Complete Chromosomal Sequences of Two Borrelia miyamotoi Samples Obtained from Ixodes ricinus Eggs in Czechia

Here, we present complete chromosome sequences of Borrelia miyamotoi samples CZ-F1E and CZ-F190E, which were obtained from Ixodes ricinus eggs from Czechia. The chromosome sequences, assembled from Illumina and Sanger sequencing data, had average coverage values of 647× and 3,216×, respectively. They belong to the European genotype, distinct from the Asian and American strains.

T he tick-borne human pathogen Borrelia miyamotoi is distributed in the Holarctic realm (1). Characterization of B. miyamotoi genome sequences, especially those from Europe, is needed since the genome sequences of only two strains from the Netherlands have recently been published (2) and several whole-genome sequences from North America and Asia have been studied (3)(4)(5)(6)(7).
In total, 416 engorged females of Ixodes ricinus were manually collected from dogs and cats by their owners in Czechia in 2018 to 2019. Ticks were stored separately in labeled tubes at 26°C and 90% relative humidity. Egg clusters were collected from 364 females promptly after oviposition and were stored in cryovials at Ϫ80°C. DNA was extracted from ϳ100 eggs per cluster with the DNeasy blood and tissue kit (Qiagen). The presence of B. miyamotoi DNA in 6 of 364 samples was confirmed by amplification of a partial sequence of the glpQ gene (8).
The ratio of B. miyamotoi to I. ricinus DNA copies in the six B. miyamotoi-positive samples was determined by nested PCR detection of the glpQ gene and I. ricinus ITS2 spacer (Table 1) in 10-fold serially diluted DNA samples (from nondiluted to 10 Ϫ6 diluted). PCR products from the second amplification step were visualized by 1.5% agarose gel electrophoresis. Two samples, designated CZ-F1E and CZ-F190E, with the highest B. miyamotoi/I. ricinus DNA ratios (i.e., 2:1 and 20:1, respectively) were subjected to whole-genome sequencing. DNA libraries were prepared using the NEBNext Ultra DNA library preparation kit (Illumina) and sequenced on the Illumina platform in paired-end mode with a read length of 150 bp (Novogene, China).
For each sample, the five longest scaffolds were manually selected from the assembly graph using Bandage (v0.8.1) (16). The average coverage values were 647ϫ and 3,216ϫ for CZ-F1E and CZ-F190E, respectively.
Gaps between the scaffolds were filled by PCR amplification using custom primers (Table 1) and Sanger sequencing. Annotation was performed using the Prokaryotic Genome Annotation Pipeline (17).
The complete chromosome sequences of 904,129 bp and 904,095 bp for CZ-F1E and CZ-F190E, respectively, with GC contents of 28.7%, contained 810 and 807 predicted protein-coding sequences, respectively, 3 rRNAs, 31 tRNAs, and 28 and 23 pseudogenes, respectively. The differences between the two genomes included 51 single-nucleotide variants, of which 35 nucleotide differences were located in coding regions, resulting in 12 amino acid differences in the predicted proteomes.

ACKNOWLEDGMENTS
This project was supported by funds provided by the Faculty of Medicine, Masaryk University, to junior researchers M.N. and P.P.
The Bioinformatics Core Facility of the Central European Institute of Technology, Masaryk University, is gratefully acknowledged for obtaining the scientific data presented in this paper. We thank the volunteers who provided ticks from their pets.