Isolates used and sequencing
For all information on isolates, including origin and source material refer to Table S1. This study utilized DNA of 136 Borreliaisolates coming from three human pathogenic species: B. afzelii(n=33), B. garinii (n=57), and B. bavariensis (n =46). Of these, 52 are novel Borrelia isolated from ticks collected either in Japan (n=43) or Germany (n=9) (see Text S1 & Tables S3, S4, S5). Additionally, 55 European isolates (B. afzelii, n=11; B. garinii , n=25; and B. bavariensis , n=19) were provided by the German National Reference Center for Borrelia at the Bavarian Food and Health Safety Authority. All isolates, expect one tick isolate, were isolated from humans. DNA for additional Japanese human and tick isolates (n = 12) was provided by the National Institute of Infectious Disease in Tokyo, Japan. Finally, previously sequenced Russian B. bavariensis (n=7) (Becker et al., 2020) and DNA from 12 additional Russian B. garinii tick isolates were included in the study (see Text S1).
Borrelia isolates were cultured either in inhouse-made MKP (Preac-Mursic, Wilske, & Schierz, 1986) (all European isolates) or inhouse-made BSK-H (Pollack, Telford, & Spielman, 1993) (all Russian and Japanese isolates) medium according to standard procedures (Pollack et al., 1993; Preac-Mursic et al., 1986) until the cultures reached a density of at least 108 cells per mL at which point whole genomic DNA was extracted. Genomic DNA from all European isolates was extracted using a Maxwell® 16 LED DNA kit (Promega, Madison, WI, USA) and from all Japanese and Russian isolates using the Wizard® Genomic DNA Purification Kit (Promega, WI, USA). DNA quality (260/280) and concentration were measured using a NanoDrop® 1000 photometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit® 3.0 fluorometer (Thermo Fisher Scientific, MA, USA), respectively.
For all samples, libraries were produced according to the Nextera XT sample preparation guide (Illumina, San Diego, CA, USA). Library quality was checked using an Agilent TapeStation 2200 (Agilent, Santa Clara, CA, USA) before being sequenced using an Ilumina MiSeq platform according to standard protocol (Illumina, USA) that produced paired end reads of 250bp.