Multi-sample long-read nanopore sequencing of Agabus bipustulatus (Coleoptera: Dytiscidae: Agabinae) mitogenome produces effectively reliable data for downstream analyses.

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.