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IntroductionSpillover events ofMycoplasma ovipneumoniaehave devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread ofM. ovipneumoniaebetween the sheep populations. Most studies involving the typing ofM. ovipneumoniaehave used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing. MethodsOur study aimed to develop and validate an MLST workflow for typing ofM. ovipneumoniaeusing 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 ofM. ovipneumoniaeusing archived DNA samples (N= 68) from nasal swabs. ResultsSequences 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. DiscussionOur proposed workflow is a convenient and effective method for strain typing ofM. ovipneumoniaeand 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.
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Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale
Abstract BackgroundIn light of the current biodiversity crisis, DNA barcoding is developing into an essential tool to quantify state shifts in global ecosystems. Current barcoding protocols often rely on short amplicon sequences, which yield accurate identification of biological entities in a community but provide limited phylogenetic resolution across broad taxonomic scales. However, the phylogenetic structure of communities is an essential component of biodiversity. Consequently, a barcoding approach is required that unites robust taxonomic assignment power and high phylogenetic utility. A possible solution is offered by sequencing long ribosomal DNA (rDNA) amplicons on the MinION platform (Oxford Nanopore Technologies). FindingsUsing a dataset of various animal and plant species, with a focus on arthropods, we assemble a pipeline for long rDNA barcode analysis and introduce a new software (MiniBar) to demultiplex dual indexed Nanopore reads. We find excellent phylogenetic and taxonomic resolution offered by long rDNA sequences across broad taxonomic scales. We highlight the simplicity of our approach by field barcoding with a miniaturized, mobile laboratory in a remote rainforest. We also test the utility of long rDNA amplicons for analysis of community diversity through metabarcoding and find that they recover highly skewed diversity estimates. ConclusionsSequencing dual indexed, long rDNA amplicons on the MinION platform is a straightforward, cost-effective, portable, and universal approach for eukaryote DNA barcoding. Although bulk community analyses using long-amplicon approaches may introduce biases, the long rDNA amplicons approach signifies a powerful tool for enabling the accurate recovery of taxonomic and phylogenetic diversity across biological communities.
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- Award ID(s):
- 1927510
- PAR ID:
- 10555364
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- GigaScience
- Volume:
- 8
- Issue:
- 5
- ISSN:
- 2047-217X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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