AUTHOR=Framst Isaac , Wolking Rebecca M. , Schonfeld Justin , Ricker Nicole , Beeler-Marfisi Janet , Chalmers Gabhan , Kamath Pauline L. , Maboni Grazieli 

TITLE=High-throughput rapid amplicon sequencing for multilocus sequence typing of Mycoplasma ovipneumoniae from archived clinical DNA samples

JOURNAL=Frontiers in Veterinary Science

VOLUME=11

YEAR=2024

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2024.1443855

DOI=10.3389/fvets.2024.1443855

ISSN=2297-1769

ABSTRACT=<sec><title>Introduction</title><p>Spillover events of <italic>Mycoplasma ovipneumoniae</italic> have devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread of <italic>M. ovipneumoniae</italic> between the sheep populations. Most studies involving the typing of <italic>M. ovipneumoniae</italic> have used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing.</p></sec><sec><title>Methods</title><p>Our study aimed to develop and validate an MLST workflow for typing of <italic>M. ovipneumoniae</italic> using Nanopore Rapid Barcoding sequencing and multiplex polymerase chain reaction (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. A multiplex PCR was optimized for four housekeeping genes of <italic>M. ovipneumoniae</italic> using archived DNA samples (<italic>N</italic> = 68) from nasal swabs.</p></sec><sec><title>Results</title><p>Sequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time and lowest cost per sample when compared with Nanopore Native Barcoding and Illumina MiSeq methods.</p></sec><sec><title>Discussion</title><p>Our proposed workflow is a convenient and effective method for strain typing of <italic>M. ovipneumoniae</italic> and can 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.</p></sec>