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核心生物指示物（bioindicators），但其形态鉴定难度大且耗时费力。下一代测序（Next Generation Sequencing, NGS）可用于微生物群落研究，因此本研究开展了针对454焦磷酸测序（454 pyrosequencing）在环境样品中估算硅藻物种名录可靠性的验证实验。本研究选用小亚基核糖体脱氧核糖核酸（SSU rDNA）、核酮糖-1,5-二磷酸羧化酶（rbcL）与细胞色素氧化酶I（COI）作为分子标记，并通过检索参考序列库，明确了种内、种间以及属内、属间遗传距离的界定阈值。基于1个模拟群落（mock community）的测试结果，我们确定以99%序列相似性作为SSU rDNA与rbcL序列的物种水平鉴定阈值，用于淡水硅藻的物种识别。我们将454焦磷酸测序应用于4组差异显著的环境样品（其中1组设置生物学重复），为环境序列标注分类单元（taxon）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。部分经形态学检出的分类单元可能来自死亡细胞残留的硅质壳（frustules）；部分分子分析检出的分类单元未被形态学观测发现，反之亦然。导致二者差异的主要原因在于DNA参考序列库的分类覆盖度不足。形态学物种名录中仅有少量物种（但几乎涵盖所有属）被收录于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%）。当物种相对丰度＞1%时，形态学与分子名录的一致性优于稀有物种。rbcL标记展现出更高的结果重现性（两组生物学重复的物种相似性达94.9%），在分子鉴定中极具应用价值，但仍需开展实验流程标准化工作。无论基于分子数据还是形态学数据计算硅藻污染敏感性指数（Pollution Sensitivity diatom index），所得站点的水质等级均保持一致。待DNA参考序列库完善后，焦磷酸测序有望成为全面检出各类物种（包括稀有物种）的有效手段。