Bielikova Olena, Vargovčík Ondrej, Čiamporová-Zaťovičová Zuzana, Čiampor Fedor
Laboratory of molecular genetic research, Institute of Fisheries of the National Academy of Agrarian Sciences of Ukraine, Kyiv, Ukraine.
ZooLab, Department of Biodiversity and Ecology, Plant Science and Biodiversity Centre, Slovak Academy of Sciences, Bratislava, Slovakia.
J Insect Sci. 2025 Jan 20;25(1). doi: 10.1093/jisesa/ieaf009.
Mitochondrial genomes are a rich source of data for various downstream analyses such as population genetics, phylogeny, and systematics. Today it is possible to assemble rapidly large numbers of mitogenomes, mainly employing next-generation sequencing and third-generation sequencing. However, verification of the correctness of the generated sequences is often lacking, especially for noncoding, length-variable parts. Here we have assembled the mitochondrial genome (mitogenome) from four specimens of Agabus bipustulatus (L.) using long-read nanopore sequence data. The use of the latest nanopore chemistry (V14) combined with a comprehensive error correction workflow enabled the generation of mitogenomes with high accuracy and reproducibility, as tested on four samples. The resulting mitogenome is 17,876 bp long, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region. Differences in the control region length between samples were minimal. The arrangement of protein-coding genes, transfer RNAs, and ribosomal RNAs is similar to that of the ancestral insect mitogenome. Finally, we used the assembled, well-supported mitogenomes in the phylogenetic analysis of a part of the Dytiscidae related to the studied species and confronted the results with previous hypotheses. Conflicting estimates of their phylogeny suggest that considerably more robust data are required for a plausible sketch of their evolutionary history. Our research has confirmed that readily available third-generation sequencing technologies, such as Oxford Nanopore Technologies, combined with long-read sequencing, offer a highly efficient, reliable, and cost-effective approach to generate complete mitogenomes and potentially other longer regions of the genome. The use of reliable data will ultimately contribute to a deeper understanding and improved conservation strategies for diving beetles and other organisms.
线粒体基因组是用于各种下游分析（如群体遗传学、系统发育和分类学）的丰富数据来源。如今，主要利用新一代测序和第三代测序技术能够快速组装大量的线粒体基因组。然而，所生成序列的正确性验证往往缺失，尤其是对于非编码的、长度可变的部分。在此，我们利用长读长纳米孔序列数据，从四斑龙虱（Agabus bipustulatus (L.)）的四个样本中组装了线粒体基因组（mitogenome）。使用最新的纳米孔化学技术（V14）并结合全面的纠错流程，能够生成具有高精度和可重复性的线粒体基因组，这在四个样本上得到了验证。所得的线粒体基因组长度为17,876 bp，包括13个蛋白质编码基因、22个转运RNA基因、2个核糖体RNA基因和一个控制区。样本之间控制区长度的差异极小。蛋白质编码基因、转运RNA和核糖体RNA的排列与原始昆虫线粒体基因组相似。最后，我们将组装好的、得到充分支持的线粒体基因组用于与所研究物种相关的部分龙虱科昆虫的系统发育分析，并将结果与先前的假设进行对比。它们系统发育的相互矛盾的估计表明，需要更可靠的数据才能勾勒出其进化历史的合理轮廓。我们的研究证实，现成的第三代测序技术，如牛津纳米孔技术，结合长读长测序，为生成完整的线粒体基因组以及潜在的基因组其他更长区域提供了一种高效、可靠且经济高效的方法。使用可靠的数据最终将有助于更深入地了解潜水甲虫和其他生物，并改进其保护策略。
