High throughput rapid amplicon sequencing for multilocus sequence typing of M. ovipneumoniae using DNA obtained from clinical samples

Background Spillover events of Mycoplasma ovipneumoniae have devastating effects on wild bighorn sheep populations. Multilocus sequence typing (MLST), a common method for tracking bacterial lineages, is used to monitor spillover events and the spread of M. ovipneumoniae between populations. Most work involving M. ovipneumoniae typing has used Sanger sequencing, however, this technology is time consuming, expensive, and is not well suited to efficient batch sample processing. Our study aimed to develop and validate a workflow for multilocus sequence typing of M. ovipneumoniae using Nanopore Rapid Barcoding sequencing and multiplex PCR. We compare the workflow with Nanopore Native Barcoding library preparation and Illumina MiSeq amplicon protocols to determine the most accurate and cost-effective method for sequencing multiplex amplicons. Results A multiplex PCR was optimized for four housekeeping genes of M. ovipneumoniae using archived DNA samples from wild sheep. Sequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time, and lowest cost per sample when compared to Nanopore Native Barcoding, and Illumina MiSeq methods. Conclusion Our proposed workflow serves as a convenient and effective diagnostic method for strain typing of M. ovipneumoniae , and could be applied to other bacterial MLST schemes. The workflow is suitable for diagnostic settings where reduced hands-on time, cost and multiplexing capabilities are important.