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

  1. Alice Salussolia
  2. Ninon Lecoquierre
  3. Fabio Stoch
  4. Jean-François Flot

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Abstract

With over 430 species currently described, the amphipod genus Niphargus Schiödte, 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.

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  1. Version published to 10.24072/pcjournal.671
  2. Peer Community In Zoology


    Thanks to technical advances in DNA sequencing methods, the scale of molecular data for evolutionary and ecological research has increased by several orders of magnitude from single genes to full genomes (e.g. Seehausen et al. 2014). However, for the majority of non-model organisms, such genomic data are not yet available. Amphipods are an important crustacean group as exemplified by their high diversity and their wide distribution in freshwater and marine habitats, including also extreme environmental conditions like the deep sea, the polar regions or groundwater. Genomic data from this group remain scarce, however. One of the reasons is their extreme variability in genome size (Rees et al. 2007; Ritchie et al. 2017), reaching up to 63-64 Gb, which are among the largest genomes in the animal kingdom, and create additional challenges to obtain chromosome-scale, high-quality genomes.
    Here, Salussolia et al. (2025) described the details of all steps for a time- and budget-efficient genome skimming protocol to generate mitochondrial genomes (mitogenomes). The study organisms were three individuals of the subterranean amphipod species Niphargus dolenianensis, which remains understudied. Genome skimming does not intend to provide full genomic data but generates DNA sequences of the most common genomic regions including mitochondrial and ribosomal regions (e.g. Hoban et al. 2022). Full mitogenomes are increasingly replacing classic DNA barcode methods because they provide better phylogenetic resolution and additional important insights into evolution as for example of gene relocations deviating from ancestral mitochondrial gene orders. By using the generated mitogenomes together with publicly available amphipod mitogenomes and comparing patterns of gene orders, this paper confirms the suitability of complete mitogenomes to study evolutionary patterns. The generated mitogenomic data could also be used in the future to test for molecular patterns of positive selection as adaptations to groundwater as in another amphipod study (Benito et al. 2024). Salussolia et al. (2025) did not check if additional nuclear regions are also covered with their genome skimming approach. This could be an important asset as evolutionary studies require molecular data from both mitochondrial and nuclear genomes. Another advantage of the presented method was the fact that nuclear copies of mitochondrial genes (numts), which can be big problem in DNA barcoding (Song et al. 2008), were not among the generated DNA sequence data. 
    This paper is not aiming to reconstruct phylogenetic relationships among amphipods at a large taxonomic scale; for this, the mitogenomic data of many more amphipod groups are required, which are right now limited to a few families. However, detailed protocols and recommendations are essential to help closing this knowledge gap in the near future.
    This is exactly what Salussolia et al. (2025) are providing, making this paper an excellent guide for other researchers aiming to generate mitogenomes from amphipods but also from other non-model organisms. The results show that Oxford Nanopore genome skimming can reconstruct entire mitogenomes but errors because of long insertions or deletions remain to be corrected, either manually or by polishing with short read Illumina techniques. Both, the price for sequencing and the turn-over time of Oxford Nanopore techniques were surprisingly small and in the range of Sanger sequencing of single genes. Salussolia et al. (2025) thus make a convincing case to apply these novel techniques instead of classic, COI-based DNA barcoding.
    I am sure that this paper will be of great value to the crustacean and evolutionary research communities to foster the wide application of genome skimming techniques.
     
    References


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  3. Version published to 10.1101/2025.09.24.678374 on bioRxiv