Draft whole-genome sequences of three Borrelia burgdorferi isolates from Western Canada

Borrelia burgdorferi sensu stricto is the predominant causative agent of Lyme disease in North America (1), the most common vector-borne disease in the region (1–3). In Canada, the highest disease risk is associated with the black-legged tick Ixodes scapularis in central to eastern provinces (4–7). B. burgdorferi is endemic but less prevalent in southern British Columbia, where it is spread primarily by the tick Ixodes pacificus (4). To investigate B. burgdorferi genome composition in Western Canada, we sequenced the whole genomes of isolates 21T 0066, 22T 0045, and 22T 0109.

The strains were cultured from three I. pacificus ticks found on humans. Strains 21T 0066 and 22T 0045 were isolated from ticks collected in Victoria (2021 and 2022, respectively), and strain 22T 0109 was isolated from a tick collected on Galiano Island (2022). Ticks were submitted to the British Columbia Center for Disease Control, surface disinfected using 10% hydrogen peroxide and 70% ethanol, and then dissected. The contents of their midgut were cultured at 34°C in modified Barbour-Stonner-Kelly medium containing kanamycin and rifampicin at concentrations of 10 µg/mL and 50 µg/mL, respectively. Cultures were checked weekly for spirochetes using dark-field microscopy, followed by quantitative PCR confirmation of B. burgdorferi using primers and probe for 23S rDNA, as previously described (8). Culturing was performed for periods up to 35 days, and positive cultures were then stored at −80°C before being shipped overnight on −80°C ice packs. Total genomic DNA was isolated from culture aliquots using the DNeasy UltraClean Microbial kit (Qiagen, Venlo, Netherlands). DNA extracts were quantified using the Qubit 2.0 fluorometer and dsDNA High Sensitivity assay kit (Thermo Fisher, Waltham, MA).

DNA sequencing libraries were prepared using Illumina DNA Prep (Illumina, CA, USA) and assumed to have an average fragment size of 600 bp. Final libraries were quantified using the NEBNext Library Quant Kit for Illumina (New England BioLabs; Ipswich, MA, USA) then normalized to 10 nM prior to pooling, diluting, and denaturing. Sequencing was performed on the Illumina MiniSeq platform using 300-cycle paired-end chemistry.

Sequence assembly was conducted entirely within BV-BRC Comprehensive Genome Analysis (9); default parameters were used at all stages of the workflow except where otherwise noted. Raw sequencing data were uploaded to BV-BRC in fastq format, and then Trim Galore (0.6.5dev) removed adapters and low-quality sequence per the “true” trim setting (10). Contigs were assembled by Unicycler (v0.4.8) (11) and then were filtered by default minimum thresholds of 300-bp length and fivefold coverage, followed by two polishing iterations using PILON (v1.23) (12). Assembly quality was assessed using QUAST (v5.2.0) (13). Genomes were subsequently annotated by the NCBI Prokaryotic Genome Annotation Pipeline (v6.5) (14); completeness assessed by CheckM (v1.2.2) (15) ranged from 99.4% to 99.7%.

Multilocus sequence typing (MLST) was performed using the PubMLST database (16), classifying isolate 21T 0066 as sequence type (ST) 13 and 22T 0045 as ST6. 22T 0109 matched ST6 at all MLST positions except for a single nucleotide variant 250G>A at the nifS locus, making 22T 0109 nearest ST6. The ospC gene encodes the primary surface antigen under immune selection pressure and so demonstrates high genetic diversity (17). Assemblies were queried against ospC reference sequences as in reference (18) using the BLASTn function in BLAST+ (v2.9.0) (19) and a >92.0% nucleotide identity threshold (20). 21T 0066 is ospC type M, while both 22T 0045 and 22T 0109 are ospC type H. Sequencing, assembly, genome quality, and annotation metrics for the three isolates are presented in Table 1.

This project was supported in part by a Natural Sciences and Engineering Research Council of Canada (NSERC) Undergraduate Student Research Award (USRA) and by Public Health Agency of Canada grant number 1920-HQ-00069.