Whole Genome Sequencing of Brucella melitensis Isolates for the Identification of Biovar, Variants and Relationship within a Biovar *Shaheed F [1], Habibi N [1], Mustafa AS [1, 2] OMICS Research Unit, Research Core Facility, Health Sciences Centre [2]; Department of Microbiology, Faculty of Medicine [2], Kuwait University INTRODUCTION RESULTS CONCLUSIONS The biovar 2 is the most prevalent biovar of B. melitensis in Kuwait. Isolate specific variations were identified, which may be useful in epidemiological investigations. Next generation sequencing along with bioinformatics can be a powerful tool not only for the identification of the biovar but also for establishing intrabiovaric relationships. Brucellosis, a highly infectious zoonotic disease, is endemic in Kuwait and the Middle East. The primary cause of Brucellosis in Kuwait is Brucella melitensis. The identification of Brucella genotypes is essential for epidemiological studies, including surveillance and contact tracing. However, the limited genetic diversity among Brucella genomes has made its genotyping a challenging task. The whole genome sequencing is emerging as a novel tool for genetic characterization of microorganisms. The aim of this study was to genotype human isolates of B. melitensis using whole genome sequencing. Variations (SNPs and inDels) were spread all over the genome; but 138 SNPs were common among the 14 isolates, supporting the same ancestral origin. In addition, SNPs (2 - 478) unique to each isolate were also identified, which divided the B. melitensis biovar 2 into two major variant groups. Table 1: Number of variants in samples observed when aligned against bivar 2 genome obtained from NCBI. METHODS Fifteen clinical isolates of B. melitensis were grown on culture plates. The bacterial colonies from individual plates were suspended in saline and heated at 95°C for 10 minutes. DNA was purified using the QIAamp DNA Mini Kit (Qiagen) and checked for quantity and purity using a spectrophotometer (Epoch) and a fluorometer (Qubit). DNA libraries were prepared using the Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using MiSeq (Illumina). The sequence files were aligned to three biovars of B. melitensis, i.e. biovar 1 str. 16M, biovar 2 str. 63/9, and biovar 3 str. Ether. The alignment and variant calling were performed using ‘bwa mem’ and SAMtools/VCFtools, respectively. Figure 2: Variant density graph for both the chromosomes of Brucella. REFERENCES   Corbel, M. J. (1997). "Brucellosis: an overview." Emerging Infectious Diseases 3(2): 213-221. Foster, J. T., et al. (2009). "Whole-Genome-Based Phylogeny and Divergence of the Genus Brucella." Journal of Bacteriology 191(8): 2864-2870. M, W. A. (2009). "Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens." Infection Genetics and Evolution 9(6): 1168-1184. Moreno, E., et al. (1990). "Brucella abortus 16S rRNA and lipid A reveal a phylogenetic relationship with members of the alpha-2 subdivision of the class Proteobacteria." Journal of Bacteriology 172(7): 3569-3576. Morgan, W. J. (1984). "Brucella classification and regional distribution." Dev Biol Stand 56: 43-53. Scholz, H. a. V., G. (2013). "Molecular characterisation of Brucella species." Rev. Sci. Tech. Off. Int. Epiz 32(1): 149-162. Figure 3: Neighbor-joining tree from the distance matrix calculated from the variations and rooted it using Sample 2 as the out-group. ACKNOWLEDGMENTS The authors would like to thank the Research Core Facility & OMICS Research Unit (Grant no. SRUL02/13) for support to carry out this work and Dr. Farhat Habib, Scientist, IISER Pune, India for his guidance in data analysis. Figure 4: Circos representation of all the variants in all the samples. Figure1: Next generation sequencing technology