High quality genome assemblies of Mycoplasma bovis using a taxon-specific Bonito basecaller for MinION and Flongle long-read nanopore sequencing

Abstract

Background Implementation of Third-Generation Sequencing approaches for Whole Genome Sequencing (WGS) all-in-one diagnostics in human and veterinary medicine, requires the rapid and accurate generation of consensus genomes. Over the last years, Oxford Nanopore Technologies (ONT) released various new devices (e.g. the Flongle R9.4.1 flow cell) and bioinformatics tools (e.g. the in 2019-released Bonito basecaller), allowing cheap and user-friendly cost-efficient introduction in various NGS workflows. While single read, overall consensus accuracies, and completeness of genome sequences has been improved dramatically, further improvements are required when working with non-frequently sequenced organisms like Mycoplasma bovis. As an important primary respiratory pathogen in cattle, rapid M. bovis diagnostics is crucial to allow timely and targeted disease control and prevention. Current complete diagnostics (including identification, strain typing, and antimicrobial resistance (AMR) detection) require combined culture-based and molecular approaches, of which the first can take 1–2 weeks. At present, cheap and quick long read all-in-one WGS approaches can only be implemented if increased accuracies and genome completeness can be obtained. Results Here, a taxon-specific custom-trained Bonito v.0.1.3 basecalling model (custom-pg45) was implemented in various WGS assembly bioinformatics pipelines. Using MinION sequencing data, we showed improved consensus accuracies up to Q45.2 and Q46.7 for reference-based and Canu de novo assembled M. bovis genomes, respectively. Furthermore, the custom-pg45 model resulted in mean consensus accuracies of Q45.0 and genome completeness of 94.6% for nine M. bovis field strains. Improvements were also observed for the single-use Flongle sequencer (mean Q36.0 accuracies and 80.3% genome completeness). Conclusions These results implicate that taxon-specific basecalling of MinION and single-use Flongle Nanopore long reads are of great value to be implemented in rapid all-in-one WGS tools as evidenced for Mycoplasma bovis as an example.

Antimicrobial Susceptibility of Mycoplasma bovis Isolates from Veal, Dairy and Beef Herds

“…Additionally, whether antimicrobial resistance is associated with specific genetic strains is not clear, as some studies observed no association between genetic subtypes based on the polC subtyping scheme [ 11 ], while others did for lincosamides and macrolides [ 9 ]. With whole genome sequencing (WGS) becoming more popular and commercially available for identification and strain typing [ 16 , 17 , 18 , 19 ], it might be helpful to determine whether phenotypic antimicrobial susceptibility patterns are associated with genomic clusters of M. bovis . Therefore, the objective of the present study was to compare the antimicrobial susceptibility results of M. bovis isolates obtained from veal calf, conventional dairy and beef herds, and to explore the association of antimicrobial resistance (AMR) with specific M. bovis genomic clusters.…”

Nanopore sequencing and its application to the study of microbial communities

“…However, both read and consensus accuracy are limited by the organisms chosen for model training, and they are drastically reduced when the basecaller is used for less frequently sequenced microbial species [140] . This issue can be addressed by developing more custom-trained basecallers, built-on taxon-specific training data so that the users can choose which basecaller most closely matches the organisms present in their samples [140] , [141] . Sequencing data throughput by single flow cell is also continuously improving, enabling the detection and sequencing of DNA/RNA from microbes even when present in very low abundance.…”

Nanopore sequencing provides rapid and reliable insight into microbial profiles of Intensive Care Units

“…Nonetheless, hospital environments such as NICUs are known to house several related species concurrently, and finding ways to accurately detect different related bacteria at species- or even strain-level is important for effective surveillance and diagnosis in healthcare settings (29,30). Recently developed tools like the Bonito basecaller promise to take on some of nanopore’s main limitations and may turn high-accuracy long-read sequencing into a reality within the next years (31).…”