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

The mitochondria! genome (mitogenome) has proven to be important for the taxonomy, systematics, and population genetics of ticks. However, current methods to generate mitogenomes can be costprohibitive 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. Twentysix 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 similar to$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.

Automated summary

A cost-effective approach for amplifying and sequencing the whole mitogenome of individual tick specimens was developed and successfully applied to 85 tick samples, including 26 species with no complete mitogenome data available on GenBank. The accuracy and cost efficiency of this method were validated, making it applicable to genetic research on ticks and other metazoans.