Journal: Frontiers in Veterinary Science
Article Title: High-throughput rapid amplicon sequencing for multilocus sequence typing of Mycoplasma ovipneumoniae from archived clinical DNA samples
doi: 10.3389/fvets.2024.1443855
Figure Lengend Snippet: Time and cost to complete Mycoplasma ovipneumoniae MLST workflows from PCR to final typing of sequences. To emphasize differences in hands-on time between methods, timeline steps are not drawn to scale. (A) Four genes are amplified using a single multiplex PCR and barcoded using Nanopore Rapid Barcoding library preparation. The subsequent library is sequenced using the minION device with a new or washed flow cell. (B) Four genes are amplified using a single multiplex PCR and barcoded using the Nanopore Native Barcoding library preparation. The subsequent library is sequenced using the minION device with a new or washed flow cell. All nanopore reads are trimmed and filtered to remove adapters and low-quality regions, and then reads are sorted by MLST loci. The resultant alignment is used to call a draft consensus and then polished to correct potential errors. (C) Four genes are amplified separately using a nested singleplex PCR assay with seven total reactions and prepared for sequencing using the Illumina 16S metagenomic sequencing library preparation with primers modified for the M. ovipneumoniae MLST scheme. A third-party laboratory sequenced the subsequent library with an Illumina MiSeq, 600-cycle flow cell. Illumina reads are trimmed and filtered and then aligned to the respective reference gene for consensus calling. (D) Four genes are amplified separately using a nested singleplex PCR assay with seven total reactions and sent for Sanger Sequencing by an offsite facility. All prices are per sample, including the cost of library preparation and flow cell, assuming multiplexed runs BioRender .
Article Snippet: ONT Experiment One determined the suitability of ONT Rapid Barcoding library preparation for multiplexed amplicons using ONT R9.4.1 flow cell, Rapid Barcoding library preparation kit, and flow cell wash kit (EXP-WSH004) (Oxford Nanopore Technologies, Oxford, UK).
Techniques: Amplification, Multiplex Assay, Sequencing, Modification
