Barbato Matteo, Kovacs Toby, Coleman Melinda A, Broadhurst Matt K, de Bruyn Mark
School of Life and Environmental Sciences The University of Sydney Sydney New South Wales Australia.
National Marine Science Centre NSW Department of Primary Industries Coffs Harbour New South Wales Australia.
Ecol Evol. 2019 Feb 1;9(5):2678-2687. doi: 10.1002/ece3.4934. eCollection 2019 Mar.
The application of high-throughput sequencing to retrieve multi-taxon DNA from different substrates such as water, soil, and stomach contents has enabled species identification without prior knowledge of taxon compositions. Here we used three minibarcodes designed to target mitochondrial COI in plankton, 16S in fish, and 16S in crustaceans, to compare ethanol- and tissue-derived DNA extraction methodologies for metabarcoding. The stomach contents of pygmy devilrays ( cf. ) were used to test whether ethanol-derived DNA would provide a suitable substrate for metabarcoding. The DNA barcoding assays indicated that tissue-derived operational taxonomic units (OTUs) were greater compared to those from extractions performed directly on the ethanol preservative. Tissue-derived DNA extraction is therefore recommended for broader taxonomic coverage. Metabarcoding applications should consider including the following: (i) multiple barcodes, both taxon specific (e.g., 12S or 16S) and more universal (e.g., COI or 18S) to overcome bias and taxon misidentification and (ii) PCR inhibitor removal steps that will likely enhance amplification yields. However, where tissue is limited or no longer available, but the ethanol-preservative medium is still available, metabarcoding directly from ethanol does recover the majority of common OTUs, suggesting the ethanol-retrieval method could be applicable for dietary studies. Metabarcoding directly from preservative ethanol may also be useful where tissue samples are limited or highly valued; bulk samples are collected, such as for rapid species inventories; or mixed-voucher sampling is conducted (e.g., for plankton, insects, and crustaceans).
高通量测序技术应用于从水、土壤和胃内容物等不同底物中检索多分类群DNA，使得无需事先了解分类群组成即可进行物种鉴定。在这里，我们使用了三种微型条形码，分别针对浮游生物中的线粒体COI、鱼类中的16S以及甲壳类动物中的16S，以比较用于宏条形码分析的乙醇衍生DNA和组织衍生DNA提取方法。侏儒魟（cf.）的胃内容物被用于测试乙醇衍生DNA是否能为宏条形码分析提供合适的底物。DNA条形码分析表明，与直接从乙醇防腐剂中提取的相比，组织衍生的可操作分类单元（OTU）更多。因此，建议采用组织衍生DNA提取方法以实现更广泛的分类覆盖。宏条形码分析应用应考虑包括以下几点：（i）多种条形码，包括分类群特异性的（如12S或16S）和更通用的（如COI或18S），以克服偏差和分类群错误识别；（ii）可能提高扩增产量的PCR抑制剂去除步骤。然而，在组织有限或不再可用但乙醇保存介质仍然可用的情况下，直接从乙醇中进行宏条形码分析确实能回收大多数常见的OTU，这表明乙醇提取方法可能适用于饮食研究。在组织样本有限或价值很高；收集大量样本（如用于快速物种清查）；或进行混合样本采样（如针对浮游生物、昆虫和甲壳类动物）的情况下，直接从保存乙醇中进行宏条形码分析也可能有用。
