We report the genomic sequence of the hard tick relapsing fever spirochete Borrelia miyamotoi strain MN18-0001. B. miyamotoi causes human illness and is geographically widespread in Ixodes spp. (Acari: Ixodidae) ticks. This is a chromosome- and plasmid-resolved genome assembly of an Am-East-2 strain type isolate from the midwestern United States

Eisen, Rebecca J

Hojgaard, Andrias

Kneubehl, Alexander R

Lopez, Job E

Osikowicz, Lynn M

Parise, Christina M

Replogle, Adam J

Van Gundy, Taylor J

English

DigitalCommons@The Texas Medical Center

The Texas Medical Center Library DigitalCommons@TMC Faculty and Staff Publications Baylor College of Medicine 11-12-2024 Complete Genome Sequence of Borrelia miyamotoi Strain MN18-0001, an Am-East-2 Strain Type Isolate Derived From an Ixodes scapularis Tick (Minnesota) Alexander R Kneubehl Lynn M Osikowicz Christina M Parise Taylor J Van Gundy Adam J Replogle See next page for additional authors Follow this and additional works at: https://digitalcommons.library.tmc.edu/baylor_docs  Part of the Biological Phenomena, Cell Phenomena, and Immunity Commons, Diseases Commons, Pediatrics Commons, and the Tropical Medicine Commons Authors Alexander R Kneubehl, Lynn M Osikowicz, Christina M Parise, Taylor J Van Gundy, Adam J Replogle, Job E Lopez, Rebecca J Eisen, and Andrias Hojgaard  | Genomics and Proteomics | AnnouncementComplete genome sequence of Borrelia miyamotoi strain MN18-0001, an Am-East-2 strain type isolate derived from an Ixodes scapularis tick (Minnesota)Alexander R. Kneubehl,1 Lynn M. Osikowicz,2 Christina M. Parise,2 Taylor J. Van Gundy,2 Adam J. Replogle,2 Job E. Lopez,1,3 Rebecca J. Eisen,2 Andrias Hojgaard2AUTHOR AFFILIATIONS See affiliation list on p. 2.ABSTRACT We report the genomic sequence of the hard tick relapsing fever spiro­chete Borrelia miyamotoi strain MN18-0001. B. miyamotoi causes human illness and is geographically widespread in Ixodes spp. (Acari: Ixodidae) ticks. This is a chromosome- and plasmid-resolved genome assembly of an Am-East-2 strain type isolate from the midwestern United States.KEYWORDS Borrelia miyamotoi, genome analysisB orrelia miyamotoi is an emerging hard (ixodid) tick-borne human pathogen that is vectored by the same ticks (Ixodes scapularis and Ixodes pacificus) that transmit the Lyme disease-causing spirochete Borrelia burgdorferi [reviewed in reference (1)]. In the United States, whole-genome sequencing of B. miyamotoi has been performed on tick-derived isolates from either the East or West Coast states but not from midwestern states. Earlier research suggested genetic variation in the US with four strain types identified: Am-East-1 and Am-East-2 in the eastern US, and Am-West-1 and Am-West-2 in the western US (2, 3). Genome assemblies of the Am-East-2 strain type are currently unavailable. The MN18-0001 genome assembly (GCF_039656655.1) is from an Am-East-2 strain type isolate and provides important data for ongoing efforts investigating the genomic diversity of this human pathogen.The isolate was obtained from transovarially-infected I. scapularis larvae. The larvae were progeny of an infected female collected by drag sampling in Morrison County, Minnesota (4). The larvae transmitted B. miyamotoi to a CD-1 mouse through blood feeding. Spirochetemic mouse blood was inoculated into BSK-R media to obtain the MN18-0001 isolate (5). Genomic DNA was purified from the second passage of B. miyamotoi MN18-0001 using phenol-chloroform, as previously described with modifi­cations (6): the starting culture was 50 mL of media, and 15 mL Qiagen MaxAttract High-Density tubes were used. Genomic DNA was sequenced without size selection using the SQK-RBK004 kit from Oxford Nanopore Technologies (ONT) following the manufacturer’s instructions with modification: 800 ng of genomic DNA in 7.5 µL of water was used with 2.5 µL of rapid barcode. The library was sequenced using an ONT Mk1B sequencer and R9.4.1 flow cell. Read data were collected using MinKnow v21.11.8 and basecalled using Guppy v6.0.1 with the SUP model. The basecalled read data were filtered with NanoFilt v2.8.0 (7) to remove reads ≤1 kb yielding 7,024 reads with an N50 of 29,397 bp. The Illumina data set was generated by SeqCenter (Pittsburgh, PA, USA) with the same stock DNA used for ONT sequencing. The Illumina library was prepared using the Illumina DNA library kit (2 × 150 bp) and sequenced on a Nextseq 2000. Adapter sequences were removed from the Illumina data using fastp v0.20.1 (8). Filtered ONT reads were assembled using Canu v2.2 (9) and manual methods (10). The assembly was November 2024  Volume 13  Issue 11 10.1128/mra.00490-24 1Editor Irene L. G. Newton, Indiana University, Bloomington, Indiana, USAAddress correspondence to Alexander R. Kneubehl, kneubehl@bcm.edu.The authors declare no conflict of interest.See the funding table on p. 3.Received 20 May 2024Accepted 30 September 2024Published 22 October 2024This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply. polished with both ONT and Illumina data sets, as previously described (10). The final assembly was assessed using Merqury v1.3 (11) to determine completeness (99.6%) and overall quality (Q44). The mean read depth of the final assembly was calculated using mosdepth v0.3.3 (12): ONT (91×) and Illumina (489×).The final assembly was 1,449,837 bp in length, consisted of a linear chromosome, five circular and 10 linear contigs matching plasmids, and had a 28.7% mean GC%. Replicons in the final assembly are described in Table 1 with their sizes, topologies, and GenBank accession numbers. Genome annotation was performed with NCBI’s Prokary­otic Genome Annotation Pipeline (PGAP) v2021-11-29.build5742 (13). A total of 1,456 coding sequences were identified as well as 67 pseudogenes. Data from this work are available on GenBank under BioProject PRJNA1088750 and BioSample SAMN40496904.ACKNOWLEDGMENTSThis work was supported by the National Institute of Allergy and Infectious Diseases grant, AI137412, to J.E.L.A.R.K.: conceptualization, data curation, formal analysis, writing-original draft; L.M.O.: writing-original draft; C.M.P., T.J.V.G., and A.J.R.: resources and writing-original draft; J.E.L., R.J.E., and A.H.: conceptualization, resources, funding acquisition, project administration, supervision, writing-original draft.AUTHOR AFFILIATIONS1Department of Pediatrics, Division of Tropical Medicine, Baylor College of Medicine, Houston, Texas, USA2Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, USA3Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USAAUTHOR ORCIDsAlexander R. Kneubehl  http://orcid.org/0000-0002-6243-4096Job E. Lopez  http://orcid.org/0000-0003-4802-5779TABLE 1 B. miyamotoi MN18-0001 repliconsReplicon Topology Size (bp) GenBank accession #Chromosome Linear 907,191 CP150054lp101 Linear 100,533 CP150055lp72 Linear 72,320 CP150056cp59 Circular 58,920 CP150057lp40 Linear 40,090 CP150058lp31 Linear 30,947 CP150059cp30-1 Circular 29,576 CP150060cp30-2 Circular 29,483 CP150061cp29 Circular 29,132 CP150062lp27 Linear 26,589 CP150063cp26 Circular 25,497 CP150064lp25 Linear 25,276 CP150065lp22 Linear 21,862 CP150066lp21 Linear 20,463 CP150067lp20 Linear 20,029 CP150068lp12 Linear 11,947 CP150069Announcement Microbiology Resource AnnouncementsNovember 2024  Volume 13  Issue 11 10.1128/mra.00490-24 2FUNDINGFunder Grant(s) Author(s)HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)AI137412 Job E. LopezAUTHOR CONTRIBUTIONSAlexander R. Kneubehl, Conceptualization, Data curation, Formal analysis, Writing – original draft | Lynn M. Osikowicz, Writing – original draft | Christina M. Parise, Resour­ces, Writing – original draft | Taylor J. Van Gundy, Resources, Writing – original draft | Adam J. Replogle, Resources, Writing – original draft | Job E. Lopez, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft | Rebecca J. Eisen, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft | Andrias Hojgaard, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draftDATA AVAILABILITYData from this work are publicly available on GenBank under BioProject PRJNA1088750 and BioSample SAMN40496904.REFERENCES1. Krause PJ, Fish D, Narasimhan S, Barbour AG. 2015. Borrelia miyamotoi infection in nature and in humans. Clin Microbiol Infect 21:631–639. https://doi.org/10.1016/j.cmi.2015.02.0062. Hojgaard A, Osikowicz LM, Maes S, Eisen L, Eisen RJ. 2021. Detection of variability in Borrelia miyamotoi (spirochaetales: spirochaetaceae) between and within the Eastern and Western United States. J Med Entomol 58:2154–2160. https://doi.org/10.1093/jme/tjab0753. Mun J, Eisen RJ, Eisen L, Lane RS. 2006. Detection of a Borrelia miyamotoi sensu lato relapsing-fever group spirochete from Ixodes pacificus in California. J Med Entomol 43:120–123. https://doi.org/10.1093/jmedent/43.1.1204. Breuner NE, Hojgaard A, Replogle AJ, Boegler KA, Eisen L. 2018. Transmission of the relapsing fever spirochete, Borrelia miyamotoi, by single transovarially-infected larval Ixodes scapularis ticks. Ticks Tick Borne Dis 9:1464–1467. https://doi.org/10.1016/j.ttbdis.2018.07.0065. Replogle AJ, Sexton C, Young J, Kingry LC, Schriefer ME, Dolan M, Johnson TL, Connally NP, Padgett KA, Petersen JM. 2021. Isolation of Borrelia miyamotoi and other borreliae using a modified BSK medium. Sci Rep 11:1926. https://doi.org/10.1038/s41598-021-81252-16. Lopez JE, Schrumpf ME, Raffel SJ, Policastro PF, Porcella SF, Schwan TG. 2008. Relapsing fever spirochetes retain infectivity after prolonged in vitro cultivation. Vector Borne Zoonotic Dis 8:813–820. https://doi.org/10.1089/vbz.2008.00337. De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34:2666–2669. https://doi.org/10.1093/bioinformatics/bty1498. Chen S, Zhou Y, Chen Y, Gu J. 2018. Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890. https://doi.org/10.1093/bioinformatics/bty5609. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722–736. https://doi.org/10.1101/gr.215087.11610. Kneubehl AR, Krishnavajhala A, Leal SM, Replogle AJ, Kingry LC, Bermúdez SE, Labruna MB, Lopez JE. 2022. Comparative genomics of the western hemisphere soft tick-borne relapsing fever borreliae highlights extensive plasmid diversity. BMC Genomics 23:1–22. https://doi.org/10.1186/s12864-022-08523-711. Rhie A, Walenz BP, Koren S, Phillippy AM. 2020. Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies. Genome Biol 21:245. https://doi.org/10.1186/s13059-020-02134-912. Pedersen BS, Quinlan AR. 2018. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics 34:867–868. https://doi.org/10.1093/bioinformatics/btx69913. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 44:6614–6624. https://doi.org/10.1093/nar/gkw569Announcement Microbiology Resource AnnouncementsNovember 2024  Volume 13  Issue 11 10.1128/mra.00490-24 3

Complete Genome Sequence of Borrelia miyamotoi Strain MN18-0001, an Am-East-2 Strain Type Isolate Derived From an Ixodes scapularis Tick (Minnesota)

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Complete Genome Sequence of Borrelia miyamotoi Strain MN18-0001, an Am-East-2 Strain Type Isolate Derived From an Ixodes scapularis Tick (Minnesota)

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