New mitochondrial primers for metabarcoding of insects, designed and evaluated using in silico methods.
New mitochondrial primers for metabarcoding of insects, designed and evaluated using in silico methods.
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DOI：
10.1111/1755-0998.12942
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发表时间：
2019-01
影响因子：
7.7
通讯作者：
Ronquist F
中科院分区：
文献类型：
--
作者：
Marquina D;Andersson AF;Ronquist F
昆虫元条形码主要基于对细胞色素氧化酶I（COI）基因的“条形码区域”内的短片段的PCR扩增。然而，由于该基因的变异性，很难设计出良好的通用PCR引物。目前使用的大多数引物都与分类覆盖率或扩增偏差中的差距有关，这使得结果不太可靠，并阻碍了样品中存在的物种的检测。我们确定新的引物昆虫metabarcoding使用计算方法（ecoprimers和degeprime）适用于最全面的参考数据库的线粒体基因组的六足目组装日期。新引物相对于先前公布的引物在分类学覆盖率和相应扩增子的分辨率方面进行了计算机模拟评价。对于后者的标准，我们提出了一个新的指数，独家分类分辨率，这是一个更有生物意义的措施比今天使用的标准指数。我们的研究结果表明，最好的标记被发现在核糖体RNA基因（12S和16S），他们解决了约90%的遗传不同的物种在参考数据库。蛋白质编码基因中的一些标记物提供类似的性能，但仅在高得多的引物简并水平下。将两个最好的个体标记组合起来，可将有效的分类分辨率提高高达10%。分辨率强烈依赖于昆虫分类：COI引物检测到40%的Hyattera，而12S引物检测到12%的弹尾目。我们的研究结果表明，昆虫样本的基于扩增子的元巴编码可以通过选择其他引物而不是目前常用的引物来改进。
Insect metabarcoding has been mainly based on PCR amplification of short fragments within the “barcoding region” of the gene cytochrome oxidase I (COI). However, because of the variability of this gene, it has been difficult to design good universal PCR primers. Most primers used today are associated with gaps in the taxonomic coverage or amplification biases that make the results less reliable and impede the detection of species that are present in the sample. We identify new primers for insect metabarcoding using computational approaches (ecoprimers and degeprime) applied to the most comprehensive reference databases of mitochondrial genomes of Hexapoda assembled to date. New primers are evaluated in silico against previously published primers in terms of taxonomic coverage and resolution of the corresponding amplicons. For the latter criterion, we propose a new index, exclusive taxonomic resolution, which is a more biologically meaningful measure than the standard index used today. Our results show that the best markers are found in the ribosomal RNA genes (12S and 16S); they resolve about 90% of the genetically distinct species in the reference database. Some markers in protein‐coding genes provide similar performance but only at much higher levels of primer degeneracy. Combining two of the best individual markers improves the effective taxonomic resolution with up to 10%. The resolution is strongly dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S primers detect 12% of Collembola. Our results indicate that amplicon‐based metabarcoding of insect samples can be improved by choosing other primers than those commonly used today.
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影响因子：
3.7
作者：
Hajibabaei M;Shokralla S;Zhou X;Singer GA;Baird DJ
通讯作者：
Baird DJ
DOI：
10.1098/rsbl.2003.0025
发表时间：
2003-08-07
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4.7
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作者：
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通讯作者：
Pompanon F