Use of the Ion Torrent PGM for Determining the Genomic Sequences of Francisella and Coxiella-Like Endosymbionts and Rickettsia Directly from Hard Ticks
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Microbiome assessments based on amplification and sequencing of their 16S rRNA genes or targeted surveys of specific agents have greatly augmented our understanding of the distribution and identity of bacteria found in ticks. The most prevalent tick bacteria include approximately 10 species, such as Coxiella-like (CLE) and Francisella-like (FLE) endosymbionts, and spotted fever group Rickettsia (SFG). However, genomes of only a few of the many tick agents that are not cultivable have been characterized by very deep next generation sequencing (Illumina HiSeq). As an alternative approach, we have assessed use of the Ion Torrent Personal Genome Machine (IT-PGM) for direct sequencing and assembly of these agent genomes. Agents were identified and quantitated in three genera of ticks (Amblyomma, Dermacentor, Rhipicephalus) by qPCR assays. Geneious, CLC Genomics Workbench, BWA, and SPAdes tools were used for read mapping and assembly of the sequences obtained from size-fractionated libraries made from DNA that was extracted from single alcohol-preserved ticks on single 318 or 314 IT-PGM chips with 200 bp chemistry. Plasmid and chromosome sequences were obtained for six SFGs, four FLEs, and three CLEs. The depth and percentage of genome coverage for symbionts and Rickettsia were enhanced by use of Qiagen Repli-G amplification of the DNA used for the libraries. The IT-PGM is a relatively inexpensive sequencing platform for initial genomic characterization of some of the abundant bacterial agents found in hard ticks.
KeywordsIon torrent PGM Hard ticks Endosymbiont Rickettsia Coxiella Francisella
The authors thank the following individuals for providing some of the individual ticks used in this study from other previous collaborative investigations: Sarah Billeter, Renjie Hu, Alece Powery, Jasmine Hensley, Lindsey Killmaster, and Amanda Jo Williams-Newkirk. We also thank Lisa Coburn, manager of the Oklahoma State University Tick Laboratory, for providing details on the origin of ticks from their tick colonies which are supported in part by NIAID BEI Resources. This research was supported in part by appointment of Arunachalam Ramaiah in the Postdoctoral Bioinformatics Fellowship Program administered by the Association of Public Health Laboratories (APHL) and funded by the Centers for Disease Control and Prevention (CDC) as part of Cooperative Agreement 1U60OE000103 (CFDA No. 93.322). The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the U.S. Centers for Disease Control and Prevention.
The Ion Torrent PGM sequence data for each sample (Table 1.1) is available at the NCBI SRA resources site under Bioproject numbers PRJNA408163 (Rhipicephalus sanguineus F23UF), PRJNA413819 (Dermacentor andersoni M9), PRJNA413914 (Dermacentor occidentalis EMCF9), PRJNA413920 (Amblyomma americanum Aam10 and Aam46), PRJNA413923 (Amblyomma maculatum Amac21, Amac50), and PRJNA413929 (Dermacentor variabilis Dvar22, Dvar200, DvarSlov, DvarF4M4, DvarFvSC4, and DvarF1SC1).
Conflict of Interest Statement
The authors declare they have no conflicts of interest affecting this work.
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