Centre for Biodiversity Genomics, University of Guelph, Guelph, Ontario, Canada.
Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada.
Mol Ecol Resour. 2019 May;19(3):711-727. doi: 10.1111/1755-0998.13008.
Although DNA metabarcoding is an attractive approach for monitoring biodiversity, it is often difficult to detect all the species present in a bulk sample. In particular, sequence recovery for a given species depends on its biomass and mitome copy number as well as the primer set employed for PCR. To examine these variables, we constructed a mock community of terrestrial arthropods comprised of 374 species. We used this community to examine how species recovery was impacted when amplicon pools were constructed in four ways. The first two protocols involved the construction of bulk DNA extracts from different body segments (Bulk Abdomen, Bulk Leg). The other protocols involved the production of DNA extracts from single legs which were then merged prior to PCR (Composite Leg) or PCR-amplified separately (Single Leg) and then pooled. The amplicons generated by these four treatments were then sequenced on three platforms (Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5). The choice of sequencing platform did not substantially influence species recovery, although the Miseq delivered the highest sequence quality. As expected, species recovery was most efficient from the Single Leg treatment because amplicon abundance varied little among taxa. Among the three treatments where PCR occurred after pooling, the Bulk Abdomen treatment produced a more uniform read abundance than the Bulk Leg or Composite Leg treatment. Primer choice also influenced species recovery and evenness. Our results reveal how variation in protocols can have substantial impacts on perceived diversity unless sequencing coverage is sufficient to reach an asymptote.
尽管 DNA 代谢组学是监测生物多样性的一种有吸引力的方法，但通常很难检测到批量样本中存在的所有物种。特别是，给定物种的序列回收取决于其生物量和线粒体拷贝数以及用于 PCR 的引物集。为了研究这些变量，我们构建了一个由 374 个物种组成的陆地节肢动物模拟群落。我们使用这个群落来研究当以四种方式构建扩增子池时，物种回收会受到怎样的影响。前两个方案涉及从不同的身体部位（Bulk Abdomen、Bulk Leg）构建批量 DNA 提取物。其他方案涉及从单个腿上提取 DNA，然后在 PCR 前合并（Composite Leg）或分别进行 PCR 扩增（Single Leg），然后再混合。然后将这四种处理方法产生的扩增子在三个平台（Illumina MiSeq、Ion Torrent PGM 和 Ion Torrent S5）上进行测序。测序平台的选择并没有显著影响物种回收，尽管 Miseq 提供了最高的序列质量。正如预期的那样，来自 Single Leg 处理的物种回收效率最高，因为扩增子丰度在分类群之间变化很小。在聚合后发生 PCR 的三种处理方法中，Bulk Abdomen 处理比 Bulk Leg 或 Composite Leg 处理产生更均匀的读长丰度。引物选择也会影响物种回收和均匀度。我们的研究结果揭示了方案的变化如何会对感知多样性产生重大影响，除非测序覆盖率足以达到渐近线。
