the sources of new infections, and allow us to monitor drug resistance. Large-scale genotyping efforts for schistosome species have been hindered by cost, limited numbers of established target loci, and the small amount of DNA obtained from miracidia, the life stage most readily acquired from humans. Here, we present a method using next generation sequencing to provide high-resolution genomic data from S. japonicum for population-based studies. We applied whole genome amplification followed by double digest restriction site associated DNA sequencing (ddRADseq) to individual S. japonicum miracidia preserved on Whatman FTA cards. We found that we could effectively and consistently survey hundreds of thousands of variants from 10,000 to 30,000 loci from archived miracidia as old as six years. An analysis of variation from eight miracidia obtained from three hosts in two villages in Sichuan showed clear population structuring by village and host even within this limited sample. This high-resolution sequencing approach yields three orders of magnitude more information than microsatellite genotyping methods that have been employed over the last decade, creating the potential to answer detailed questions about the sources of human infections and to monitor drug resistance. Costs per sample range from $50-$200, depending on the amount of sequence information desired, and we expect these costs can be reduced further given continued reductions in sequencing costs, improvement of protocols, and parallelization. This approach provides new promise for using modern genome-scale sampling to S. japonicum surveillance, and could be applied to other schistosome species and other parasitic helminthes. Schistosomiasis is a devastating tropical disease that affects more than 200 million people worldwide. Over the past several decades, transmission control strategies implemented in China have reduced the prevalence and morbidity of Schistosoma japonicum in many areas. Infections still persist, however, and it is therefore of great interest to determine the sources of recurring infections. Genetic analysis is a promising means to achieve this. Towards this aim, we conducted a pilot study to assess the feasibility of using high-throughput sequencing to assess the geographic distribution of schistosome genetic variants. Because DNA yields from miracidia, the most easily accessible life stage, are insufficient for high throughput sequencing, we first employed whole genome amplification to obtain sufficient quantities of DNA. We then employed a technique that reproducibly sequences the same fraction of a genome across numerous samples. We successfully sequenced 6-year old S. japonicum samples from Sichuan Province, China, easily and economically identifying tens of thousands of variable loci, a sufficient number to discriminate fine-scale population structure. Further population sampling will help answer important questions concerning the persistence of infections, the sources of new infections, and whether parasite populations have undergone incipient evolution of drug resistance.