2.2. Sequencing

We integrated a set of 3 amplification and sequencing protocols into the algorithm to (1) putatively identify Borrelia-positive samples that test negative for B. burgdorferi s.s., B. mayonii, and B. miyamotoi, (2) putatively identify B. burgdorferi s.l. fliD-positive samples that test negative for B. burgdorferi s.s. and B. mayonii, (3) verify B. mayonii-positive samples, and (4) confirm the presence of B. burgdorferi s.s. in samples with suspect real-time PCR results (Fig. 1). All 3 protocols employed (semi-)nested PCR to facilitate specific amplification of potentially scarce Borrelia DNA from tick material. Primer sequences and per-reaction concentrations appear in Table 1.

To identify borreliae in ticks infected with a single B. burgdorferi s.l. species, we amplified and sequenced segments of the housekeeping genes encoding Clp protease subunit A (clpA) or Dipeptidyl aminopeptidase (pepX) as described at pubMLST (http://pubmlst.org/borrelia/ ; Margos et al., 2015) and elsewhere (Margos et al., 2008; Wang et al., 2014) with minor modifications. Briefly, each 25-μl outer reaction included 1X HotStar Taq Master Mix (Qiagen, Valencia, CA, USA), primers clpAF1237 and clpAR2218 or pepXF449 and pepXR1172, 0.5 μl 25mM MgCl₂ to bring the final concentration to 2mM MgCl₂, and 5–10 μl template. Touchdown cycling conditions for outer reactions were as described at PubMLST, but with annealing temperatures of 60 °C-52 °C for the first set of pepX amplification cycles and 52 °C for the second set of pepX amplification cycles as suggested by Wang et al. (2014). Each 50-μl inner reaction included 1X HotStar Taq Master Mix (Qiagen), inner primers clpAF1255 and clpAR2104 or pepX449 and pepXR1115, 1 μl 25mM MgCl₂ to bring the final concentration to 2mM MgCl₂, and 5–10 μl of the outer reaction product. Cycling conditions were as described at PubMLST, but with a 15 min initial activation at 95 °C and an annealing temperature of 52 °C for pepX.

Because the clpA and pepX primers generate amplicons from all B. burgdorferi s.l. species, they do not allow for verification of B. mayonii in samples that are co-infected with another B. burgdorferi s.l. species, e.g., B. burgdorferi s.s. We therefore developed an amplification and sequencing protocol targeting a B. mayonii-specific segment of circular plasmid 26 (cp26) between Bmayo_06250 and Bmayo_06255 (Kingry et al., 2016). Each 25-μl outer reaction and each 50-μl inner reaction included 1X HotStar Taq Master Mix (Qiagen), 900 nM each primer (outer reaction: Bm_cp26_OF, Bm_cp26_OR; inner reaction: Bm_cp26_IF, Bm_cp26_IR), 0.5 μl 25mM MgCl₂ to bring the final MgCl₂ concentration to 2mM per reaction, and 5–10 μl DNA (outer reaction) or 5–10 μl outer reaction product (inner reaction). Cycling conditions included a 15 min initial activation at 95 °C followed by 30 (outer reaction) or 40 (inner reaction) cycles of 94 °C for 30 s, 56 °C (outer reaction) or 55 °C (inner reaction) for 30 s and 72 °C for 1 min, and a final 10 min extension at 72 °C.

Before sequencing, we visualized inner products on a 1% agarose gel to verify the presence of an approximately 850-nucleotide (nt) (clpA), 668-nt (pepX) or 337-nt (cp26) amplicon. The remaining product was purified using the QIAquick PCR Purification Kit (Qiagen). We sequenced each product using the inner amplification primers and BigDye Terminator v3.1 Ready Reaction Mix, removed unincorporated dyes with the BigDye Xterminator Kit (ThermoFisher Scientific Inc., Waltman, MA, USA), and analyzed the samples on an ABI 3130XL genetic analyzer. Using Lasergene 12 software (DNASTAR, Madison, WI, USA), we aligned forward and reverse sequences to generate a consensus sequence with at least 2-fold coverage of every nt. We manually trimmed poor or ambiguous sequence and any primer sequence from either end of the consensus sequence. We then used the Basic Local Alignment Search Tool (BLAST) to identify similar sequences in GenBank. We also queried the PubMSLT database using the clpA and pepX consensus sequences to identify similar alleles.