Nanopore genome skimming with Illumina polishing yields highly accurate mitogenome sequences: a case study of Niphargus amphipods

Abstract

With over 430 species currently described, the amphipod genus Niphargus Schiodte, 1849 is the most species-rich crustacean genus in subterranean waters. Previous phylogenetic studies of this genus have relied mainly on mitochondrial COI and nuclear 28S sequences, which do not resolve all the nodes in its phylogeny. As a first step towards a mitogenome-based phylogeny of niphargids, we present here the first complete mitogenome sequence of Niphargus. To obtain high-accuracy mitogenome sequences and annotations, genome skimming of three individuals of Niphargus dolenianensis Lorenzi, 1898 was performed using both short, accurate reads (Illumina) and long, noisier reads (nanopore). Whereas the direct assembly of Illumina sequences yielded structurally incorrect mitogenome sequences, the assembly of nanopore reads produced highly accurate sequences that were corroborated by the mapping of Illumina reads. Polishing the nanopore consensus using Illumina reads corrected a handful of errors at the homopolymer level. The resulting mitogenome sequences ranged from 14,956 to 15,199 bp and shared the same arrangement of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a putative control region. Phylogenetic analyses based on protein-coding genes confirmed that the Niphargidae family is sister to Pseudoniphargidae, resolving their relationships with other amphipod families. This highlights the utility of mtDNA genome sequences for studying the evolution of this groundwater genus, and the refinement of new methodological approaches, such as nanopore sequencing, is promising for the study of its origin and diversification.