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Cryptosporidium in fish: alternative sequencing approaches and analyses at multiple loci to resolve mixed infections

机译：鱼中的脊髓孢子虫：多个基因座的替代测序方法和分析以解决混合感染

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Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger - and next-generation - sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68.9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections.
机译：目前，人们对鱼类隐孢子虫的系统学、生物学和流行病学了解甚少。在这里，我们比较了鱼隐孢子虫在18S rRNA和肌动蛋白基因上的Sanger和下一代测序（NGS）。寄主包括11种观赏鱼，跨越四目八科。目的是：（i）确认寄生虫的丰富遗传多样性和混合感染的高频率；以及（ii）探索在复杂遗传混合物存在的情况下NGS的潜力。通过Sanger测序，在肌动蛋白基因座上获得了四种主要基因型，而在18S基因座上，鉴定了七种基因型。在这两个基因座上，NGS都显示出频繁的混合感染，包括一个高度显性的变种加上相当罕见的基因型。这两种测序方法在两个位点都检测到了新的隐孢子虫基因型，包括通过Sanger测序和NGS鉴定的一种新的高度丰富的肌动蛋白基因型。重要的是，该基因型占所有样本所有NGS读数的68.9%（249 585/362 372）。目前的研究证实，水族馆鱼类可以容纳大量未经探索的隐孢子虫遗传多样性。尽管巢式PCR常用于分子寄生虫学研究，但当后一种方法用于调查多种感染时，巢式PCR阻止了定量比较，并阻碍了NGS的优势。

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