Amplification and sequencing of entire tick mitochondrial genomes for a phylogenomic analysis

Abstract

The mitochondrial genome (mitogenome) has proven to be important for the taxonomy, systematics, and population genetics of ticks. However, current methods to generate mitogenomes can be cost-prohibitive at scale. To address this issue, we developed a cost-effective approach to amplify and sequence the whole mitogenome of individual tick specimens. Using two different primer sites, this approach generated two full-length mitogenome amplicons that were sequenced using the Oxford Nanopore Technologies’ Mk1B sequencer. We used this approach to generate 85 individual tick mitogenomes from samples comprised of the three tick families, 11 genera, and 57 species. Twenty-six of these species did not have a complete mitogenome available on GenBank prior to this work. We benchmarked the accuracy of this approach using a subset of samples that had been previously sequenced by low-coverage Illumina genome skimming. We found our assemblies were comparable or exceeded the Illumina method, achieving a median sequence concordance of 99.98%. We further analyzed our mitogenome dataset in a mitophylogenomic analysis in the context of all three tick families. We were able to sequence 72 samples in one run and achieved a cost/sample of ~ $10 USD. This cost-effective strategy is applicable for sample identification, taxonomy, systematics, and population genetics for not only ticks but likely other metazoans; thus, making mitogenome sequencing equitable for the wider scientific community.

Description of a new Pavlovskyella species (Acari: Argasidae) from Chile

“…Mitogenome sequencing and analyses were performed similar to the methods used by Kneubehl et al (2022). DNA was extracted from an unfed female using the Qiagen DNeasy Blood and Tissue kit following manufacturer's instructions with minor modifications.…”

“…From mitochondrial genome sequencing, Mans et al hypothesized that there are currently 15 different genera and 9 subgenera in the Argasidae family (Mans et al 2021). Moreover, phylogenetic analyses of tick mitogenomes including multiple species from South America support the reinstatement of old genera like Alectorobius Pocock, 1909 (Kneubehl et al 2022). Yet one conflict that arises while adding species to the Argasidae tree is that Pavlovskyella appears as a paraphyletic group composed of 4 clearly defined clades: the Afrotropical, Nearctic, Neotropical, and Palearctic (Mans et al 2019, 2021, Kneubehl et al 2022).…”

“…Moreover, phylogenetic analyses of tick mitogenomes including multiple species from South America support the reinstatement of old genera like Alectorobius Pocock, 1909 (Kneubehl et al 2022). Yet one conflict that arises while adding species to the Argasidae tree is that Pavlovskyella appears as a paraphyletic group composed of 4 clearly defined clades: the Afrotropical, Nearctic, Neotropical, and Palearctic (Mans et al 2019, 2021, Kneubehl et al 2022). From a morphological perspective, while adults and nymphs of Pavlovskyella spp.…”

Genome Skimming with Nanopore Sequencing Precisely Determines Global and Transposon DNA Methylation in Vertebrates

“…There are only five reports of nanopore sequencing used for genome skimming in animals at the time of publication, none of which examine the epigenome. 4,[23][24][25][26] In only one case has genome skimming been used to reconstruct mitochondria with a methylation-aware base caller but they did not assess either mitogenome or nuclear DNA methylation. 23 The generation of long-reads is crucial to improve alignment over traditional short reads.…”

Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes

“…Vossen & Buermans [ 26 ] employed a PCR-based indexing approach to sequence a human mitogenome in two slightly overlapping fragments, but did not pool multiple samples together. During revision of this manuscript, Kneubehl et al [ 27 ] introduced an ONT approach to sequence 85 tick mitochondrial genomes using a similar long amplicon approach as we present here. We sought to leverage these new developments to highly multiplex mitogenomes using long-amplicon sequencing.…”