Abstract
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.
Funder
Federaal Agentschap Voor de Veiligheid Van de Voedselketen
Publisher
Springer Science and Business Media LLC
Subject
Applied Mathematics,Computer Science Applications,Molecular Biology,Biochemistry,Structural Biology
Reference41 articles.
1. Leggett RM, Alcon-Giner C, Heavens D, Caim S, Brook TC, Kujawska M, et al. Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens. Nat Microbiol. 2020;5:430–42. https://doi.org/10.1038/s41564-019-0626-z.
2. Goldstein S, Beka L, Graf J, Klassen JL. Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing. BMC Genomics. 2019;20:1–17.
3. Golparian D, Donà V, Sánchez-Busó L, Foerster S, Harris S, Endimiani A, et al. Antimicrobial resistance prediction and phylogenetic analysis of Neisseria gonorrhoeae isolates using the Oxford Nanopore MinION sequencer. Sci Rep. 2018;8:1–12.
4. Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet. 2016;17:333–51.
5. Jain M, Tyson JR, Loose M, Ip CLC, Eccles DA, O’Grady J, et al. MinION Analysis and Reference Consortium: Phase 2 data release and analysis of R9.0 chemistry. F1000Research. 2017;6:1–18.
Cited by
34 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献