Data from: A next-generation sequencing approach to river biomonitoring using benthic diatoms

Diatoms are main bioindicators used to assess the ecological quality of rivers, but their identification is difficult and time-consuming. Next Generation Sequencing (NGS) can be used to study communities of microorganisms, so we carried out a test of the reliability of 454 pyrosequencing for estimating diatom inventories in environmental samples. We used small subunit ribosomal deoxyribonucleic acid (SSU rDNA), ribulose-1, 5-bisphosphate carboxylase (rbcL), and cytochrome oxidase I (COI) markers and examined reference libraries to define thresholds between the intra- and interspecific and intra- and intergeneric genetic distances. Based on tests of 1 mock community, we used a threshold of 99% identity for SSU rDNA and rbcL sequences to study freshwater diatoms at the species level. We applied 454 pyrosequencing to 4 contrasting environmental samples (with one in duplicate), assigned taxon names to environmental sequences, and compared the qualitative and quantitative molecular inventories to those obtained by microscopy. Species richness detected by microscopy was always higher than that detected by pyrosequencing. Some morphologically detected taxa may have been persistent frustules from dead cells. Some taxa detected by molecular analysis were not detected by morphology and vice versa. The main source of divergence appears to be inadequate taxonomic coverage in DNA reference libraries. Only a small percentage of species (but almost all genera) in morphological inventories were included in DNA reference libraries. DNA reference libraries contained a smaller percentage of species from tropical (27.1–38.1%) than from temperate samples (53.7–77.8%). Agreement between morphological and molecular inventories was better for species with relative abundance >1% than for rare species. The rbcL marker appeared to provide more reproducible results (94.9% species similarity between the 2 duplicates) and was very useful for molecular identification, but procedural standardization is needed. The water-quality ranking assigned to a site via the Pollution Sensitivity diatom index was the same whether calculated with molecular or morphological data. Pyrosequencing is a promising approach for detecting all species, even rare ones, once reference libraries have been developed.硅藻（Diatoms）是用于评估河流生态质量的主要生物指示物（bioindicators），但其物种鉴定工作难度高且耗时耗力。下一代测序（Next Generation Sequencing，NGS）技术可用于微生物群落研究，本研究据此验证了454焦磷酸测序（454 pyrosequencing）在环境样品硅藻物种名录评估中的可靠性。研究选取小亚基核糖体脱氧核糖核酸（small subunit ribosomal deoxyribonucleic acid，SSU rDNA）、核酮糖-1,5-二磷酸羧化酶（ribulose-1,5-bisphosphate carboxylase，rbcL）与细胞色素氧化酶I（cytochrome oxidase I，COI）作为分子标记，并通过参考序列库明确了种内、种间及属内、属间的遗传距离阈值。基于1个模拟群落（mock community）的测试结果，我们将SSU rDNA与rbcL序列的物种级鉴定阈值设定为99%序列一致性，用于淡水硅藻的物种水平分析。我们对4组具有显著差异的环境样品（其中1组设置了生物学重复）开展454焦磷酸测序，为环境序列匹配对应分类单元名称，并将定性与定量的分子物种名录与显微镜观测结果进行对比。显微镜观测到的物种丰富度始终高于焦磷酸测序结果。部分形态学检出的分类单元可能来自死亡细胞残留的硅质壳体（frustules）。部分分子分析检出的分类单元未被形态学方法观测到，反之亦然。二者产生差异的核心原因在于DNA参考序列库的分类学覆盖度不足：形态学名录中仅有极小比例的物种（但几乎覆盖所有检出属）被收录至DNA参考序列库。热带样品中被DNA参考序列库收录的物种占比（27.1%~38.1%）低于温带样品（53.7%~77.8%）。相对丰度大于1%的物种，其形态学与分子名录的一致性优于稀有物种。rbcL标记的实验结果重现性更佳（两组重复样品的物种相似性达94.9%），且可有效用于分子鉴定，但仍需开展实验流程标准化工作。无论采用分子数据还是形态学数据计算污染敏感性硅藻指数（Pollution Sensitivity diatom index），所得到的点位水质等级结果完全一致。一旦完善DNA参考序列库，焦磷酸测序将成为全面检测所有物种（包括稀有物种）的极具应用前景的技术手段。