Use of the Ion Torrent PGM for Determining the Genomic Sequences of Francisella and Coxiella-Like Endosymbionts and Rickettsia Directly from Hard Ticks

Abstract

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.