A total 94 out of 120 ticks were randomly selected for sequencing. The total DNA from individual homogenates was extracted with the ZymoBiomics® DNA Miniprep kit (Zymo Research, CA, USA) following the manufacturer's protocol. As control we used sterile deionized water for sample preparation containing DNA from E. faecalis V583 extracted using the same DNA extraction protocol as that for ticks. The DNA was quantified using a spectrophotometer (NanoDrop 2000, Thermo Scientific, USA) and a fluorometer (PicoGreen, Invitrogen, MA, USA). To assess the quantity of 16S rDNA, serial dilutions of a known amount of tick DNA were used for quantitative PCR (qPCR) standardization curves using the single copy gene of the tick V-ATPase subunit C. We used similar DNA template amounts (between 5 and 20 ng across samples) for both genes in qPCR. The qPCR reaction was prepared using the 2× Luna® Universal qPCR Master mix following the manufacturer's instructions. A single copy gene from A. americanum (V-ATPase subunit C) representing the quantity of tick chromosome, was used to normalize the bacterial 16S copy number. (primers: 894F: 5′-CCC TGA GGC TTT TTG TTG AG-3′ and 1043R: 5′ CCT GGG CAA TGC TTG TGT-3′). For quantification of the 16S rRNA gene, the V4 region amplification with universal eubacterial primers 515F: 5′-GTG YCA GCM GCC GCG GTA A-3′ and 806R: 5′-GGA CTA CNV GGG TWT CTA AT-3′ (modified from Caporaso et al. [27]) (Fig. (Fig.1)1) was used. Delta Ct values were calculated by the difference in qPCR Ct values of the 16S rDNA and the tick V-ATPase subunit C.

Library preparation and sequencing of the V3 and V4 region of the 16S rRNA gene (341F and 806R) (Fig. (Fig.1)1) were performed at the Genome Sequencing Core of the University of Kansas. Libraries were generated using unique dual indexing (UDI) and prepared using the Nextera XT index kit. Sequencing was conducted using the MiSeq Next Generation Sequencer. Raw sequence reads were analyzed using the Mothur software package (version 1.39.5, [28]). Paired-end sequences for 300 nt were joined, and sequence reads with low quality (q < 25), ambiguous base, and ambiguous length (< 100 and > 450 bp) were removed. All sequences other than that of E. faecalis from the positive control sample were also filtered out. High-quality sequences were aligned with SSU rRNA SILVA reference alignment [29] using the Needleman-Wunsch global alignment algorithm [30]. Chimeric sequences were checked using UCHIME [31] and removed. Non-E. faecalis sequences from the positive control sample were also removed. Sequence reads were then clustered into OTUs using the average neighbor algorithm with the 97% sequence similarity criterion. For each OTU, taxonomy was assigned using the naïve Bayesian classifier algorithm [25]. Low abundance and erroneous OTUs (abundance ≤ 0.005% of total abundance) were filtered out as described previously [32]. Furthermore, to lower the bias due to variation in sequence numbers across the samples, the OTU table was normalized by subsampling to equal sequence numbers (15,613) per sample. Rarefaction curves show that full richness of a community has been reached showing a good sequencing depth (Additional file 1: Fig. S1). OTUs with the same taxonomic identification were grouped into same genera for further analysis at the genus level, and taxa with relative abundance < 0.005% were grouped under the “others” category.

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