Computational protocol: Draft Genome Sequences of Three Strains of Ehrlichia ruminantium, a Tick-Borne Pathogen of Ruminants, Isolated from Zimbabwe, The Gambia, and Ghana

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Protocol publication

[…] Heartwater is a fatal disease of ruminants caused by an obligate intracellular bacterium Ehrlichia ruminantium. This rickettsial pathogen is transmitted by ticks of the genus Amblyomma () and is distributed in nearly all the countries of sub-Saharan Africa and on neighboring islands (). The disease has also become established on some islands of the Caribbean, to which infected ticks were introduced through the livestock trade. Although a high level of genetic diversity was found among the strains in Africa by several genotyping methods (, ), only a limited number of strains have been sequenced so far (, ).The following three E. ruminantium strains were sequenced in the present study: the Crystal Springs strain from Zimbabwe (), the Kerr Seringe strain from The Gambia (), and the Sankat 430 strain from Ghana (). All strains were cultured in bovine aorta endothelial cells. When cell monolayers showed 70 to 90% lysis, the supernatant containing the free elementary bodies (EBs) was collected and centrifuged at 20,000 × g for 30 min. The pellet containing the EBs was then resuspended in phosphate-buffered saline, and the solution was filtered through a 5-µm membrane filter (Millipore, Bedford, MA, USA). The filtrate was treated with DNase I to remove any contaminating host-cell DNA. Bacterial DNA was then extracted using the NucleoSpin Tissue XS kit (Macherey-Nagel, Düren, Gemany) according to the manufacturer’s instructions.The genomes were sequenced on the MiSeq platform (Illumina, San Diego, CA, USA) using a paired-end library with a 300-bp read length. After mapping the reads against each genome (phiX, Bos taurus, and Mycoplasma spp.) to remove contaminated data, de novo assembly was performed using Velvet version 1.2.10 () or CLC genomics workbench version 8.5.1 (Qiagen, Valencia, CA, USA). The assembled contigs were ordered to the Welgevonden (Erwo) strain using Mauve version 2.3.1 (). Draft genome sequences of strains Crystal Springs, Kerr Seringe, and Sankat 430 comprised 34, 118, and 183 contigs (>500 bp), respectively (). The estimated genome sizes ranged from approximately 1,454 to 1,481 kb, and the N50 statistics ranged from 13,071 to 80,453 bp. The G+C content of each genome was calculated to be 27.5%.Prediction of protein-coding sequences (CDSs) and annotation were performed by the Microbial Genome Annotation Pipeline (http://www.migap.org). The number of CDSs varied between 961 and 1,039 (). All three strains possess a complete set of the major antigenic protein (map1) gene family, which has been associated with bacterial adaptation to mammalian hosts and vector ticks (). The data presented here will facilitate comparative genomic analysis and expand our understanding of the genetic diversity of E. ruminantium circulating in the African continent, which is useful for the appropriate formulation of the vaccine against heartwater. […]

Pipeline specifications

Software tools Velvet, CLC Genomics Workbench, Mauve
Application Nucleotide sequence alignment
Organisms Ehrlichia ruminantium