Department of Entomology and Nematology, University of Florida, Gainesville, Florida, USA.
Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, USA.
Mol Ecol Resour. 2023 Jul;23(5):975-989. doi: 10.1111/1755-0998.13761. Epub 2023 Feb 22.
DNA barcoding approaches have greatly increased our understanding of biodiversity on the planet, and metabarcoding is widely used for classifying members of the phylum Nematoda. However, loci typically utilized in metabarcoding studies are often unable to resolve closely related species or are unable to recover all taxa present in a sample due to inadequate PCR primer binding. Mitochondrial metagenomics (mtMG) is an alternative approach utilizing shotgun sequencing of total DNA to recover the mitochondrial genomes of all species present in samples. However, this approach requires a comprehensive reference database for identification and currently available mitochondrial sequences for nematodes are highly dominated by sequences from the order Rhabditida, and excludes many clades entirely. Here, we analysed the efficacy of mtMG for the recovery of nematode taxa and the generation of mitochondrial genomes. We first developed a curated reference database of nematode mitochondrial sequences and expanded it with 40 newly sequenced taxa. We then tested the mito-metagenomics approach using a series of nematode mock communities consisting of morphologically identified nematode species representing various feeding traits, life stages, and phylogenetic relationships. We were able to identify all but two species through the de novo assembly of COX1 genes. We were also able to recover additional mitochondrial protein coding genes (PCGs) for 23 of the 24 detected species including a full array of 12 PCGs from five of the species. We conclude that mtMG offers a potential for the effective recovery of nematode biodiversity but remains limited by the breadth of the reference database.
DNA 条形码方法极大地提高了我们对地球上生物多样性的理解，代谢条形码广泛用于分类线虫门的成员。然而，代谢条形码研究中常用的基因座通常无法解决密切相关的物种，或者由于 PCR 引物结合不足而无法恢复样品中存在的所有分类群。线粒体宏基因组学（mtMG）是一种替代方法，利用总 DNA 的鸟枪法测序来恢复样品中存在的所有物种的线粒体基因组。然而，这种方法需要一个全面的参考数据库进行鉴定，目前可用的线虫线粒体序列高度由 Rhabditida 目序列主导，并且完全排除了许多进化枝。在这里，我们分析了 mtMG 用于回收线虫分类群和生成线粒体基因组的功效。我们首先开发了一个经过精心整理的线虫线粒体序列参考数据库，并通过 40 个新测序的分类群对其进行了扩展。然后，我们使用一系列由形态学鉴定的线虫物种组成的线虫模拟群落来测试 mito-metagenomics 方法，这些线虫物种代表各种摄食特征、生活阶段和系统发育关系。我们能够通过 COX1 基因的从头组装识别除两种物种之外的所有物种。我们还能够恢复 24 个检测到的物种中的 23 个额外的线粒体蛋白编码基因（PCGs），包括五个物种的 12 个 PCGs 全数组。我们得出结论，mtMG 为有效回收线虫生物多样性提供了潜力，但仍然受到参考数据库广度的限制。
