BACKGROUND Owing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA. However, sWGA has had limited success in generating reliable sequencing data from low parasitaemia samples. In this study, enzymatic digestion with MspJI prior to sWGA and whole genome sequencing was evaluated to determine whether this approach improved genome coverage compared to sWGA alone. The potential of sWGA to cause amplification bias in polyclonal infections was also examined. METHODS DNA extracted from laboratory-created dried blood spots was treated with a modification-dependent restriction endonuclease, MspJI, and filtered via vacuum filtration. Samples were then selectively amplified using a previously reported sWGA protocol and subjected to WGS. Genome coverage statistics were compared between the optimized sWGA approach and the previously reported sWGA approach performed in parallel. Differential amplification by sWGA was assessed by comparing WGS data generated from lab-created mixtures of parasite isolates, from the same geographical region, generated with or without sWGA. RESULTS MspJI digestion did not enrich for parasite DNA. Samples that underwent vacuum filtration (without MspJI digestion) prior to sWGA had the highest parasite DNA concentration and displayed greater genome coverage compared to MspJI + sWGA and sWGA alone, particularly for low parasitaemia samples. The optimized sWGA (filtration + sWGA) approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasites did not show evidence of differential amplification of parasite strains compared to directly sequenced samples. CONCLUSION This optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitaemia samples. The absence of amplification bias in data generated from mixtures of isolates from the same geographic region suggests that this approach can be appropriately used for molecular epidemiological studies.

中文翻译：

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优化寄生虫DNA富集方法以从低寄生虫血症样品中生成恶性疟原虫的全基因组测序数据。

背景技术由于临床样品中含有大量宿主DNA，因此要生成高质量的恶性疟原虫全基因组测序（WGS）数据，需要对寄生虫DNA进行富集。富集通常是通过在存储之前清除受感染血液的白细胞来实现的。但是，在资源匮乏的情况下白细胞耗竭是困难的，并且限制了对预期收集样品的分析。结果，诸如选择性全基因组扩增（sWGA）之类的方法被用于富集寄生虫DNA。但是，sWGA在从低寄生虫血症样品中生成可靠的测序数据方面取得的成功有限。在这项研究中，评估了在sWGA之前使用MspJI进行的酶消化和全基因组测序，以确定与单独的sWGA相比，该方法是否提高了基因组覆盖率。还检查了sWGA在多克隆感染中引起扩增偏倚的潜力。方法用实验室修饰的限制性内切核酸酶MspJI处理从实验室干血斑中提取的DNA，并通过真空过滤进行过滤。然后使用先前报道的sWGA协议选择性扩增样品，并进行WGS处理。比较了优化的sWGA方法和先前报告的并行执行的sWGA方法之间的基因组覆盖率统计数据。通过比较由实验室创建的来自相同地理区域的有或没有sWGA的寄生虫分离物混合物产生的WGS数据来评估sWGA的差异扩增。结果MspJI消化未富集寄生虫DNA。与单独的MspJI + sWGA和sWGA相比，在sWGA之前经过真空过滤（无MspJI消化）的样品具有最高的寄生虫DNA浓度并显示出更大的基因组覆盖范围，尤其是对于低寄生虫血症的样品。优化的sWGA（过滤+ sWGA）方法已成功用于从马拉维的218个非白细胞耗竭的野外样本中生成WGS数据。与直接测序的样品相比，实验室创建的寄生虫混合物的序列没有显示出寄生虫菌株差异扩增的证据。结论该优化的sWGA方法是一种可靠的方法，可从非白细胞耗竭的低寄生虫血症样本中获取WGS数据。