Non-specific amplification compromises environmental DNA metabarcoding with COI
Non-specific amplification compromises environmental DNA metabarcoding with COI
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DOI：
10.1111/2041-210x.13276
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发表时间：
2019-09-25
影响因子：
6.6
通讯作者：
Mariani, Stefano
中科院分区：
文献类型：
--
作者：
Collins, Rupert A.;Bakker, Judith;Mariani, Stefano
环境样品中生物体外DNA的元条形码编码是目前水生生物监测和生态系统健康评价的关键技术。在设计实验时，特别是在制定社区标准和立法框架时，关键的考虑因素是遗传标记和引物组的选择。线粒体细胞色素c氧化酶亚基I（COI）是动物的标准DNA条形码标记，具有广泛的参考文库、分类学鉴别能力和可预测的序列变异，是许多元条形码应用的自然选择。然而，针对环境样品中的特定分类群体，COI的效用尚未得到充分审查。在这里，通过使用来自不列颠群岛的海洋和淡水鱼类的案例研究，我们量化了来自四个线粒体基因座- COI，细胞色素B，12 S和16 S-的12对引物在参考文库覆盖率，分类判别力和引物通用性方面的计算机性能。随后，我们在体外测试四个引物对-三个COI和一个12 S-他们的特异性，再现性和一致性与独立的数据集来自传统的调查方法在五个河口和沿海网站周围的英吉利海峡和北海。我们的研究结果表明，在低模板浓度下，对于含水的生物体外DNA，后生动物靶向和鱼类靶向COI引物与12 S相比表现不佳，由于原核和非靶真核DNA的非特异性扩增而表现出低水平的再现性。一个理想的元条形码应该有一个广泛的参考库，在此基础上可以设计定制引物，用于广泛的生物多样性评估或分类单元特定的调查。这样的数据库可用于COI，但低引物特异性阻碍了实际应用，而相反，12 S引物提供高特异性，但缺乏足够的参考。然而，后者可以通过扩展DNA条形码的概念来减轻，以包括通过基因组撇取现有组织集合产生的整个线粒体基因组。
Metabarcoding extra-organismal DNA from environmental samples is now a key technique in aquatic biomonitoring and ecosystem health assessment. Of critical consideration when designing experiments, and especially so when developing community standards and legislative frameworks, is the choice of genetic marker and primer set. Mitochondrial cytochrome c oxidase subunit I (COI), the standard DNA barcode marker for animals, with its extensive reference library, taxonomic discriminatory power and predictable sequence variation, is the natural choice for many metabarcoding applications. However, for targeting specific taxonomic groups in environmental samples, the utility of COI has yet to be fully scrutinized. Here, by using a case study of marine and freshwater fishes from the British Isles, we quantify the in silico performance of twelve primer pairs from four mitochondrial loci - COI, cytochrome b, 12S and 16S - in terms of reference library coverage, taxonomic discriminatory power and primer universality. We subsequently test in vitro four primer pairs - three COI and one 12S - for their specificity, reproducibility, and congruence with independent datasets derived from traditional survey methods at five estuarine and coastal sites around the English Channel and North Sea. Our results show that for aqueous extra-organismal DNA at low template concentrations, both metazoan-targeted and fish-targeted COI primers perform poorly in comparison to 12S, exhibiting low levels of reproducibility due to non-specific amplification of prokaryotic and non-target eukaryotic DNAs. An ideal metabarcode would have an extensive reference library upon which custom primers could be designed, either for broad assessments of biodiversity, or taxon specific surveys. Such a database is available for COI, but low primer specificity hinders practical application, while conversely, 12S primers offer high specificity, but lack adequate references. The latter, however, can be mitigated by expanding the concept of DNA barcodes to include whole mitochondrial genomes generated by genome-skimming existing tissue collections.