Dataset Information

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

ABSTRACT:

Introduction

Spillover events of Mycoplasma ovipneumoniae have devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread of M. ovipneumoniae between the sheep populations. Most studies involving the typing of M. ovipneumoniae have used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing.

Methods

Our study aimed to develop and validate an MLST workflow for typing of M. ovipneumoniae 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 M. ovipneumoniae using archived DNA samples (N = 68) from nasal swabs.

Results

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.

Discussion

Our proposed workflow is a convenient and effective method for strain typing of M. ovipneumoniae 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.

SUBMITTER: Framst I

PROVIDER: S-EPMC11322507 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

High-throughput rapid amplicon sequencing for multilocus sequence typing of <i>Mycoplasma ovipneumoniae</i> from archived clinical DNA samples.

Framst Isaac I Wolking Rebecca M RM Schonfeld Justin J Ricker Nicole N Beeler-Marfisi Janet J Chalmers Gabhan G Kamath Pauline L PL Maboni Grazieli G

Frontiers in veterinary science 20240731

<h4>Introduction</h4>Spillover events of <i>Mycoplasma ovipneumoniae</i> have devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread of <i>M. ovipneumoniae</i> between the sheep populations. Most studies involving the typing of <i>M. ovipneumoniae</i> have used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing.<h4>Methods</h4>Our stu ...[more]

PMID: 39144078

Similar Datasets

Project description:ObjectivesIn this cross-sectional epidemiological study we aimed to identify molecular profiles for Trichomonas vaginalis and to determine how these molecular profiles were related to patient demographic and clinical characteristics.SettingMolecular typing methods previously identified two genetically distinct subpopulations for T. vaginalis; however, few molecular epidemiological studies have been performed. We now increased the sensitivity of a previously described multilocus sequence typing (MLST) tool for T. vaginalis by using nested PCR. This enabled the typing of direct patient samples.ParticipantsFrom January to December 2014, we collected all T. vaginalis positive samples as detected by routine laboratory testing. Samples from patients either came from general practitioners offices or from the sexually transmitted infections (STI) clinic in Amsterdam. Epidemiological data for the STI clinic patients were retrieved from electronic patient files.Primary and secondary outcome measuresThe primary outcome was the success rate of genotyping direct T. vaginalis positive samples. The secondary outcome was the relation between T. vaginalis genotypes and risk factors for STI.ResultsAll 7 MLST loci were successfully typed for 71/87 clinical samples. The 71 typed samples came from 69 patients, the majority of whom were women (n=62; 90%) and half (n=34; 49%) were STI clinic patients. Samples segregated into a two population structure for T. vaginalis representing genotypes I and II. Genotype I was most common (n=40; 59.7%). STI clinic patients infected with genotype II reported more sexual partners in the preceding 6 months than patients infected with genotype I (p=0.028). No other associations for gender, age, ethnicity, urogenital discharge or co-occurring STIs with T. vaginalis genotype were found.ConclusionsMLST with nested PCR is a sensitive typing method that allows typing of direct (uncultured) patient material. Genotype II is possibly more prevalent in high-risk sexual networks.