Whole-Genome Sequencing of Six Borrelia miyamotoi Clinical Strains Isolated in Russia Konstantin V. Kuleshov, a,e Joris Koetsveld, b Irina A. Goptar, a,g Mikhail L. Markelov, g Nadezhda M. Kolyasnikova, a,f Denis S. Sarksyan, a,c Marina G. Toporkova, a,d Nina P. Kirdyashkina, g German A. Shipulin, a Joppe W. Hovius, b Alexander E. Platonov a a Central Research Institute of Epidemiology, Moscow, Russia b Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands c Izhevsk State Medical Academy, Izhevsk, Russia d Medical Association “Novaya Bolnitsa,” Yekaterinburg, Russia e Kovalenko All-Russian Research Institute for Experimental Veterinary Medicine, Moscow, Russia f Chumakov Federal Scientific Center for Research and Development of Immunobiological Products of Russian Academy of Sciences, Moscow, Russia g Research Institute of Occupational Health, Moscow, Russia ABSTRACT Here, we report the whole-genome sequence of six clinical Borrelia miy- amotoi isolates from the Russian Federation. Using two independent next-generation sequencing platforms, we determined the complete sequence of the chromosome and several plasmids. All strains have an Asian genotype with 99.8% chromosome nucleotide similarity with B. miyamotoi strain FR64b. S tudies on the emerging human tick-borne pathogen, Borrelia miyamotoi, are cum- bersome, since only a limited set of strains are available and sequenced to date (1–4). We previously isolated six clinical strains from blood of Russian patients with acute Borrelia miyamotoi disease in Izhevsk City (strains Izh-4, Izh-5, Izh-14, and Izh-16) and Yekaterinburg City (strains Yekat-1 and Yekat-6) in 2016 (5). Low-passage isolates were grown in vitro and total DNA was extracted using the DNeasy blood and tissue kit and Qiagen Tip-100 prep (Qiagen) for sequencing using MiSeq and MinION platforms, respectively. In addition, to increase the reliability of the assembly of individual plasmids, we separated DNA by pulsed-field gel electrophoresis (PFGE); 8 to 11 extrachromosomal fragments per isolate—ranging from 5 to 73 kb— were cut out from gels and dissolved in Agarose Dissolving Buffer (Zymoresearch). DNA was extracted using the DNeasy blood and tissue kit, and DNA libraries were prepared using the NexteraXT DNA library kit (Illumina), with a distinct barcode for each fragment. Next, DNA libraries were sequenced using the MiSeq platform and 500-cycle V2 reagent kit (Illumina). Adapter sequences were removed from the Illumina reads by BBTools (https://sourceforge.net/projects/bbmap/). The Native barcoding kit 1D (EXP-NBD103) was used together with the Ligation sequencing kit (SQK-LSK108) to prepare Nanopore sequencing libraries from total DNA. One R9.4 MinION flow cell was used for six multiplexed DNA samples. Base calling of MinION sequences was performed using Albacore v1.1.0, and adapters were removed by Porechop (https:// github.com/rrwick/Porechop). The hybrid de novo assembly of Illumina and Nanopore reads was done using Unicycler v0.3.1 (6). We performed separate assemblies of Illumina reads relating to a particular PFGE fragment, aided by the long corresponding Nanopore reads. Subse- quently, the contigs from independent assemblies of each isolate were compared and clustered (when 98% similar) by CD-HIT-EST (https://github.com/weizhongli/cdhit) in order to leave only one representative contig from each cluster. The accuracy of our Received 14 November 2017 Accepted 15 November 2017 Published 4 January 2018 Citation Kuleshov KV, Koetsveld J, Goptar IA, Markelov ML, Kolyasnikova NM, Sarksyan DS, Toporkova MG, Kirdyashkina NP, Shipulin GA, Hovius JW, Platonov AE. 2018. Whole-genome sequencing of six Borrelia miyamotoi clinical strains isolated in Russia. Genome Announc 6:e01424-17. https://doi.org/10.1128/genomeA .01424-17. Copyright © 2018 Kuleshov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Konstantin V. Kuleshov, konstantinkul@gmail.com. PROKARYOTES crossm Volume 6 Issue 1 e01424-17 genomea.asm.org 1