Characterization of the family-level Borreliaceae pan-genome and development of an episomal typing protocol

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Socarras, Mary C. Marino, Joshua P. Earl, Rachel L. Ehrlich, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4491589/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The Borreliaceae family includes many obligate parasitic bacterial species which are etiologically associated with a myriad of zoonotic borrelioses including Lyme disease and vector-borne relapsing fevers. Infections by the Borreliaceae are difficult to detect by both direct and indirect methods, often leading to delayed and missed diagnoses. Efforts to improve diagnoses center around the development of molecular diagnostics (MDx), but due to deep tissue sequestration of the causative spirochaetes and the lack of persistent bacteremias, even MDx assays suffer from a lack of sensitivity. Additionally, the highly extensive genomic heterogeneity among isolates, even within the same species, contributes to the lack of assay sensitivity as single target assays cannot provide universal coverage. This within-species heterogeneity is partly due to differences in replicon repertoires and genomic structures that have likely arisen to support the complex Borreliaceae lifecycle in which these parasites have to survive in multiple hosts each with unique immune responses. Results We constructed a Borreliaceae family-level pangenome and characterized the phylogenetic relationships among the constituent taxa which supports the recent taxonomy of splitting the family into at least two genera. Gene content profiles were created for the majority of the Borreliaceae replicons, providing for the first time their unambiguous molecular typing. Conclusion Our characterization of the Borreliaceae pan-genome supports the splitting of the former Borrelia genus into two genera and provides for the phylogenetic placement of several non-species designated isolates. Mining this family-level pangenome will enable precision diagnostics corresponding to gene content-driven clinical outcomes while also providing targets for interventions. Spirochete distributed genome hypothesis pan-genome supragenome comparative genomics phylogenetics Borrelia Borreliella Borreliaceae taxonomy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Background Our development of the distributed genome hypothesis ( 1 – 4 ) led directly to the concept of the bacterial species-level supragenome/pan-genome ( 5 – 16 ). These types of analyses have been extended to the genus level ( 17 – 21 ), and recently to the family level ( 22 , 23 ). The identification of novel distributed genes and their association with phenotypic characteristics provides for precision diagnostics and targeted characterization of components of the genomic dark matter ( 19 , 24 , 25 ). Despite the progress made in comparative bacterial genomics, there have been relatively few reports examining the agents of spirochetoses including the Borreliaceae ( 26 , 27 ). Spirochetoses are characterized as chronic, invasive, debilitating diseases caused by parasitic bacteria within the taxonomic order spirochaetales. Among these diderm helical microbes, the most genomically complex in terms of replicon numbers and heterogeneity are those in the Borreliaceae family. Due to climate change and other anthropomorphic environmental disruptions, the Borreliaceae have greatly expanded beyond their traditional geographic epidemiological zones and have developed diverse symbiotic interactions with multiple alternative hosts ( 28 ). The true taxonomy of the Borrelia genus has been a subject of interest due to the genomic and pathogenic disparities among its member species. A recent taxonomic revision led to the proposed division of the Borrelia genus into two distinct genera, the Borreliella and Borrelia ( 29 ). In this revised classification, the relapsing fever agents retain the original Borrelia taxonomy due to their priority of discovery and the Lyme borreliosis agents became known as Borreliella spirochetes. This work did not include a novel subset of isolates known as the reptile-associated Borrelia , which to date have not been well characterized. It is vital to note that this reclassification has not been without controversy ( 30 – 32 ). In the years since the original proposal, some authors have argued that data other than differences in average nucleotide identity (ANI) do not support the split ( 30 ). However, these latter authors were themselves criticized for not fully examining the multi-omic data that provided extensive quantitative data in support of the division ( 33 , 34 ), thus the controversy continues. Early genomic studies on Borreliaceae spirochetes revealed that they possess a complex genome of a single, linear chromosome terminating in covalently bound hairpin ends, along with multiple single-copy episomes that exist as circular or hairpinned-linear molecules ( 35 , 36 ). While the chromosome contains conserved genes linked to essential functions and survival, the individual episomal elements vary widely in size, structure (linear and circular), and gene content, and facilitate Borreliella host adaptation and/or virulence. Historically, the smaller replicons were found to not be as conserved and were not always retained in long-time cultures. The smaller episomes that were retained or found biologically important were principally categorized by molecular size and structure with only minimal use of gene content. Despite these limitations, previous comparative genomic studies led to the establishment of a B. burgdorferi sensu lato pangenome ( 37 ). This study utilized 23 Borreliella genomes, most of which were B. burgdorferi , and were of varying assembly quality and plasmid content. This first attempt to create a genus-level pangenome showed that additional isolates would be necessary to understand the degree of variation among Borreliella . However, until recently, there have been very few pan-genomic publications on these clinically important taxa ( 38 , 39 ). Moreover, there has not been a pangenome constructed for the Borrelia genus nor for the entire Borreliaceae family. It is essential to better understand these pathogens through the construction of a family-level pangenome using the latest error-correcting long-read DNA sequencing methodologies to ensure capture and single-contig assembly of all genomic elements. Results Study design All available complete or near-complete high-quality Borreliaceae genomes, as of the commencement of this study (n = 69) with sequencing coverage greater than 30X and an average N50 of 900 Kb or above were downloaded from the NCBI prokaryotic genome database and subjected to additional QC analyses for completeness. These genomes were then combined with 39 newly sequenced Borreliaceae genomes chosen to fill in gaps within the taxonomic coverage of the family. All pan-genome analyses were conducted from this final curated database of 108 strains ( Table 1 ). Pacific Biosciences Sequel I whole-genome sequencing and validation A modified gel-plug DNA isolation and pulse field gel electrophoresis (PFGE) analysis was performed on selected Borreliaceae isolates ( Borreliella burgdorferi strain B31 and Borrelia hermsii strain HS1) to determine the number of replicons and to serve as a control for the extraction and sequencing of the multiple replicons that make up the Borreliaceae genomes ( 40 ). Using the same DNAs, we performed whole genome sequencing on a Pacific Biosciences (PacBio) Sequel I using single molecule error-correcting circular consensus sequencing. Quality assurance measures of the WGS’s included assembling the linear chromosomes into a single contig ≥ 0.9 Mb and identifying each of the other assembled contigs (9 Kb to 200 Kb) as corresponding to one of the PFGE replicons. We first performed comparative analyses of our lab-sequenced genomes of the Borreliella burgdorferi B31 and Borrelia hermsii HS1 strains with their respective NCBI reference genomes to determine if all replicons of both Borreliaceae species representing both genera were present and fully sequenced ( 41 , 42 ). We confirmed that our whole genome sequencing was comparable to previous work by aligning a reference and our sequence of B. hermsii strain HS1 ( Fig. 1 a ) . Using a progressive Mauve alignment of the reference strain and our sequenced B. hermsii strain HS1d, multiple locally colinear blocks (LCB) were noted across the entire length of the genome. The largest LCB showed high homology between our sequence and the reference for the linear chromosome and the large linear plasmid. The linear chromosomes contained 100 single nucleotide polymorphisms (SNPs) and the large linear plasmids had 5 SNPs. The smaller contigs from both genomes were either partially aligned or had no clear matches, likely in part due to progressive Mauve being unable to track duplications and the shared gene content between some plasmids ( 43 ). The estimated total number of SNPs between the two HS1 genomes was ~ 1,000. These differences likely stem from small duplications near the telomeres of the linear plasmids since progressive Mauve is ill-suited to handle them. Additionally, novel plasmids within the newly sequenced B. hermsii strain HS1d may have arisen from recombination events to which Borreliaceae plasmids are prone ( 38 , 44 ), or alternatively due to plasmid loss during the culturing of the original HS1 isolate. A second alignment using D-Genies corroborated these findings (Fig. 1 b). Finally, whole genome alignments of the reference B. hermsii strain HS1 and our sequenced B. hermsii strain HS1d confirmed their similarity ( Fig. S1 ) but also demonstrated that they are not identical, particularly with respect to the episomal elements. Some differences in the episomal elements may be accounted for by genes encoding proteins undergoing antigenic variation or by the variable cassettes which drive genetic conversion. Coverage of covalently bound hairpin ends of Borreliaceae episomes In our alignments, several reference B. hermsii strain HS1 plasmids exhibited homology to the middle of our sequenced HS1d contig pairs (Fig. 1 a). This could be an artifact of how these contigs were parsed and assembled. We identified short flanking inverted repeats at the ends of many HS1d contigs that were not included in an LCB with the HS1 reference (Fig. 1 and Fig. 2 ). These inverted repeats stem from the hairpin present within the ends of all linear Borreliaceae replicons or because of artifacts of long-read sequencing ( 35 , 38 , 45 ). To confirm which regions of the B. hermsii strain HS1 and HS1d plasmids were inverted, we paired the contigs and aligned them using the D-Genies program (Fig. 1 b). This analysis identified regions with inverted repeats present at the very ends of some HS1d contigs that were termini of linear replicons. Further inspection of the B. hermsii strain HS1d contigs using minimap2 and subsequent plotting with pafr confirmed this artifact on both ends of suspected linear plasmids (Fig. 2 ) ( 46 , 47 ). The presence of these inverted repeats results from a specific artifact that arises due to a combination of the actual hairpin topology of linear Borreliaceae replicons and the stem-loop adapters used for PacBio long-read sequencing (Fig. 3 ) ( 35 , 38 , 45 ). The heterogeneous ends of these contigs were closely inspected for the presence of the telomere resolvase (ResT) Box 3 TATA motif found in Borrelia species (Fig. 3 ) ( 48 , 49 ). These motifs were identified solely on linear B. hermsii strain HS1d contigs as well as within our sequenced Borreliaceae cohort. To ensure that the inverted repeat sequence in each contig for all the Borreliaceae assembly would not skew downstream comparative genomic analyses, each contig that contained a ResT Box 3 motif was trimmed down. Average Nucleotide Identity Analyses Support a Digeneric Borreliaceae The ANI among all Borreliaceae genome assemblies were computed using the pyani average nucleotide identity blast method (BLASTN+). These analyses revealed that the species members of the original single Borrelia genus were clearly separated into two groups corresponding to the proposed ( 50 ) Borrelia and Borreliella genera (Fig. 4 ). Thus, our findings concur with the division of the former Borrelia genus into two genera and henceforth we will refer to the Lyme borreliosis-causing spirochetes as Borreliella and the relapsing fever-causing spirochetes as Borrelia . Additionally, we included in the analysis two non-species designated Borrelia spirochetes, B_sp_HM_thM16W and B_sp_FGy1, which are thought to be part of the reptile-associated group ( 51 – 55 ) that were demonstrated to be sui generis , but clustered more closely with the Borreliella genus than the Borrelia . Further inspection of the 65 Borreliella strains included in the analyses, covering nine named species, demonstrated that the vast majority have been placed within the correct taxa with essentially all pairwise strain analyses within a species showing > 96% ANI (Fig. 5 ). All the B. burgdorferi and B. afzelii were correctly assigned. One of the two strains typed as B. bavariensis clustered within the B. garinii . The other typed with one of the two strains typed as B. valaisiana , forming a small sui generis group which may correspond to a hybrid species between B. garinii and B. afzelii as the hierarchical clustering placed them between the proposed parent species. These findings are in line with previous reports that had suggested that B. bavariensis was a subspecies of B. garinii . B. maritima was equidistant between B. afzelii and B. burgdorferi. In addition, we characterized the phylogenetic relationship of several novel isolates included in this study. These included the lagomorph isolate, Borreliella andersonii , which at the time of its discovery was misclassified as a B. burgdorferi strain ( 56 ) but was later reclassified as B. andersonii ( 57 ). In our ANI analyses, the B. andersonii strain MOK_3a clustered closely to B. burgdorferi and B. maritima . Of the remaining three Borreliella species ( B. andersonii , B. bissetii , and B. mayonii ), all were most closely related to B. burgdorferi but each were sufficiently distant to warrant their species designations. Compared to the Borreliella , the Borrelia spirochetes displayed greater genetic heterogeneity. The B. miyamotoi strains clustered strongly together, displaying an average pairwise ANI of 97% (Fig. 6 ) but were distinct from the cluster of species containing B. hermsii , B. parkeri , and B. turicatae . The reptile-associated Borrelia species were both remarkably distant from these other major relapsing fever spirochetes and highly divergent among themselves with only B_sp_FGy1 and B. turcica strains sharing any substantial similarity at 94% (Fig. 7 ). Through ANIb (blast), we determined that these two genomes bracket the Borrelia genera (Fig. 4 ). The isolate B_sp_HM_thM16w was also divergent from the group and was closest to B. recurrentis (Fig. 4 , 6 ). Following our initial ANI analysis, the evolutionary relatedness of these spirochetes was checked further by constructing a family phylogeny (Fig. 7 ). This revealed both a division amongst the Borreliaceae spirochetes and the same clustering for Borreliella and Borrelia spirochetes. Interestingly, we observed that some more recently identified Borreliella and Borrelia isolates cluster within their respective genera. This is most clearly illustrated with B. andersonii which, based on the single-copy core phylogeny, was most closely related to B. burgdorferi and B. bissettii . Other novel genomes, like those seen within the reptile-associated Borrelia spirochetes, stood apart from the majority of the Borrelia genus as had been previously seen in the ANI. Only the novel B. sp strain thHMw intercalated within B. miyamotoi and B. hermsii strains. The rest of the reptile-associated spirochetes formed a separate clade within Borrelia spirochetes. Borreliaceae family-level pangenome Following our initial ANI analyses, we wanted to understand how similar the genera were based on their overall gene similarities. The percentage of shared genes within the family was approximately 50% (Fig. 9 ). To begin to understand which genes are shared between the different genera, the Borreliaceae family-level pangenome was multiply calculated at 5% intervals of increasing similarity (Fig. 9 ). At 75% BlastP, clear separation between the core and cloud components of the Borreliaceae pangenome is seen (Fig. 9 ). At the 75% BlastP threshold, there are 322 core genes, 8 soft-core genes, 1,352 genes in the shell, and 3,424 genes in the cloud. There are 1,253 unique genes for all 109 isolates included in this study. Interestingly, the Borreliaceae pangenome had a different composition when analyzed with the EggNOG algorithm. Through this second approach, EggNOG estimates that Borreliaceae contains 997 unique genes for 109 isolates. Furthermore, its pangenome distribution consists of 577 core, 30 soft-core, 204 shell, and 213 cloud genes. Although each of these pangenome analysis tools were given the same dataset, they yielded different results. These discrepancies are due to underlying differences in the alignment methods used by the algorithms. EggNOG relies upon alignment to a database of proteins which have been pre-clustered, rather than relying on a specific blast threshold. Additionally, EggNOG largely ignores genes of unknown function rather than labeling it as “unknown,” which artificially decreases the number of unique genes. Previously, a Borreliella genospecies complex pangenome was constructed through the use of 22 genomes comprised of B. burgdorferi, B. afzelii, and B. garinii ( 37 ). In this work, they found that the Borreliella pangenome was open. To ascertain if this was true within our own curated collection of Borreliaceae genomes, we next tested for changes in the pangenome using both data acquired from Roary at 75% BlastP and EggNOG (Fig. 10 ). It is evident that the number of gene clusters present within each pangenome compartment is subject to change at the incorporation of a novel pangenome. This suggests that the family-level pangenome is open with a growing distributed component reminiscent of what was found within the early Borreliella comparative genomic study. Next, we visualized the Borreliaceae pangenome using a heat map for all included isolates (Fig. 11 ). The family-level core genome consists of 488 genes possessed by all isolates in both genera. Interestingly, the family-level distributed pangenome is fragmented into multiple components. This division reveals two additional groups of genes that on further inspection were identified as the core genes of either the Borreliella or Borrelia genera. The Borreliaceae episomes can be grouped through gene content Borreliaceae spirochetes thrive throughout their complex lifecycles during which they undergo multiple transmissions between phylogenetically distinct hosts including arthropod vectors and multiple classes of vertebrates. Their ability to survive in various hosts is dependent upon a large set of distributed genes that are mostly encoded on episomal replicons. These episomes have traditionally been typed primarily based on molecular size, structure (linear or circular), and alleles of proteins in the PFam32 family ( 58 ). To ensure proper episomal identification, this system needs to be expanded to include information on the entire gene repertoire of all available episomes. To this end, we ran a pyani ANIb on approximately 1,800 Borreliaceae replicons from our curated genome collection. As pyani ANIb may not factor all gene content within the replicons, we also employed a Hadamard matrix (which interprets identity and coverage simultaneously) for this purpose. In the Hadamard matrix heatmap, multiple subgroups for nearly all of the Borreliaceae family replicons were identified (Fig. 12 ). The most similar episomes clustered closely together. The three most conserved groups are the Borreliella chromosome, cp26, and lp54. Other Borreliella episomal groups with relatively higher percentages of similarity were lp25 and lp36. Interestingly, there was very broad and diffuse clustering for the clinically significant family of cp32 plasmids which are known to contain genes encoding host-specific proteins, indicating both their relatedness and the very high degrees of heterogeneity necessary to ensure survival during a lifecycle that includes multiple hosts. Unlike cp32, the lp28 episomes do not likely represent a single family of replicons as they did not all cluster together and instead separated out into smaller clusters. For the Borrelia genus, a high degree of similarity was seen amongst the chromosomes as well as for the large linear plasmids. Additionally, species-specific groupings amongst the episomes were also observed. This is best illustrated in B. miyamotoi strains lp6 and lp12. It is important to note that while the reptile-associated Borrelia spirochetes were included in these analyses, they were the only isolates to have extraordinarily dissimilar replicons. This is likely attributable to the incompleteness of the B_sp_A_FGy1 genome as indicated by its large number of constituent contigs. Discussion The debilitating diseases caused by Borreliaceae spirochetes have remained on the periphery of societal memory for centuries. Despite our current knowledge of the etiological agents of these spirochetoses, there is still a paucity of information on how each disease is caused and why their presentations are so variable among individuals. In the work presented in this study, multiple pressing points within the field of Borreliaceae comparative genomics were addressed to help provide a framework going forward to be able to associate microbial genotypes with disease phenotypes in the host. The first topic we addressed was the composition of the Borreliaceae family. It was recently proposed that the previous single genus, Borrelia , be divided into two genera based on differences in overall genomic content ( 29 , 34 ). The well-studied and more widely known Lyme borreliosis spirochetes were given the genus name of Borreliella , while the older and less clinically studied species complex comprising the relapsing fever spirochetes retained the Borrelia designation. Since the establishment of this proposed dichotomy, there has been animated discussion among scientists both for and against the split ( 30 – 34 ). Pairwise ANI analyses of all 108 sequenced isolates (N = 11556 genome comparisons) in this study revealed a sharp division between the two proposed genera indicating that the recent taxonomic re-classification is correct. Even isolates from within each of the genera often did not share sequence similarities above 85%. While it may be true that the ANI cut-off can vary based on species as seen with Strenotrophomonas maltophila, Escherichia spp, or Lactobacillus spp., our results do not support including the Lyme borreliosis spirochetes and relapsing fever spirochetes within a single genus ( 59 , 60 ). Additional analyses revealed that the recently sequenced novel spirochaetal species B_sp_FGy1 and B_sp_HM_thM16w clustered most closely with B. anserina and B. recurrensis , respectively. These two genomes clustered within the known reptile- associated Borreliaceae . As these isolates are novel, it is unknown if they are pathogenic to humans. We constructed a family-level pan-genome to characterize the similarities and differences between the two genera concerning gene content. In doing so, we found that the family-level Borreliaceae core genome was very small, consisting of only 488 genes, but that each of the genus-level core genomes were each more than double that size and that the family-level pangenome is still open. This indicates that there are large numbers of distributed genes still to be discovered. The number of core genes possessed by both genera were similar. It was previously known that there is some similarity between Borreliella and Borrelia replicons with the Borrelia replicons containing regions of similarity to the Borreliella cp26 and lp54 ( 27 , 44 , 61 , 62 ). To better place the various replicons within a gene-functional framework, we established a system for typing the Borreliaceae plasmids. Previously, these episomes have been typed based on size, structure, and the presence of specific alleles of particular protein families found on many of the plasmids ( 26 , 58 ). While this technique helped somewhat in the identification of the many Borreliaceae episomes, it did little to identify them by the biological functions they encode. In our work, all of the available episomes for each of the isolates in our data set were used in an ANI analysis via pyani. Doing so for over 1,800 replicons resulted in a complex matrix that did little to clarify how these episomes related to each other (Fig. 12 ). However, in building a Hadamard matrix from these analyses it was possible to visualize each of the known plasmids with respect to their overall hierarchical clustering, even for the most heterogenous replicon, cp32. Thus, we have established a universal Borreliaceae plasmid typing system based on gene content that captures all of the replicons produced via our PacBio-based long-read sequencing protocols. Finally, we identified an artifact of sequencing linear bacterial replicons with hairpin termini with the PacBio SMRTbell technology. In our sequencing, it was noted that some of the many linear episomes had regions of heterogeneity. After closer inspection, it became apparent that these regions were inverted repeats that could emerge as artifacts in long-read sequencing platforms ( 38 , 63 ). These regions were identified as the telomeric ends of all linear Borreliaceae replicons, a feature not unlike those seen in higher eukaryotes ( 41 , 45 , 64 , 65 ). To prevent false inflation in the genes within our data set, we searched for and confirmed the location of the ResT Box 3 site ( 49 , 65 ). From this, we have begun to build the first system to handle these Borreliaceae long-read sequencing artifacts that could be applied to any replicon with similar features. These data also suggest that raw PacBio circular consensus sequences may contain hidden information about hairpin and cruciform DNA structures in other genomes. This work on the Borreliaceae family-wide pangenome is pivotal and the first of its kind. More sequencing is required to ensure that the pangenome is truly reflective of all Borreliaceae spirochetes. Furthermore, more work is required for the typing of all Borreliaceae episomes. This family-level pangenome analysis provides new tools and information in a field that requires more insight on how to handle such evasive and persistent pathogens. More importantly, this work can later culminate into effective therapeutics. Conclusions Through the application of multiple comparative genomic methods including phylogenetics, ANI, gene content, and core genome analyses, we demonstrate that the Borreliaceae are composed of at least two genera. We also developed a gene-based plasmid typing protocol to replace the current confusing method that relies solely on size and topography. This approach to episomal typing revealed an extremely high degree of allelic heterogeneity in the plasmid(s) that contain the genes that encode the major host-interacting proteins which are reflective of the extremely broad host-range of these parasitic bacteria. Additional analyses with a larger number of genomes will be required to determine if the reptile-associated strains and other underrepresented groups in the current analysis define additional genera. Methods Borreliaceae pangenome project design This study included all available reference Borreliaceae genomes obtained from NCBI RefSeq on October 17th, 2021. The downloaded genomes were checked for completeness, sequencing platform, sequencing coverage, and if the average number of contigs greatly superseded the number of included replicons. For example, selected genomes were disqualified if more than 2 or 3 contigs were needed for a single replicon. Additionally, they were run through a QC pipeline to ensure usability in later studies. The remainder of the genomes were used henceforth. Novel isolates collected by collaborators at Virginia Commonwealth University were then sequenced at the Center for Genomic Sciences core facility at Drexel University College of Medicine. These newly sequenced members of the Borreliaceae are included in BioProject PRJNA861274. Borreliaceae culturing All Borreliella and Borrelia isolates were cultivated in 50 mL of BSK-II media supplemented with 6 or 12% rabbit serum (Sigma, MA), respectively. The culture was then monitored using wet-mounts and dark-field microscopy. Once spirochetes grew to the concentration of 5 x 10^7 cells/mL at late log phase, they were harvested by centrifugation, resuspended in 1X sterile PBS, and then recovered by centrifugation. Pulse-Field Gel plasmid verification DNA isolation of selected Borreliaceae isolates was done through a modified BioNano plug prep (Bionano Genomics, Bionano Prep cell Culture DNA Isolation Protocol) (Bionano Genomics, CA). Cultured Borreliaceae were pelleted at 5,000 xg for 10 mins and sent overnight to the Center for Genomic Sciences at Drexel University College of Medicine. Received pellets were immediately thawed, resuspended with a total volume of 66 µL of cell buffer, and then 40 µL of 2% agarose was added. The contents were mixed 10 times via pipette and then 100 µL of each sample was aliquoted into a plug mold. The filled plug mold was incubated at 4°C for 15 mins before starting protein digestion. This process was done by placing the plug into a solution of 167 µL proteinase K (Qiagen, DE) and 2.5 mL Lysis buffer, then incubated for 2 hours at 50°C in a thermomixer at intermittent mixing. After incubation, the digestion solution was replaced and incubated overnight on the thermomixer as described above. Following the overnight incubation, plugs were allowed to cool at room temp for 5 mins and then we added 50 µL of RNAse A before placing the plugs in the thermomixer at 37°C with intermittent mixing. Plugs were then washed 3 times with 10 mL 1X Wash buffer before being washed once more with 10 mL and set on an orbital shaker for 15 mins at 180 RPM. This latter step was repeated 3 more times. DNA recovery was initiated by adding 10 mL of 1X TE buffer to wash plugs and shaking at 180 RPM for 15 mins a total of 5 times. Cleaned plugs were then inserted into a 1% low melt agarose gel and run on a pulse-field gel at 5V for 21 hrs. Pulse field gel was stained with ethidium bromide and visualized under UV. In silico Borreliaceae replicon verification The Borreliaceae genome assembly pairs were aligned through progressiveMauve (v.2.4.0), NUCmer (MUMer 3.0) and D-Genies (v1.4.0) ( 43 , 66 , 67 ). Additionally, each spirochete genome FASTA file that had been used for the above initial validation work was split by contig, imported into R, and then aligned to itself using pafr (v 0.0.2) ( 46 ). Borreliaceae DNA extraction Once received, frozen Borreliaceae pellets were thawed on ice and centrifuged at 13,000 rpm for 1 min. Following pelleting, the remaining solvent was discarded before the pellets were resuspended in 200 µL of 1X PBS, pipette mixed, and transferred to a 2 mL bead beating tube (Matrix E) (MP Biomedicals, CA). Following the transfer, 20 µL of Proteinase K (Qiagen, DE) was added, and cells were homogenized using a SPEX 1600 MiniG (Fisher Scientific, MA) for 1 min at 1500 Hz. Subsequent DNA extraction was performed using the Qiagen™ DNeasy Blood & Tissue Kit according to the manufacturer’s instructions (Qiagen, DE). DNA preparation and sequencing Extracted Borreliaceae DNA was quantified following ThermoFisher Scientific™ 1X dsDNA HS kit per the manufacturer’s instructions (Biotium Inc, CA) on Qubit. DNA from each Borrelial/Borrelia specimen was prepped with the SMRTbell Template Prep Kit 2.0 (Pacific Biosciences, CA) to make PacBio SMRTbell libraries with barcodes sourced from the Barcoded Overhang Adaptor Kit 8A and 8B (Pacific Biosciences, CA). The sequencing primers were then annealed and bound to Polymerase 3.0 using the Sequel Binding Kit 3.0 (Pacific Biosciences, CA). The final bound complex was then purified and later sequenced on PacBio Sequel I using the SMRT Cell M1 v3 tray (Pacific Biosciences, CA). The spike-in controls for each PacBio Sequel I run were from the Internal Control Kit 3.0 (Pacific Biosciences, CA). Genome assembly, annotation, and pangenome construction Borreliaceae isolates that were sequenced on the PacBio Sequel I platform were processed using pbcromwell (v 1.0.4) (Pacific Biosciences, CA). All the barcoded data were demultiplexed using pb_demux_subreads and assembled using pb_assembly_microbial. The base modification motifs for each assembly were computed using pb_basemods. The species of each genome was verified using taxator and GTDB-Tk (v1.7.0) with refpack (v r202). All reference and sequenced Borreliaceae assemblies were annotated with Prokka (v 1.11) and then homologous genes were clustered with Roary (v 3.5.1) ( 68 , 69 ). The BlastP threshold was calculated consecutively at 5% intervals to determine the threshold. The final BlastP threshold, 75%, was selected based on the separation of the core pangenome from the cloud and accessory pangenomes. All CDS identified by Prokka were also annotated using EggNOG ( 70 ). Phylogenetic tree construction Single-copy core genes of each Borreliaceae genome were aligned using MAFFTT (v7.490-1) and then the concatenated alignment was run through Randomized Accelerated Maximum Likelihood (RaxML) to generate a Newick string ( 71 , 72 ). The resulting file was then ported into RStudio (v2022.07.2 + 576), a tree was generated using ape and then visualized using ggtree (v3.15) R packages ( 73 , 74 ). Average Nucleotide Identity and percent similarity or difference analysis All pairs of Borreliaceae genomes, both sequenced and downloaded were checked for overall similarity by ANI using the pyani (v2.12) with the ANIb method ( 75 ). The resulting whole-genome ANI matrix (or Hadamard matrix) was read into RStudio (v.4.1.1), clustered with adapted RaxML phylogeny, and visualized with pheatmap (v1.0.12). The Borreliaceae percent gene similarity was calculated using RStudio in a pairwise fashion for each isolate in this study. The final list of shared percent identity was then pivoted wider to generate a matrix. Any pairs that did not have a gene percent similarity value were assigned a value of 0. The final matrix was then clustered with hclust and visualized with pheatmap (v1.0.12). Borreliaceae pangenome mapping To find pangenome distribution, all Roary at BlastP 75% or EggNOG gene clusters were imported into R ( 70 ). Then each gene was associated with a corresponding strain and/or replicon before being used to construct a presence-absence matrix. This matrix was then visualized in pheatmap to see the gene presence and absence across all species used in this study. To determine if the Borreliaceae pangenome was either open or closed, the composition was checked by shuffling strains, incorporating them one at a time into the analysis, and then calculating the core/distributed component. The combinations of strains incorporated were shifted prior to being plotted in each iteration. Declarations Funding This work was supported by grants from the Bill and Marian Cook Foundation; the Oskar Fischer Project, a James Truchard Philanthropy; Cure; the Coulter-Drexel Translational Research Partnership; and NIH grant Nos: DC 02148 and DK082316 to GDE. This work was also supported by NIH grants 1R01AI165876-01 and 1R01AI141801-01A1 to RTM. Availability of data and materials The sequencing data used in this study is available under Bioproject PRJNA1026537. Competing interests (NONE) Author details (NA) Author Contribution The main manuscript text was written by KMS, GDE, and MCM. Study strategy was conceived by GDE. Study design was conceived by KMS, JPE, JCM, RLE, RTM, and GDE. Experimental work was performed by KMS, SB, AA, NAC, RLE, JPE, and JCM. Data analysis was performed by KMS, JPE, RLE, and JCM. Figure construction was performed by KMS, JPE, JCM, and MCM. Funding was provided through grants awarded to GDE and RTM. References Shen K, Wang X, Post JC, Ehrlich GD. 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Paradis E, Claude J, Strimmer K. APE: Analyses of Phylogenetics and Evolution in R language. Bioinformatics. 2004;20(2):289–90. Yu G. Using ggtree to Visualize Data on Tree-Like Structures. Curr Protoc Bioinf. 2020;69(1):e96. Pritchard LGR, Humphris S, Elphinstone JG, Toth IK. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. Anal Methods. 2016(1). Additional Declarations No competing interests reported. Supplementary Files FigureS1.png TableS1.docx TableS2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4491589","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311820092,"identity":"8888f700-f285-4d11-8e4e-e917d9d8aa52","order_by":0,"name":"Kayla M. 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Alignments of B. hermsii strain HS1 show nearly identical genomes. Similar pairwise locally colinear blocks are indicated in the same colors (pink to pink, blue to blue etc.). Contig breaks are indicated by red lines. b D-Genies dot plot of whole-genome complete reference B. hermsii strain HS1 (query, x-axis) to our sequenced B. hermsii strain HS1d (target, y-axis). Overall percent similarity was indicated from red (low = 0) to green (high = 100%). All contigs of the two whole genome sequences are portrayed in the figure. Each contig pair is separated by a dotted line. The position of the matches between strains is indicated by colored dots and lines (yellow, orange, green).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/33d6a3e70994db626780a225.png"},{"id":58145023,"identity":"48e2b8be-a1f1-40eb-8bde-be8644b4ab0d","added_by":"auto","created_at":"2024-06-11 18:28:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":499600,"visible":true,"origin":"","legend":"\u003cp\u003eSelf-alignment of B. hermsii strain HS1d contigs reveals inverted repeats at linear contig ends. A faceted pafr dot plot of each contig within the B. hermsii strain HS1d whole-genome sequence. The overall percent similarity was indicated from light blue (low = 0) to dark blue (high = 100%). All contigs were represented within each individual facet of the plot. Within each plot X and Y-axes are the overall lengths of the compared contigs.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/e29e356611a4c437cbd1c339.png"},{"id":58142984,"identity":"99b7368b-8650-493b-b6c5-7eacb19ee693","added_by":"auto","created_at":"2024-06-11 18:12:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":350038,"visible":true,"origin":"","legend":"\u003cp\u003eBlast search of B. hermsii strain HS1 linear contigs reveals ResT site located on ends of sequenced replicons. A graphic depiction of Pacific Biosciences long-read sequencing platform and modified prepared Borreliaceae template. Alongside sequencing schematic is one of the faceted plots of B. hermsii strain HS1d (contig 3). This plot shows the percent similarity of replicon along its entire length with regions of similarity removed. Perpendicular lines are inverted repeats. The overall percent similarity was indicated from light blue (low = 0) to dark blue (high = 100%). All contigs were represented within each individual facet of the plot.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/f7eca45f10e9d398f5f591db.png"},{"id":58146351,"identity":"141d2fbc-5199-4797-9d62-d64763e91fd6","added_by":"auto","created_at":"2024-06-11 18:36:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1094134,"visible":true,"origin":"","legend":"\u003cp\u003eAverage Nucleotide Identity of all Borreliaceae species and strains examined demonstrated genera-specific clustering. The range of ANI is from least (yellow) to greatest (blue). Each species is represented on the x- and y-axes. The diagonal line represents identity. The dendrograms were made with complete-linkage hierarchical clustering. The key color is noted on the right-hand side. The annotations indicate the generic groupings.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/c0edd2036eba3625c11036e8.png"},{"id":58145021,"identity":"af158af6-a88d-4f94-aea7-ef2c417fa9b5","added_by":"auto","created_at":"2024-06-11 18:28:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":557461,"visible":true,"origin":"","legend":"\u003cp\u003eAverage Nucleotide Identity analyses of Borreliella genomes shows a high degree of similarity amongst the major species of the genus. The range of ANI is from least (yellow) to greatest (blue). Each of the Borreliella spirochetes are represented on the x- and y-axes. The diagonal line represents identities. The dendrograms were made with complete-linkage hierarchical clustering. The color bar key is noted on the right-hand side. The annotations indicate the species group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/a7766909c84cb8ae0629451d.png"},{"id":58143273,"identity":"2c58b7af-28c1-4cca-b0bd-bc4d9d1bfdda","added_by":"auto","created_at":"2024-06-11 18:20:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":287358,"visible":true,"origin":"","legend":"\u003cp\u003eAverage Nucleotide Identity of Borrelia genome spirochete shows separation of major species. The range of ANI is from least (yellow) to greatest (blue). Each Borrelia spirochete was represented on both axes. The diagonal line is where sample similarity values are to itself and beyond said line is for other genus members. Complete-linkage hierarchical clustering. The color bar key is noted on the right-hand side. The annotations indicate the species group.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/44f3fdaea89a75616178cdee.png"},{"id":58142988,"identity":"4666c7f5-c1c3-485a-9f38-fecd8e29261c","added_by":"auto","created_at":"2024-06-11 18:12:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":172860,"visible":true,"origin":"","legend":"\u003cp\u003eAverage Nucleotide Identity of reptile-associated Borrelia spirochetes suggests placement of non-species designated isolates. The range of ANI is from least (yellow) to greatest (blue). Each isolate is represented on the x- and y-axis. The diagonal line is where sample similarity values are to itself and beyond said line is for other genus members. Complete-linkage hierarchical clustering. The annotation color bars for each spirochete are noted on the right-hand side.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/35b29a8c54efc81e14fc69f9.png"},{"id":58142991,"identity":"0ea66847-643e-4fcb-9201-f619edf48ede","added_by":"auto","created_at":"2024-06-11 18:12:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1637842,"visible":true,"origin":"","legend":"\u003cp\u003eMidpoint rooted single-copy core gene tree of Borreliaceae shows the division between the Borreliella and Borrelia genera. The origin of the spirochete is annotated in blue (VCU) or red (NCBI) spheres at the nodes. The annotation bar indicates the species.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/2b46e258dc768beac494b357.png"},{"id":58143274,"identity":"d0dedf54-9ee6-4559-837b-21a8b4585be0","added_by":"auto","created_at":"2024-06-11 18:20:13","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":473393,"visible":true,"origin":"","legend":"\u003cp\u003eThe gene cluster frequency of the Borreliaceae family pangenome reveals a large pangenome. Each colored circle signifies different components of the pangenome [core (blue), soft-core (orange), shell (green), and cloud (red)]. X-axis signifies the BlastP threshold. Y-axis signifies the number of genes within the pangenome.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/bc1b96147de596beffd4daaf.png"},{"id":58142995,"identity":"96591db1-27d2-440b-a357-a92efcd6d739","added_by":"auto","created_at":"2024-06-11 18:12:13","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":500101,"visible":true,"origin":"","legend":"\u003cp\u003eRoary and EggNOG depict an expanding Borreliaceae pangenome over the number of included isolate genomes. \u0026nbsp;X- axis represents the number of isolates and Y-axis represents the number of gene clusters within pangenome. Each panel-faceted plot is dedicated to different components of the pangenome. \u003cstrong\u003ea\u003c/strong\u003eRoary BlastP 75% was chosen as the threshold. Each light blue dot indicates an incorporated isolate. \u003cstrong\u003eb\u003c/strong\u003e EggNOG depiction of pangenome fluctuation with each incorporated genome.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/c3f71010aa9e5ffba6ea065d.png"},{"id":58142999,"identity":"476661d1-a488-4d0d-a93d-b7e1c59f3256","added_by":"auto","created_at":"2024-06-11 18:12:13","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":591098,"visible":true,"origin":"","legend":"\u003cp\u003eBorreliaceae gene presence/absence heatmap. Genes are colored blue if present and white if absent. Homologs were defined as having Roary BlastP of ³75%. Y-axis represents gene clusters within the Borreliaceae pangenome. X-axis depicts all Borreliaceae isolates. The red annotations indicate the gene groups. The Borreliaceae pangenome has a relatively small core with each of the two genera-defined core genomes being more than twice as large. Only the most prevalent of the distributed genes (not present in either of the three core genomes) are included in the heatmap.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/12f323ee72b77780e6b967df.png"},{"id":58142994,"identity":"3dd644f2-c0e7-4907-972f-4dfd97046c81","added_by":"auto","created_at":"2024-06-11 18:12:13","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":808544,"visible":true,"origin":"","legend":"\u003cp\u003eLarge-scale average nucleotide identity of Borreliaceae replicons reveals definitive groupings across the family. The range of ANI is from least (yellow) to greatest (blue). Each of the Borreliaceae replicons is represented on the x- and y-axes based on complete-linkage hierarchical clustering. The annotations indicate the replicon group.\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/33305126ec8614a34c895a8b.png"},{"id":58643428,"identity":"0279960c-ae49-40b6-8f40-77ebf065b522","added_by":"auto","created_at":"2024-06-19 08:24:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6105820,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/feac5aaa-eb0f-457e-b262-93f46cbafde1.pdf"},{"id":58142998,"identity":"a6c09eae-43be-4ff3-ab7b-449d05e3a119","added_by":"auto","created_at":"2024-06-11 18:12:13","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":374055,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.png","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/916dd05d536b8a0ee3b38918.png"},{"id":58143001,"identity":"e1a247e9-8d1b-4da7-84cf-f4d0e09c14fb","added_by":"auto","created_at":"2024-06-11 18:12:14","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":52730,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/1071a0faff2800d3a38ad6d1.docx"},{"id":58142997,"identity":"aea70433-5111-41bf-9bdc-25d891f80003","added_by":"auto","created_at":"2024-06-11 18:12:13","extension":"docx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":45029,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4491589/v1/90a53a53a6761f41350b0860.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of the family-level Borreliaceae pan-genome and development of an episomal typing protocol","fulltext":[{"header":"Background","content":"\u003cp\u003eOur development of the distributed genome hypothesis (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) led directly to the concept of the bacterial species-level supragenome/pan-genome (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). These types of analyses have been extended to the genus level (\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), and recently to the family level (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The identification of novel distributed genes and their association with phenotypic characteristics provides for precision diagnostics and targeted characterization of components of the genomic dark matter (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Despite the progress made in comparative bacterial genomics, there have been relatively few reports examining the agents of spirochetoses including the \u003cem\u003eBorreliaceae\u003c/em\u003e (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpirochetoses are characterized as chronic, invasive, debilitating diseases caused by parasitic bacteria within the taxonomic order spirochaetales. Among these diderm helical microbes, the most genomically complex in terms of replicon numbers and heterogeneity are those in the \u003cem\u003eBorreliaceae\u003c/em\u003e family. Due to climate change and other anthropomorphic environmental disruptions, the \u003cem\u003eBorreliaceae\u003c/em\u003e have greatly expanded beyond their traditional geographic epidemiological zones and have developed diverse symbiotic interactions with multiple alternative hosts (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The true taxonomy of the \u003cem\u003eBorrelia\u003c/em\u003e genus has been a subject of interest due to the genomic and pathogenic disparities among its member species. A recent taxonomic revision led to the proposed division of the \u003cem\u003eBorrelia\u003c/em\u003e genus into two distinct genera, the \u003cem\u003eBorreliella\u003c/em\u003e and \u003cem\u003eBorrelia\u003c/em\u003e (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In this revised classification, the relapsing fever agents retain the original \u003cem\u003eBorrelia\u003c/em\u003e taxonomy due to their priority of discovery and the Lyme borreliosis agents became known as \u003cem\u003eBorreliella\u003c/em\u003e spirochetes. This work did not include a novel subset of isolates known as the reptile-associated \u003cem\u003eBorrelia\u003c/em\u003e, which to date have not been well characterized.\u003c/p\u003e \u003cp\u003eIt is vital to note that this reclassification has not been without controversy (\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In the years since the original proposal, some authors have argued that data other than differences in average nucleotide identity (ANI) do not support the split (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). However, these latter authors were themselves criticized for not fully examining the multi-omic data that provided extensive quantitative data in support of the division (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), thus the controversy continues.\u003c/p\u003e \u003cp\u003eEarly genomic studies on \u003cem\u003eBorreliaceae\u003c/em\u003e spirochetes revealed that they possess a complex genome of a single, linear chromosome terminating in covalently bound hairpin ends, along with multiple single-copy episomes that exist as circular or hairpinned-linear molecules (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). While the chromosome contains conserved genes linked to essential functions and survival, the individual episomal elements vary widely in size, structure (linear and circular), and gene content, and facilitate \u003cem\u003eBorreliella\u003c/em\u003e host adaptation and/or virulence. Historically, the smaller replicons were found to not be as conserved and were not always retained in long-time cultures. The smaller episomes that were retained or found biologically important were principally categorized by molecular size and structure with only minimal use of gene content.\u003c/p\u003e \u003cp\u003eDespite these limitations, previous comparative genomic studies led to the establishment of a \u003cem\u003eB. burgdorferi sensu lato\u003c/em\u003e pangenome (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). This study utilized 23 \u003cem\u003eBorreliella\u003c/em\u003e genomes, most of which were \u003cem\u003eB. burgdorferi\u003c/em\u003e, and were of varying assembly quality and plasmid content. This first attempt to create a genus-level pangenome showed that additional isolates would be necessary to understand the degree of variation among \u003cem\u003eBorreliella\u003c/em\u003e. However, until recently, there have been very few pan-genomic publications on these clinically important taxa (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Moreover, there has not been a pangenome constructed for the \u003cem\u003eBorrelia\u003c/em\u003e genus nor for the entire \u003cem\u003eBorreliaceae\u003c/em\u003e family. It is essential to better understand these pathogens through the construction of a family-level pangenome using the latest error-correcting long-read DNA sequencing methodologies to ensure capture and single-contig assembly of all genomic elements.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eAll available complete or near-complete high-quality \u003cem\u003eBorreliaceae\u003c/em\u003e genomes, as of the commencement of this study (n\u0026thinsp;=\u0026thinsp;69) with sequencing coverage greater than 30X and an average N50 of 900 Kb or above were downloaded from the NCBI prokaryotic genome database and subjected to additional QC analyses for completeness. These genomes were then combined with 39 newly sequenced \u003cem\u003eBorreliaceae\u003c/em\u003e genomes chosen to fill in gaps within the taxonomic coverage of the family. All pan-genome analyses were conducted from this final curated database of 108 strains (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePacific Biosciences Sequel I whole-genome sequencing and validation\u003c/h2\u003e \u003cp\u003eA modified gel-plug DNA isolation and pulse field gel electrophoresis (PFGE) analysis was performed on selected \u003cem\u003eBorreliaceae\u003c/em\u003e isolates (\u003cem\u003eBorreliella burgdorferi\u003c/em\u003e strain B31 and \u003cem\u003eBorrelia hermsii\u003c/em\u003e strain HS1) to determine the number of replicons and to serve as a control for the extraction and sequencing of the multiple replicons that make up the \u003cem\u003eBorreliaceae\u003c/em\u003e genomes (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Using the same DNAs, we performed whole genome sequencing on a Pacific Biosciences (PacBio) Sequel I using single molecule error-correcting circular consensus sequencing. Quality assurance measures of the WGS\u0026rsquo;s included assembling the linear chromosomes into a single contig \u0026ge; 0.9 Mb and identifying each of the other assembled contigs (9 Kb to 200 Kb) as corresponding to one of the PFGE replicons.\u003c/p\u003e \u003cp\u003eWe first performed comparative analyses of our lab-sequenced genomes of the \u003cem\u003eBorreliella burgdorferi\u003c/em\u003e B31 and \u003cem\u003eBorrelia hermsii\u003c/em\u003e HS1 strains with their respective NCBI reference genomes to determine if all replicons of both \u003cem\u003eBorreliaceae\u003c/em\u003e species representing both genera were present and fully sequenced (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). We confirmed that our whole genome sequencing was comparable to previous work by aligning a reference and our sequence of \u003cem\u003eB. hermsii\u003c/em\u003e strain HS1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Using a progressive Mauve alignment of the reference strain and our sequenced \u003cem\u003eB. hermsii\u003c/em\u003e strain HS1d, multiple locally colinear blocks (LCB) were noted across the entire length of the genome. The largest LCB showed high homology between our sequence and the reference for the linear chromosome and the large linear plasmid. The linear chromosomes contained 100 single nucleotide polymorphisms (SNPs) and the large linear plasmids had 5 SNPs. The smaller contigs from both genomes were either partially aligned or had no clear matches, likely in part due to progressive Mauve being unable to track duplications and the shared gene content between some plasmids (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The estimated total number of SNPs between the two HS1 genomes was ~\u0026thinsp;1,000. These differences likely stem from small duplications near the telomeres of the linear plasmids since progressive Mauve is ill-suited to handle them. Additionally, novel plasmids within the newly sequenced \u003cem\u003eB. hermsii\u003c/em\u003e strain HS1d may have arisen from recombination events to which \u003cem\u003eBorreliaceae\u003c/em\u003e plasmids are prone (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), or alternatively due to plasmid loss during the culturing of the original HS1 isolate. A second alignment using D-Genies corroborated these findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eFinally, whole genome alignments of the reference \u003cem\u003eB. hermsii\u003c/em\u003e strain HS1 and our sequenced \u003cem\u003eB. hermsii\u003c/em\u003e strain HS1d confirmed their similarity (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) but also demonstrated that they are not identical, particularly with respect to the episomal elements. Some differences in the episomal elements may be accounted for by genes encoding proteins undergoing antigenic variation or by the variable cassettes which drive genetic conversion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCoverage of covalently bound hairpin ends of\u003c/b\u003e \u003cb\u003eBorreliaceae\u003c/b\u003e \u003cb\u003eepisomes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn our alignments, several reference \u003cem\u003eB. hermsii\u003c/em\u003e strain HS1 plasmids exhibited homology to the middle of our sequenced HS1d contig pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This could be an artifact of how these contigs were parsed and assembled. We identified short flanking inverted repeats at the ends of many HS1d contigs that were not included in an LCB with the HS1 reference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These inverted repeats stem from the hairpin present within the ends of all linear \u003cem\u003eBorreliaceae\u003c/em\u003e replicons or because of artifacts of long-read sequencing (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo confirm which regions of the \u003cem\u003eB. hermsii\u003c/em\u003e strain HS1 and HS1d plasmids were inverted, we paired the contigs and aligned them using the D-Genies program (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). This analysis identified regions with inverted repeats present at the very ends of some HS1d contigs that were termini of linear replicons. Further inspection of the \u003cem\u003eB. hermsii\u003c/em\u003e strain HS1d contigs using minimap2 and subsequent plotting with pafr confirmed this artifact on both ends of suspected linear plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe presence of these inverted repeats results from a specific artifact that arises due to a combination of the actual hairpin topology of linear \u003cem\u003eBorreliaceae\u003c/em\u003e replicons and the stem-loop adapters used for PacBio long-read sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The heterogeneous ends of these contigs were closely inspected for the presence of the telomere resolvase (ResT) Box 3 TATA motif found in \u003cem\u003eBorrelia\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). These motifs were identified solely on linear \u003cem\u003eB. hermsii\u003c/em\u003e strain HS1d contigs as well as within our sequenced \u003cem\u003eBorreliaceae\u003c/em\u003e cohort. To ensure that the inverted repeat sequence in each contig for all the \u003cem\u003eBorreliaceae\u003c/em\u003e assembly would not skew downstream comparative genomic analyses, each contig that contained a ResT Box 3 motif was trimmed down.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAverage Nucleotide Identity Analyses Support a Digeneric\u003c/b\u003e \u003cb\u003eBorreliaceae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe ANI among all \u003cem\u003eBorreliaceae\u003c/em\u003e genome assemblies were computed using the pyani average nucleotide identity blast method (BLASTN+). These analyses revealed that the species members of the original single \u003cem\u003eBorrelia\u003c/em\u003e genus were clearly separated into two groups corresponding to the proposed (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBorreliella\u003c/em\u003e genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Thus, our findings concur with the division of the former \u003cem\u003eBorrelia\u003c/em\u003e genus into two genera and henceforth we will refer to the Lyme borreliosis-causing spirochetes as \u003cem\u003eBorreliella\u003c/em\u003e and the relapsing fever-causing spirochetes as \u003cem\u003eBorrelia\u003c/em\u003e. Additionally, we included in the analysis two non-species designated \u003cem\u003eBorrelia\u003c/em\u003e spirochetes, B_sp_HM_thM16W and B_sp_FGy1, which are thought to be part of the reptile-associated group (\u003cspan additionalcitationids=\"CR52 CR53 CR54\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) that were demonstrated to be \u003cem\u003esui generis\u003c/em\u003e, but clustered more closely with the \u003cem\u003eBorreliella\u003c/em\u003e genus than the \u003cem\u003eBorrelia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFurther inspection of the 65 \u003cem\u003eBorreliella\u003c/em\u003e strains included in the analyses, covering nine named species, demonstrated that the vast majority have been placed within the correct taxa with essentially all pairwise strain analyses within a species showing\u0026thinsp;\u0026gt;\u0026thinsp;96% ANI (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All the \u003cem\u003eB. burgdorferi\u003c/em\u003e and \u003cem\u003eB. afzelii\u003c/em\u003e were correctly assigned. One of the two strains typed as \u003cem\u003eB. bavariensis\u003c/em\u003e clustered within the \u003cem\u003eB. garinii\u003c/em\u003e. The other typed with one of the two strains typed as \u003cem\u003eB. valaisiana\u003c/em\u003e, forming a small \u003cem\u003esui generis\u003c/em\u003e group which may correspond to a hybrid species between \u003cem\u003eB. garinii\u003c/em\u003e and \u003cem\u003eB. afzelii\u003c/em\u003e as the hierarchical clustering placed them between the proposed parent species. These findings are in line with previous reports that had suggested that \u003cem\u003eB. bavariensis\u003c/em\u003e was a subspecies of \u003cem\u003eB. garinii\u003c/em\u003e. \u003cem\u003eB. maritima\u003c/em\u003e was equidistant between \u003cem\u003eB. afzelii\u003c/em\u003e and \u003cem\u003eB. burgdorferi.\u003c/em\u003e In addition, we characterized the phylogenetic relationship of several novel isolates included in this study. These included the lagomorph isolate, \u003cem\u003eBorreliella andersonii\u003c/em\u003e, which at the time of its discovery was misclassified as a \u003cem\u003eB. burgdorferi\u003c/em\u003e strain (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) but was later reclassified as \u003cem\u003eB. andersonii\u003c/em\u003e (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). In our ANI analyses, the \u003cem\u003eB. andersonii\u003c/em\u003e strain MOK_3a clustered closely to \u003cem\u003eB. burgdorferi and B. maritima\u003c/em\u003e. Of the remaining three \u003cem\u003eBorreliella\u003c/em\u003e species (\u003cem\u003eB. andersonii\u003c/em\u003e, \u003cem\u003eB. bissetii\u003c/em\u003e, and \u003cem\u003eB. mayonii\u003c/em\u003e), all were most closely related to \u003cem\u003eB. burgdorferi\u003c/em\u003e but each were sufficiently distant to warrant their species designations.\u003c/p\u003e \u003cp\u003eCompared to the \u003cem\u003eBorreliella\u003c/em\u003e, the \u003cem\u003eBorrelia\u003c/em\u003e spirochetes displayed greater genetic heterogeneity. The \u003cem\u003eB. miyamotoi\u003c/em\u003e strains clustered strongly together, displaying an average pairwise ANI of 97% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) but were distinct from the cluster of species containing \u003cem\u003eB. hermsii\u003c/em\u003e, \u003cem\u003eB. parkeri\u003c/em\u003e, and \u003cem\u003eB. turicatae\u003c/em\u003e. The reptile-associated \u003cem\u003eBorrelia\u003c/em\u003e species were both remarkably distant from these other major relapsing fever spirochetes and highly divergent among themselves with only B_sp_FGy1 and \u003cem\u003eB. turcica\u003c/em\u003e strains sharing any substantial similarity at 94% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Through ANIb (blast), we determined that these two genomes bracket the \u003cem\u003eBorrelia\u003c/em\u003e genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The isolate B_sp_HM_thM16w was also divergent from the group and was closest to \u003cem\u003eB. recurrentis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFollowing our initial ANI analysis, the evolutionary relatedness of these spirochetes was checked further by constructing a family phylogeny (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This revealed both a division amongst the \u003cem\u003eBorreliaceae\u003c/em\u003e spirochetes and the same clustering for \u003cem\u003eBorreliella\u003c/em\u003e and \u003cem\u003eBorrelia\u003c/em\u003e spirochetes. Interestingly, we observed that some more recently identified \u003cem\u003eBorreliella and Borrelia\u003c/em\u003e isolates cluster within their respective genera. This is most clearly illustrated with \u003cem\u003eB. andersonii\u003c/em\u003e which, based on the single-copy core phylogeny, was most closely related to \u003cem\u003eB. burgdorferi\u003c/em\u003e and \u003cem\u003eB. bissettii\u003c/em\u003e. Other novel genomes, like those seen within the reptile-associated \u003cem\u003eBorrelia\u003c/em\u003e spirochetes, stood apart from the majority of the \u003cem\u003eBorrelia\u003c/em\u003e genus as had been previously seen in the ANI. Only the novel \u003cem\u003eB. sp strain thHMw\u003c/em\u003e intercalated within \u003cem\u003eB. miyamotoi\u003c/em\u003e and \u003cem\u003eB. hermsii\u003c/em\u003e strains. The rest of the reptile-associated spirochetes formed a separate clade within \u003cem\u003eBorrelia\u003c/em\u003e spirochetes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBorreliaceae\u003c/b\u003e \u003cb\u003efamily-level pangenome\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFollowing our initial ANI analyses, we wanted to understand how similar the genera were based on their overall gene similarities. The percentage of shared genes within the family was approximately 50% (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). To begin to understand which genes are shared between the different genera, the \u003cem\u003eBorreliaceae\u003c/em\u003e family-level pangenome was multiply calculated at 5% intervals of increasing similarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). At 75% BlastP, clear separation between the core and cloud components of the \u003cem\u003eBorreliaceae\u003c/em\u003e pangenome is seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). At the 75% BlastP threshold, there are 322 core genes, 8 soft-core genes, 1,352 genes in the shell, and 3,424 genes in the cloud. There are 1,253 unique genes for all 109 isolates included in this study. Interestingly, the \u003cem\u003eBorreliaceae\u003c/em\u003e pangenome had a different composition when analyzed with the EggNOG algorithm. Through this second approach, EggNOG estimates that \u003cem\u003eBorreliaceae\u003c/em\u003e contains 997 unique genes for 109 isolates. Furthermore, its pangenome distribution consists of 577 core, 30 soft-core, 204 shell, and 213 cloud genes. Although each of these pangenome analysis tools were given the same dataset, they yielded different results. These discrepancies are due to underlying differences in the alignment methods used by the algorithms. EggNOG relies upon alignment to a database of proteins which have been pre-clustered, rather than relying on a specific blast threshold. Additionally, EggNOG largely ignores genes of unknown function rather than labeling it as \u0026ldquo;unknown,\u0026rdquo; which artificially decreases the number of unique genes.\u003c/p\u003e \u003cp\u003ePreviously, a \u003cem\u003eBorreliella\u003c/em\u003e genospecies complex pangenome was constructed through the use of 22 genomes comprised of \u003cem\u003eB. burgdorferi, B. afzelii, and B. garinii\u003c/em\u003e (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In this work, they found that the \u003cem\u003eBorreliella\u003c/em\u003e pangenome was open. To ascertain if this was true within our own curated collection of \u003cem\u003eBorreliaceae\u003c/em\u003e genomes, we next tested for changes in the pangenome using both data acquired from Roary at 75% BlastP and EggNOG (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). It is evident that the number of gene clusters present within each pangenome compartment is subject to change at the incorporation of a novel pangenome. This suggests that the family-level pangenome is open with a growing distributed component reminiscent of what was found within the early \u003cem\u003eBorreliella\u003c/em\u003e comparative genomic study.\u003c/p\u003e \u003cp\u003eNext, we visualized the \u003cem\u003eBorreliaceae\u003c/em\u003e pangenome using a heat map for all included isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). The family-level core genome consists of 488 genes possessed by all isolates in both genera. Interestingly, the family-level distributed pangenome is fragmented into multiple components. This division reveals two additional groups of genes that on further inspection were identified as the core genes of either the \u003cem\u003eBorreliella\u003c/em\u003e or \u003cem\u003eBorrelia\u003c/em\u003e genera.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003eBorreliaceae\u003c/b\u003e \u003cb\u003eepisomes can be grouped through gene content\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eBorreliaceae\u003c/em\u003e spirochetes thrive throughout their complex lifecycles during which they undergo multiple transmissions between phylogenetically distinct hosts including arthropod vectors and multiple classes of vertebrates. Their ability to survive in various hosts is dependent upon a large set of distributed genes that are mostly encoded on episomal replicons. These episomes have traditionally been typed primarily based on molecular size, structure (linear or circular), and alleles of proteins in the PFam32 family (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). To ensure proper episomal identification, this system needs to be expanded to include information on the entire gene repertoire of all available episomes. To this end, we ran a pyani ANIb on approximately 1,800 \u003cem\u003eBorreliaceae\u003c/em\u003e replicons from our curated genome collection. As pyani ANIb may not factor all gene content within the replicons, we also employed a Hadamard matrix (which interprets identity and coverage simultaneously) for this purpose. In the Hadamard matrix heatmap, multiple subgroups for nearly all of the \u003cem\u003eBorreliaceae\u003c/em\u003e family replicons were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe most similar episomes clustered closely together. The three most conserved groups are the \u003cem\u003eBorreliella\u003c/em\u003e chromosome, cp26, and lp54. Other \u003cem\u003eBorreliella\u003c/em\u003e episomal groups with relatively higher percentages of similarity were lp25 and lp36. Interestingly, there was very broad and diffuse clustering for the clinically significant family of cp32 plasmids which are known to contain genes encoding host-specific proteins, indicating both their relatedness and the very high degrees of heterogeneity necessary to ensure survival during a lifecycle that includes multiple hosts. Unlike cp32, the lp28 episomes do not likely represent a single family of replicons as they did not all cluster together and instead separated out into smaller clusters.\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003eBorrelia\u003c/em\u003e genus, a high degree of similarity was seen amongst the chromosomes as well as for the large linear plasmids. Additionally, species-specific groupings amongst the episomes were also observed. This is best illustrated in \u003cem\u003eB. miyamotoi\u003c/em\u003e strains lp6 and lp12. It is important to note that while the reptile-associated \u003cem\u003eBorrelia\u003c/em\u003e spirochetes were included in these analyses, they were the only isolates to have extraordinarily dissimilar replicons. This is likely attributable to the incompleteness of the B_sp_A_FGy1 genome as indicated by its large number of constituent contigs.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe debilitating diseases caused by \u003cem\u003eBorreliaceae\u003c/em\u003e spirochetes have remained on the periphery of societal memory for centuries. Despite our current knowledge of the etiological agents of these spirochetoses, there is still a paucity of information on how each disease is caused and why their presentations are so variable among individuals. In the work presented in this study, multiple pressing points within the field of \u003cem\u003eBorreliaceae\u003c/em\u003e comparative genomics were addressed to help provide a framework going forward to be able to associate microbial genotypes with disease phenotypes in the host.\u003c/p\u003e \u003cp\u003eThe first topic we addressed was the composition of the \u003cem\u003eBorreliaceae\u003c/em\u003e family. It was recently proposed that the previous single genus, \u003cem\u003eBorrelia\u003c/em\u003e, be divided into two genera based on differences in overall genomic content (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The well-studied and more widely known Lyme borreliosis spirochetes were given the genus name of \u003cem\u003eBorreliella\u003c/em\u003e, while the older and less clinically studied species complex comprising the relapsing fever spirochetes retained the \u003cem\u003eBorrelia\u003c/em\u003e designation. Since the establishment of this proposed dichotomy, there has been animated discussion among scientists both for and against the split (\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePairwise ANI analyses of all 108 sequenced isolates (N\u0026thinsp;=\u0026thinsp;11556 genome comparisons) in this study revealed a sharp division between the two proposed genera indicating that the recent taxonomic re-classification is correct. Even isolates from within each of the genera often did not share sequence similarities above 85%. While it may be true that the ANI cut-off can vary based on species as seen with \u003cem\u003eStrenotrophomonas maltophila, Escherichia\u003c/em\u003e spp, or \u003cem\u003eLactobacillus\u003c/em\u003e spp., our results do not support including the Lyme borreliosis spirochetes and relapsing fever spirochetes within a single genus (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditional analyses revealed that the recently sequenced novel spirochaetal species B_sp_FGy1 and B_sp_HM_thM16w clustered most closely with \u003cem\u003eB. anserina\u003c/em\u003e and \u003cem\u003eB. recurrensis\u003c/em\u003e, respectively. These two genomes clustered within the known reptile- associated \u003cem\u003eBorreliaceae\u003c/em\u003e. As these isolates are novel, it is unknown if they are pathogenic to humans.\u003c/p\u003e \u003cp\u003eWe constructed a family-level pan-genome to characterize the similarities and differences between the two genera concerning gene content. In doing so, we found that the family-level \u003cem\u003eBorreliaceae\u003c/em\u003e core genome was very small, consisting of only 488 genes, but that each of the genus-level core genomes were each more than double that size and that the family-level pangenome is still open. This indicates that there are large numbers of distributed genes still to be discovered. The number of core genes possessed by both genera were similar.\u003c/p\u003e \u003cp\u003eIt was previously known that there is some similarity between \u003cem\u003eBorreliella\u003c/em\u003e and \u003cem\u003eBorrelia\u003c/em\u003e replicons with the \u003cem\u003eBorrelia\u003c/em\u003e replicons containing regions of similarity to the \u003cem\u003eBorreliella\u003c/em\u003e cp26 and lp54 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). To better place the various replicons within a gene-functional framework, we established a system for typing the \u003cem\u003eBorreliaceae\u003c/em\u003e plasmids. Previously, these episomes have been typed based on size, structure, and the presence of specific alleles of particular protein families found on many of the plasmids (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). While this technique helped somewhat in the identification of the many \u003cem\u003eBorreliaceae\u003c/em\u003e episomes, it did little to identify them by the biological functions they encode. In our work, all of the available episomes for each of the isolates in our data set were used in an ANI analysis via pyani. Doing so for over 1,800 replicons resulted in a complex matrix that did little to clarify how these episomes related to each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). However, in building a Hadamard matrix from these analyses it was possible to visualize each of the known plasmids with respect to their overall hierarchical clustering, even for the most heterogenous replicon, cp32. Thus, we have established a universal \u003cem\u003eBorreliaceae\u003c/em\u003e plasmid typing system based on gene content that captures all of the replicons produced via our PacBio-based long-read sequencing protocols.\u003c/p\u003e \u003cp\u003eFinally, we identified an artifact of sequencing linear bacterial replicons with hairpin termini with the PacBio SMRTbell technology. In our sequencing, it was noted that some of the many linear episomes had regions of heterogeneity. After closer inspection, it became apparent that these regions were inverted repeats that could emerge as artifacts in long-read sequencing platforms (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). These regions were identified as the telomeric ends of all linear \u003cem\u003eBorreliaceae\u003c/em\u003e replicons, a feature not unlike those seen in higher eukaryotes (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). To prevent false inflation in the genes within our data set, we searched for and confirmed the location of the ResT Box 3 site (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). From this, we have begun to build the first system to handle these \u003cem\u003eBorreliaceae\u003c/em\u003e long-read sequencing artifacts that could be applied to any replicon with similar features. These data also suggest that raw PacBio circular consensus sequences may contain hidden information about hairpin and cruciform DNA structures in other genomes.\u003c/p\u003e \u003cp\u003eThis work on the \u003cem\u003eBorreliaceae\u003c/em\u003e family-wide pangenome is pivotal and the first of its kind. More sequencing is required to ensure that the pangenome is truly reflective of all \u003cem\u003eBorreliaceae\u003c/em\u003e spirochetes. Furthermore, more work is required for the typing of all \u003cem\u003eBorreliaceae\u003c/em\u003e episomes. This family-level pangenome analysis provides new tools and information in a field that requires more insight on how to handle such evasive and persistent pathogens. More importantly, this work can later culminate into effective therapeutics.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThrough the application of multiple comparative genomic methods including phylogenetics, ANI, gene content, and core genome analyses, we demonstrate that the \u003cem\u003eBorreliaceae\u003c/em\u003e are composed of at least two genera. We also developed a gene-based plasmid typing protocol to replace the current confusing method that relies solely on size and topography. This approach to episomal typing revealed an extremely high degree of allelic heterogeneity in the plasmid(s) that contain the genes that encode the major host-interacting proteins which are reflective of the extremely broad host-range of these parasitic bacteria. Additional analyses with a larger number of genomes will be required to determine if the reptile-associated strains and other underrepresented groups in the current analysis define additional genera.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eBorreliaceae\u003c/b\u003e \u003cb\u003epangenome project design\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study included all available reference \u003cem\u003eBorreliaceae\u003c/em\u003e genomes obtained from NCBI RefSeq on October 17th, 2021. The downloaded genomes were checked for completeness, sequencing platform, sequencing coverage, and if the average number of contigs greatly superseded the number of included replicons. For example, selected genomes were disqualified if more than 2 or 3 contigs were needed for a single replicon. Additionally, they were run through a QC pipeline to ensure usability in later studies. The remainder of the genomes were used henceforth. Novel isolates collected by collaborators at Virginia Commonwealth University were then sequenced at the Center for Genomic Sciences core facility at Drexel University College of Medicine. These newly sequenced members of the \u003cem\u003eBorreliaceae\u003c/em\u003e are included in BioProject PRJNA861274.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBorreliaceae\u003c/b\u003e \u003cb\u003eculturing\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAll \u003cem\u003eBorreliella\u003c/em\u003e and \u003cem\u003eBorrelia\u003c/em\u003e isolates were cultivated in 50 mL of BSK-II media supplemented with 6 or 12% rabbit serum (Sigma, MA), respectively. The culture was then monitored using wet-mounts and dark-field microscopy. Once spirochetes grew to the concentration of 5 x 10^7 cells/mL at late log phase, they were harvested by centrifugation, resuspended in 1X sterile PBS, and then recovered by centrifugation.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePulse-Field Gel plasmid verification\u003c/h2\u003e \u003cp\u003eDNA isolation of selected \u003cem\u003eBorreliaceae\u003c/em\u003e isolates was done through a modified BioNano plug prep (Bionano Genomics, Bionano Prep cell Culture DNA Isolation Protocol) (Bionano Genomics, CA). Cultured \u003cem\u003eBorreliaceae\u003c/em\u003e were pelleted at 5,000 xg for 10 mins and sent overnight to the Center for Genomic Sciences at Drexel University College of Medicine. Received pellets were immediately thawed, resuspended with a total volume of 66 \u0026micro;L of cell buffer, and then 40 \u0026micro;L of 2% agarose was added. The contents were mixed 10 times via pipette and then 100 \u0026micro;L of each sample was aliquoted into a plug mold. The filled plug mold was incubated at 4\u0026deg;C for 15 mins before starting protein digestion. This process was done by placing the plug into a solution of 167 \u0026micro;L proteinase K (Qiagen, DE) and 2.5 mL Lysis buffer, then incubated for 2 hours at 50\u0026deg;C in a thermomixer at intermittent mixing. After incubation, the digestion solution was replaced and incubated overnight on the thermomixer as described above.\u003c/p\u003e \u003cp\u003eFollowing the overnight incubation, plugs were allowed to cool at room temp for 5 mins and then we added 50 \u0026micro;L of RNAse A before placing the plugs in the thermomixer at 37\u0026deg;C with intermittent mixing. Plugs were then washed 3 times with 10 mL 1X Wash buffer before being washed once more with 10 mL and set on an orbital shaker for 15 mins at 180 RPM. This latter step was repeated 3 more times. DNA recovery was initiated by adding 10 mL of 1X TE buffer to wash plugs and shaking at 180 RPM for 15 mins a total of 5 times. Cleaned plugs were then inserted into a 1% low melt agarose gel and run on a pulse-field gel at 5V for 21 hrs. Pulse field gel was stained with ethidium bromide and visualized under UV.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn silico Borreliaceae\u003c/b\u003e \u003cb\u003ereplicon verification\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBorreliaceae\u003c/em\u003e genome assembly pairs were aligned through progressiveMauve (v.2.4.0), NUCmer (MUMer 3.0) and D-Genies (v1.4.0) (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). Additionally, each spirochete genome FASTA file that had been used for the above initial validation work was split by contig, imported into R, and then aligned to itself using pafr (v 0.0.2) (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBorreliaceae\u003c/b\u003e \u003cb\u003eDNA extraction\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOnce received, frozen \u003cem\u003eBorreliaceae\u003c/em\u003e pellets were thawed on ice and centrifuged at 13,000 rpm for 1 min. Following pelleting, the remaining solvent was discarded before the pellets were resuspended in 200 \u0026micro;L of 1X PBS, pipette mixed, and transferred to a 2 mL bead beating tube (Matrix E) (MP Biomedicals, CA). Following the transfer, 20 \u0026micro;L of Proteinase K (Qiagen, DE) was added, and cells were homogenized using a SPEX 1600 MiniG (Fisher Scientific, MA) for 1 min at 1500 Hz. Subsequent DNA extraction was performed using the Qiagen\u0026trade; DNeasy Blood \u0026amp; Tissue Kit according to the manufacturer\u0026rsquo;s instructions (Qiagen, DE).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA preparation and sequencing\u003c/h3\u003e\n\u003cp\u003eExtracted \u003cem\u003eBorreliaceae\u003c/em\u003e DNA was quantified following ThermoFisher Scientific\u0026trade; 1X dsDNA HS kit per the manufacturer\u0026rsquo;s instructions (Biotium Inc, CA) on Qubit. DNA from each Borrelial/Borrelia specimen was prepped with the SMRTbell Template Prep Kit 2.0 (Pacific Biosciences, CA) to make PacBio SMRTbell libraries with barcodes sourced from the Barcoded Overhang Adaptor Kit 8A and 8B (Pacific Biosciences, CA). The sequencing primers were then annealed and bound to Polymerase 3.0 using the Sequel Binding Kit 3.0 (Pacific Biosciences, CA). The final bound complex was then purified and later sequenced on PacBio Sequel I using the SMRT Cell M1 v3 tray (Pacific Biosciences, CA). The spike-in controls for each PacBio Sequel I run were from the Internal Control Kit 3.0 (Pacific Biosciences, CA).\u003c/p\u003e\n\u003ch3\u003eGenome assembly, annotation, and pangenome construction\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eBorreliaceae\u003c/em\u003e isolates that were sequenced on the PacBio Sequel I platform were processed using pbcromwell (v 1.0.4) (Pacific Biosciences, CA). All the barcoded data were demultiplexed using pb_demux_subreads and assembled using pb_assembly_microbial. The base modification motifs for each assembly were computed using pb_basemods. The species of each genome was verified using taxator and GTDB-Tk (v1.7.0) with refpack (v r202). All reference and sequenced \u003cem\u003eBorreliaceae\u003c/em\u003e assemblies were annotated with Prokka (v 1.11) and then homologous genes were clustered with Roary (v 3.5.1) (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). The BlastP threshold was calculated consecutively at 5% intervals to determine the threshold. The final BlastP threshold, 75%, was selected based on the separation of the core pangenome from the cloud and accessory pangenomes. All CDS identified by Prokka were also annotated using EggNOG (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic tree construction\u003c/h2\u003e \u003cp\u003eSingle-copy core genes of each \u003cem\u003eBorreliaceae\u003c/em\u003e genome were aligned using MAFFTT (v7.490-1) and then the concatenated alignment was run through Randomized Accelerated Maximum Likelihood (RaxML) to generate a Newick string (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). The resulting file was then ported into RStudio (v2022.07.2\u0026thinsp;+\u0026thinsp;576), a tree was generated using ape and then visualized using ggtree (v3.15) R packages (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAverage Nucleotide Identity and percent similarity or difference analysis\u003c/h2\u003e \u003cp\u003eAll pairs of \u003cem\u003eBorreliaceae\u003c/em\u003e genomes, both sequenced and downloaded were checked for overall similarity by ANI using the pyani (v2.12) with the ANIb method (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). The resulting whole-genome ANI matrix (or Hadamard matrix) was read into RStudio (v.4.1.1), clustered with adapted RaxML phylogeny, and visualized with pheatmap (v1.0.12).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBorreliaceae\u003c/em\u003e percent gene similarity was calculated using RStudio in a pairwise fashion for each isolate in this study. The final list of shared percent identity was then pivoted wider to generate a matrix. Any pairs that did not have a gene percent similarity value were assigned a value of 0. The final matrix was then clustered with hclust and visualized with pheatmap (v1.0.12).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBorreliaceae\u003c/b\u003e \u003cb\u003epangenome mapping\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo find pangenome distribution, all Roary at BlastP 75% or EggNOG gene clusters were imported into R (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). Then each gene was associated with a corresponding strain and/or replicon before being used to construct a presence-absence matrix. This matrix was then visualized in pheatmap to see the gene presence and absence across all species used in this study. To determine if the \u003cem\u003eBorreliaceae\u003c/em\u003e pangenome was either open or closed, the composition was checked by shuffling strains, incorporating them one at a time into the analysis, and then calculating the core/distributed component. The combinations of strains incorporated were shifted prior to being plotted in each iteration.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Bill and Marian Cook Foundation; the Oskar Fischer Project, a James Truchard Philanthropy; Cure; the Coulter-Drexel Translational Research Partnership; and NIH grant Nos: DC 02148 and DK082316 to GDE. This work was also supported by NIH grants 1R01AI165876-01 and 1R01AI141801-01A1 to RTM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequencing data used in this study is available under Bioproject PRJNA1026537.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(NONE)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(NA)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe main manuscript text was written by KMS, GDE, and MCM. Study strategy was conceived by GDE. Study design was conceived by KMS, JPE, JCM, RLE, RTM, and GDE. Experimental work was performed by KMS, SB, AA, NAC, RLE, JPE, and JCM. Data analysis was performed by KMS, JPE, RLE, and JCM. Figure construction was performed by KMS, JPE, JCM, and MCM. Funding was provided through grants awarded to GDE and RTM.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShen K, Wang X, Post JC, Ehrlich GD. Molecular and Translational Research Approaches for the study of Bacterial Pathogenesis in Otitis Media. In: Rosenfeld R, Bluestone CD, editors. Evidence-based Otitis Media. 2nd ed. Hamilton, London: B.C. Decker Inc.; 2003. pp. 91\u0026ndash;119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEhrlich GD, Hu ZF, Post JC. Role for Biofilms in Infectious Disease. In: Ghannoum M, O\u0026rsquo;Toole GA, editors. Microbial Biofilms. Washington, D.C.: ASM; 2004. pp. 332\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEhrlich GD, Hu FZ, Shen K, Stoodley P, Post JC. Bacterial plurality as a general mechanism driving persistence in chronic infections. 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Anal Methods. 2016(1).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spirochete, distributed genome hypothesis, pan-genome, supragenome, comparative genomics, phylogenetics, Borrelia, Borreliella, Borreliaceae, taxonomy","lastPublishedDoi":"10.21203/rs.3.rs-4491589/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4491589/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eBorreliaceae\u003c/em\u003e family includes many obligate parasitic bacterial species which are etiologically associated with a myriad of zoonotic borrelioses including Lyme disease and vector-borne relapsing fevers. Infections by the \u003cem\u003eBorreliaceae\u003c/em\u003e are difficult to detect by both direct and indirect methods, often leading to delayed and missed diagnoses. Efforts to improve diagnoses center around the development of molecular diagnostics (MDx), but due to deep tissue sequestration of the causative spirochaetes and the lack of persistent bacteremias, even MDx assays suffer from a lack of sensitivity. Additionally, the highly extensive genomic heterogeneity among isolates, even within the same species, contributes to the lack of assay sensitivity as single target assays cannot provide universal coverage. This within-species heterogeneity is partly due to differences in replicon repertoires and genomic structures that have likely arisen to support the complex \u003cem\u003eBorreliaceae\u003c/em\u003e lifecycle in which these parasites have to survive in multiple hosts each with unique immune responses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe constructed a \u003cem\u003eBorreliaceae\u003c/em\u003e family-level pangenome and characterized the phylogenetic relationships among the constituent taxa which supports the recent taxonomy of splitting the family into at least two genera. Gene content profiles were created for the majority of the \u003cem\u003eBorreliaceae\u003c/em\u003e replicons, providing for the first time their unambiguous molecular typing.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur characterization of the \u003cem\u003eBorreliaceae\u003c/em\u003e pan-genome supports the splitting of the former \u003cem\u003eBorrelia\u003c/em\u003e genus into two genera and provides for the phylogenetic placement of several non-species designated isolates. Mining this family-level pangenome will enable precision diagnostics corresponding to gene content-driven clinical outcomes while also providing targets for interventions.\u003c/p\u003e","manuscriptTitle":"Characterization of the family-level Borreliaceae pan-genome and development of an episomal typing protocol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 18:12:08","doi":"10.21203/rs.3.rs-4491589/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"de368f1e-82e0-4b1c-a3ef-b4cbea9aee03","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-23T05:46:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-11 18:12:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4491589","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4491589","identity":"rs-4491589","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}