Leibniz Institute for Zoo and Wildlife Research, Department of Ecological Dynamics, Alfred-Kowalke-Str. 17, 10315 Berlin, Germany.
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang East Rd, Kunming, Yunnan 650223, China.
Gigascience. 2019 Apr 1;8(4). doi: 10.1093/gigascience/giz029.
The use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation.
Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S,16S,CytB); (iii) a "twin-tagging," 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples.
Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.
通过 metabarcoding 利用环境 DNA 进行物种检测的应用正在迅速发展。我们提出了一个经过共同设计的实验室工作流程和生物信息学管道，以减轻环境 DNA 使用过程中两个最重要的风险：样品污染和分类学误配。这些风险源于聚合酶链反应 (PCR) 扩增以检测痕量 DNA 的需要，再加上由于 DNA 降解，必须使用短的目标区域。
我们的高通量工作流程通过以下四个步骤来最大限度地降低这些风险：(i)使用 2 个 PCR 重复和 2 个提取重复进行技术复制；(ii)使用多标记物（12S、16S、CytB）；(iii)“双标记”两步 PCR 方案；(iv)使用概率分类分配方法 PROTAX，该方法可以解释不完整的参考数据库。由于参考序列中的注释错误可能导致分类学误配，因此我们提供了一个用于管理序列数据集的协议。对于一些分类群和一些标记物，由于以下原因，从公共参考数据库中删除了超过 50%的序列：(i)我们的目标扩增子和参考序列之间的重叠有限；(ii)参考序列的标记错误；(iii)冗余。最后，我们提供了一个生物信息学管道来处理扩增子并进行 PROTAX 分配，并在来自马来西亚沙巴的 1532 只水蛭的无脊椎动物衍生 DNA 数据集上对其进行了测试。双标记允许我们检测和排除不匹配标记的序列。所有样本中，最小的 DNA 片段（16S）扩增最频繁，但在物种级别上的区分能力较弱。使用严格和宽松的接受标准，我们在 95 个（严格）和 109 个（宽松）水蛭样本中分别发现了 162 个（严格）和 190 个（宽松）脊椎动物检测。
我们的 metabarcoding 工作流程应该有助于研究小组提高结果的稳健性，从而促进对环境和无脊椎动物衍生 DNA 的更广泛使用，这些 DNA 正在成为四足动物生态和保护信息的宝贵来源。
