Validation of tick cell line origin by sequencing and assembly of complete mitogenomes

<ns5:p> Background Tick cell lines are increasingly widely-used tools for the study of tick biology and interactions with tick-borne pathogens. While cell lines cannot fully recapitulate the natural history of ticks, they fill a valuable niche for laboratories that lack access to tick colonies, and reduce dependence on laboratory animals for tick lifecycle maintenance. Equally as for cell lines derived from any other organism, validating their species identity and origin is of critical importance in maintaining the reliability and reproducibility of research using these tools. Here, we report the complete mitogenomes from nine cell lines (derived from seven ixodid species: <ns5:italic>Rhipicephalus sanguineus</ns5:italic> , <ns5:italic>Rhipicephalus appendiculatus</ns5:italic> , <ns5:italic>Rhipicephalus microplus</ns5:italic> , <ns5:italic>Amblyomma variegatum</ns5:italic> , <ns5:italic>Hyalomma anatolicum</ns5:italic> , <ns5:italic>Ixodes ricinus</ns5:italic> and <ns5:italic>Ixodes scapularis</ns5:italic> ) maintained in the Tick Cell Biobank. Methods We use both short- and long-read sequencing technologies to obtain the mitochondrial assemblies from the indicated cell lines. Mitochondrial assemblies were generated using MitoHiFi from long reads and NOVOPlasty from short reads. Gene annotations were performed using MitoHiFi or Mitos2. Results We confirmed the original presumptive species of each cell line in all cases, including RSE/PILS35 as derived from <ns5:italic>R. sanguineus sensu stricto</ns5:italic> , an important result in light of the recent reclassification of species within the <ns5:italic>R. sanguineus</ns5:italic> complex. Analysis of mitogenomic nucleotide diversity in the <ns5:italic>R. sanguineus</ns5:italic> and <ns5:italic>R. microplus</ns5:italic> species complexes identified regions of high variability that may be useful for future targeted assessment of molecular systematics and population structure. Conclusions This study highlights the tractability of mitogenome assembly for confirmation of cell line species origin and enables phylogenetic placement of these cell lines among the expanding mitogenomic resources for ticks worldwide. </ns5:p>