A broadly applicable COI primer pair and an efficient single-tube amplicon library preparation protocol for metabarcoding
A broadly applicable COI primer pair and an efficient single-tube amplicon library preparation protocol for metabarcoding
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DOI：
10.1002/ece3.4520
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发表时间：
2018-12-01
影响因子：
2.6
通讯作者：
Traugott, Michael
中科院分区：
文献类型：
--
作者：
Rubbmark, Oskar Rennstam;Sint, Daniela;Traugott, Michael
细胞色素c氧化酶亚基I（COI）基因的核苷酸变异使其成为物种序列分配的理想选择。然而，这种可变性也使得难以设计真正通用的引物。在这里，我们提出了正向引物“Sauron-S878”，专门设计用于促进元巴编码的文库制备。与针对水生系统优化的引物mCOIintF相比，该引物被修饰以提高陆生物种的覆盖率，其将计算机模拟覆盖率从可用NCBI序列的74.4%提高到98.3%（3 '区域中完全匹配，剩余引物中多达三个错配）。当与反向引物“jgHCO 2198”（片段长度与313 bp相似）配对时，这些引物对255个来自不同分类群的DNA提取物的扩增率为98.4%，优于许多其他常见的COI条形码引物。此外，开发了单管方案，其中这些引物扩增靶基因，并在一个反应中连接MID和Illumina测序衔接子。这消除了在文库制备期间对再扩增或酶促连接的需要，同时保持了模块化地组合联合收割机引物和MID的灵活性。使用单管方法，在MiSeq平台上对三个模拟样品的三个重复进行测序，与商业Nextera索引试剂盒相比没有不利影响。从这次运行中，75%的所有包括分类群可以回收，没有相当大的偏见之间的分类组。尽管98.4%的提取物被证实在体外扩增，但这一数字低于预期。这种差异的一个原因是模板中特定DNA类型的相对浓度与该DNA的返回读数数量之间存在明显的联系。我们认为，这种偏见可能是特别有问题的元条码，其中样本通常含有微量DNA在未知的数量。然而，这如何影响元条形码结果的完整性还没有得到很好的研究。
The nucleotide variation in the cytochrome c oxidase subunit I (COI) gene makes it ideal for assigning sequences to species. However, this variability also makes it difficult to design truly universal primers. Here, we present the forward primer "Sauron-S878," specifically designed to facilitate library preparation for metabarcoding. This primer is modified to improve the coverage of terrestrial species compared to the primer mCOIintF, optimized for aquatic systems, which raised the in silico coverage from 74.4% to 98.3% of available NCBI sequences (perfect match in 3 ' region, up to three mismatches in remaining primer). When paired with the reverse primer "jgHCO2198" (fragment length similar to 313 bp), these primers amplified 98.4% of 255 tested DNA extracts from various taxa, which are better than many other common COI barcoding primers. Furthermore, a single-tube protocol was developed, wherein these primers amplify the target gene, and attach MIDs and Illumina sequencing adapters in one reaction. This eliminates the need for re-amplification or enzymatic ligation during library preparation while keeping the flexibility to modularly combine primers and MIDs. Using the single-tube approach, three replicates of three mock samples were sequenced on a MiSeq platform with no adverse effects compared to commercial Nextera indexing kits. From this run, 75% of all included taxa could be recovered, with no considerable bias among taxonomic groups. Despite the fact that 98.4% of the extracts were confirmed to amplify in vitro, this number was lower than expected. A reason for this discrepancy was a clear link between the relative concentration of a specific DNA type in the template and the number of returned reads for this DNA. We would argue that such a bias may be especially problematic in metabarcoding where samples usually contain trace DNA in unknown amounts. However, how this affects the completeness of metabarcoding results has yet been poorly investigated.