Next-generation sequencing to inventory taxonomic diversity in eukaryotic communities: a test for freshwater diatoms
Next-generation sequencing to inventory taxonomic diversity in eukaryotic communities: a test for freshwater diatoms
复制标题
DOI：
10.1111/1755-0998.12105
复制
发表时间：
2013-07-01
影响因子：
7.7
通讯作者：
Bouchez, A.
中科院分区：
文献类型：
--
作者：
Kermarrec, L.;Franc, A.;Bouchez, A.
最近出现的条形码方法与下一代测序（NGS）相结合，为研究环境群落提出了新的视角。在这个框架内，我们测试了从焦磷酸测序输出与可用的DNA参考文库中获得准确的硅藻群落库存的可能性。我们使用了三个分子标记，针对核，叶绿体和线粒体基因组（SSU rDNA，rbcL和cox 1）和三个样本的模拟社区组成的30个已知的硅藻菌株属于21个物种。在检测方法学偏倚的目标中，一个样本直接由合并的培养物构成，而其他样本由合并的PCR产物组成。首先将通过焦磷酸测序（Roche 454）获得的NGS读段与包括用于构成模拟群落的所有物种的序列的DNA参考文库进行比较，其次与具有较大分类学覆盖的完整DNA参考文库进行比较。一个严格的分类分配给库存进行比较，以真实的之一。我们检测到由于DNA提取和PCR扩增导致假阴性检测的偏倚。相反，焦磷酸测序错误似乎会产生假阳性，特别是在密切相关的物种的情况下。DNA参考文库的分类覆盖率似乎是最关键的因素，再加上标记多态性，这是必不可少的，以确定分类群在物种水平。RbcL提供了高分辨率和大型DNA参考文库。虽然需要进一步优化，焦磷酸测序是适合于确定硅藻组合，并可能会发现在淡水生物监测领域的应用。
The recent emergence of barcoding approaches coupled to those of next-generation sequencing (NGS) has raised new perspectives for studying environmental communities. In this framework, we tested the possibility to derive accurate inventories of diatom communities from pyrosequencing outputs with an available DNA reference library. We used three molecular markers targeting the nuclear, chloroplast and mitochondrial genomes (SSU rDNA, rbcL and cox1) and three samples of a mock community composed of 30 known diatom strains belonging to 21 species. In the goal to detect methodological biases, one sample was constituted directly from pooled cultures, whereas the others consisted of pooled PCR products. The NGS reads obtained by pyrosequencing (Roche 454) were compared first to a DNA reference library including the sequences of all the species used to constitute the mock community, and second to a complete DNA reference library with a larger taxonomic coverage. A stringent taxonomic assignation gave inventories that were compared to the real one. We detected biases due to DNA extraction and PCR amplification that resulted in false-negative detection. Conversely, pyrosequencing errors appeared to generate false positives, especially in case of closely allied species. The taxonomic coverage of DNA reference libraries appears to be the most crucial factor, together with marker polymorphism which is essential to identify taxa at the species level. RbcL offers a high resolving power together with a large DNA reference library. Although needing further optimization, pyrosequencing is suitable for identifying diatom assemblages and may find applications in the field of freshwater biomonitoring.