基于高通量测序的表层水体纤毛虫多样性评估方法比较与优化

基于扩增子的高通量测序技术广泛应用于微型生物多样性的研究, 不同的测序手段和数据分析流程, 影响着微型生物多样性与群落结构的分析结果。本研究以易于形态鉴定的砂壳纤毛虫作为研究对象, 比较DNA测序、RNA测序和形态学方法检获的多样性和物种组成等, 探究序列分析流程中关键步骤: 嵌合体处理、可操作分类单元分析方法选择、合并相似分类单元以及去除稀有类群等对多样性结果的影响。研究结果显示无论基于DNA还是RNA的分子手段与形态学方法检获的主要物种基本一致, 与DNA测序相比, RNA测序检获的物种数少, 但差异不显著。基于97%以上相似度聚类和单核苷酸变异所得群落结构相似, 无显著差异; 且所有分析方法都能在一定程度上反映出自然界中不同类群的相对丰度。相较于单核苷酸变异和其他相似度阈值, 99%相似度下聚类所得多样性更为接近形态学结果。去除嵌合体和稀有类群(去除阈值: DNA测序0.05%; RNA测序0.07%), 可明显改善分子多样性虚高的问题。本研究为纤毛虫等真核微生物的分子多样性研究提供了科学的数据分析流程, 对未来真核微生物多样性研究具有指导意义。

Comparison and optimization of high-throughput sequence processing strategies: a case study regarding ciliate diversity in surface seawater

High-throughput amplicon sequencing has been widely employed for studying microbial diversity. Different sequencing methods and data analysis processes influence the analysis results of microbial biodiversity and community structure. Therefore, this study aimed to investigate the effects of different methods, the key steps involved in data analysis of the chimera removal and rare taxa, and the merging of similar taxonomic units. Results revealed that the main species detected by DNA sequencing, RNA sequencing, and morphological methods were similar. Moreover, fewer species were detected by RNA sequencing than by DNA sequencing; however, the difference was not significant. Community structural analysis showed a similar community structure based on the operational taxonomic unit (OTU) clustering on a similarity of 97%, 98%, 99%, and 100%. Both methods of the single- nucleotide variation and cluster based on sequence similarity were able to detect a similar relative abundance of different taxa to that by the morphological method. The species richness detected by OTU clustering under 99% similarity was similar to that detected by morphological results. Furthermore, chimera removal and rare taxa (removal thresholds: 0.05% for DNA sequencing and 0.07% for RNA sequencing) can improve the issue of inflated molecular diversity. Thus, this study provides a scientific analysis process to investigate ciliate molecular diversity and provides guidelines for future research on eukaryotic microbial diversity.