Improved 18S and 28S rDNA primer sets for NGS-based parasite detection
Improved 18S and 28S rDNA primer sets for NGS-based parasite detection
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DOI：
10.1038/s41598-019-52422-z
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发表时间：
2019-10-31
影响因子：
4.6
通讯作者：
Kikuchi, Taisei
中科院分区：
文献类型：
--
作者：
Kounosu, Asuka;Murase, Kazunori;Kikuchi, Taisei
新一代测序（NGS）的发展和应用使得通过广泛的并行测序对微生物群落进行全面分析成为可能。目前对真核微生物群落的分析主要基于 18S rRNA 基因 (rDNA) 片段的聚合酶链反应扩增。我们发现广泛使用的 18S rDNA 引物可以扩增细菌 16S rRNA 基因的大量片段，从而阻碍了稀有真核物种的高通量检测，特别是在粪便材料等富含细菌的样品中。在这项研究中，我们对粪便样本进行了计算机模拟和基于 NGS 的分析，以评估针对真核生物 18S 和 28S rDNA 的现有引物，以避免细菌读数污染并提高真核生物的分类覆盖率，特别是寄生虫类群。我们的研究结果表明，新选择的引物组可以实现这些目标，代表了 NGS 的替代策略。
The development and application of next-generation sequencing (NGS) have enabled comprehensive analyses of the microbial community through extensive parallel sequencing. Current analyses of the eukaryotic microbial community are primarily based on polymerase chain reaction amplification of 18S rRNA gene (rDNA) fragments. We found that widely-used 18S rDNA primers can amplify numerous stretches of the bacterial 16S rRNA gene, preventing the high-throughput detection of rare eukaryotic species, particularly in bacteria-rich samples such as faecal material. In this study, we employed in silico and NGS-based analyses of faecal samples to evaluated the existing primers targeting eukaryotic 18S and 28S rDNA in terms of avoiding bacterial read contamination and improving taxonomic coverage for eukaryotes, with a particular emphasis on parasite taxa. Our findings revealed that newly selected primer sets could achieve these objectives, representing an alternative strategy for NGS.