Adaptation ofandassays for diversity profiling by Illumina sequencing to identifyand.

Abstract

Bartonellosis is an emerging zoonotic disease caused by bacteria of the genus. Mixedinfections are a well-documented phenomenon in mammals and their ectoparasites. The accurate identification ofspecies in single and mixed infections is valuable, as differentspecies have varying impacts on infected hosts.Current diagnostic methods are inadequate at identifying thespecies present in mixed infections.The aim of this study was to adopt a Next Generation Sequencing (NGS) approach using Illumina sequencing technology to identifyspecies and demonstrate that this approach can resolve mixedinfections.We used Illumina PCR amplicon NGS to target theandgenes ofin fleas collected from cats, dogs and a hedgehog in Israel. We included artificially mixedsamples to demonstrate the ability for NGS to resolve mixed infections and we compared NGS to traditional Sanger sequencing.In total, we identified 74, twotwoand threefleas. Real-time PCR of a subset of 48 fleas revealed that twelve were positive for, all of which were cat fleas. Sanger sequencing of theandgenes confirmed the presence of,and. Illumina NGS ofandamplicons further confirmed thespecies identity in all 12 flea samples and unambiguously resolved the artificially mixedsamples.The adaptation and multiplexing of existing PCR assays for diversity profiling via NGS is a feasible approach that is superior to traditional Sanger sequencing forspeciation and resolving mixedinfections. The adaptation of other PCR primers for Illumina NGS will be useful in future studies where mixed bacterial infections may be present.