Selective whole genome amplification of Plasmodium malariae DNA from clinical samples reveals insights into population structure
Selective whole genome amplification of Plasmodium malariae DNA from clinical samples reveals insights into population structure
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DOI：
10.1038/s41598-020-67568-4
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发表时间：
2020-07-02
影响因子：
4.6
通讯作者：
Campino, Susana
中科院分区：
文献类型：
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作者：
Ibrahim, Amy;Benavente, Ernest Diez;Campino, Susana
三日疟原虫疟疾寄生虫的基因组多样性研究不足，部分原因是受感染的个体往往存在低寄生虫密度，导致难以获得足够的寄生虫DNA进行基因组分析。选择性全基因组扩增（SWGA）增加了临床样品中病原体DNA的相对水平，但尚未适用于三日疟原虫寄生虫。在这里，我们设计了定制的SWGA引物，其成功地扩增了直接从来自六个国家的患有疟疾疟原虫单感染的患者的未处理的临床血液样品中提取的三日疟原虫DNA，并进一步测试了SWGA对与其他疟原虫属的混合感染的功效。SWGA能够成功地对具有低寄生虫密度的样品进行全基因组测序（即，具有0.0064%寄生虫血症的一个样品导致>= 5个读段覆盖44%的基因组），当与未扩增的样品相比时，导致基因组覆盖率平均增加14倍。我们总共鉴定了868，476个全基因组SNP，其中194，709个在18个高质量分离株中是独特的。在排除高变亚端粒区域后，定义了29，899个独特SNP的高质量核心子集。群体遗传学分析表明，三日疟原虫寄生虫显示出明显的大陆地理分离。此外，SWGA成功地扩增了感兴趣的遗传区域，例如恶性疟原虫耐药相关基因座的直系同源物（Pfdhfr、Pfdhps、Pfcrt、Pfk13和Pfmdr1），并且在这些基因中检测到几种非同义SNP。总之，我们已经建立了一个强大的SWGA方法，可以帮助全基因组测序的三日疟原虫，从而促进实施急需的大规模多人口的基因组研究，这种被忽视的疟疾寄生虫。正如在其他疟原虫中所证明的那样，这种遗传多样性研究可以深入了解这种疾病的生物学基础，并为疟疾监测和控制措施提供信息。
The genomic diversity of Plasmodium malariae malaria parasites is understudied, partly because infected individuals tend to present with low parasite densities, leading to difficulties in obtaining sufficient parasite DNA for genome analysis. Selective whole genome amplification (SWGA) increases the relative levels of pathogen DNA in a clinical sample, but has not been adapted for P. malariae parasites. Here we design customized SWGA primers which successfully amplify P. malariae DNA extracted directly from unprocessed clinical blood samples obtained from patients with P. malariae-mono-infections from six countries, and further test the efficacy of SWGA on mixed infections with other Plasmodium spp. SWGA enables the successful whole genome sequencing of samples with low parasite density (i.e. one sample with a parasitaemia of 0.0064% resulted in 44% of the genome covered by >= 5 reads), leading to an average 14-fold increase in genome coverage when compared to unamplified samples. We identify a total of 868,476 genome-wide SNPs, of which 194,709 are unique across 18 high-quality isolates. After exclusion of the hypervariable subtelomeric regions, a high-quality core subset of 29,899 unique SNPs is defined. Population genetic analysis suggests that P. malariae parasites display clear geographical separation by continent. Further, SWGA successfully amplifies genetic regions of interest such as orthologs of P. falciparum drug resistance-associated loci (Pfdhfr, Pfdhps, Pfcrt, Pfk13 and Pfmdr1), and several non-synonymous SNPs were detected in these genes. In conclusion, we have established a robust SWGA approach that can assist whole genome sequencing of P. malariae, and thereby facilitate the implementation of much-needed large-scale multi-population genomic studies of this neglected malaria parasite. As demonstrated in other Plasmodia, such genetic diversity studies can provide insights into the biology underlying the disease and inform malaria surveillance and control measures.