Ryan Robert Wick

Oxford Nanopore Technologies (ONT) sequencing is widely used in bacterial genomics, particularly for reference-based variant calling and de novo assembly. Both analyses routinely start with quality control (QC) preprocessing, but as ONT basecalling accuracy has improved, its necessity and downstream trade-offs remain poorly defined. We benchmarked 17 preprocessing combinations, built from three adapter trimmers (Dorado, Porechop ABI, Barbell) and three quality-based processing tools (Filtlong, Chopper, SeqKit) with untrimmed and unprocessed controls, plus Fastplong, which performs both. Each combination was evaluated across 13 bacterial isolates at 100× and 20× depth under the HAC and SUP basecalling models. Variant calling was assessed by F1 score. Assemblies were scored on sequence accuracy, contiguity, replicon recovery and residual adapter contamination, then aggregated into a weighted arithmetic mean using weights from a survey of the microbial genomics community. With SUP basecalling, raw reads with no trimming or quality-based processing produced accurate SNP calls and outperformed several pipelines that applied both operations; adapter trimming alone left SNP accuracy unchanged. Subsetting reads by combined length and quality with Filtlong gave the highest SNP F1 scores at both depths, except with Barbell at 20×, where it ranked second lowest. As is often the case in benchmarking studies, assembly results involved trade-offs, and the best tool depends on which aspects of performance matter most for a given use. Filtlong produced the lowest error rates, but every Filtlong pipeline lost three small plasmids at both depths. Barbell trimming raised error rates and reduced contiguity at 20×, while skipping adapter trimming left residual adapter and barcode sequences in assemblies. Under the community-derived weights, Porechop ABI followed by Chopper had the highest overall score at both depths, without winning any single metric. QC tool choice should therefore be guided by the downstream analysis goal rather than applied indiscriminately across all workflows. Author summary: Oxford Nanopore reads are routinely processed to remove adapter and barcode sequences introduced during library preparation and discard low-quality reads or read segments. As sequencing accuracy improves, the benefits and costs of these steps need reassessment. We compared 17 preprocessing combinations across 13 bacterial isolates for detecting genetic differences (variant calling) and reconstructing genomes (genome assembly). Most combinations produced similar results to unprocessed reads, but some affected the genetic information recovered. Filtlong, which selects reads by length and quality, improved detection of single-nucleotide variants in several combinations and reduced assembly errors. However, it removed reads supporting three small plasmids, preventing their assembly. Missing small plasmids in bacterial assemblies can have a large impact, as they are known to carry important genes (e.g., antibiotic resistance). Adapter trimming offered little benefit for single-nucleotide variant detection, but omitting it left adapter and barcode sequences in some assemblies. These findings support choosing preprocessing tools according to the intended analysis. We surveyed researchers to weight four aspects of assembly quality: sequence accuracy, contiguity, chromosome and plasmid recovery, and residual adapter contamination. Our interactive dashboard lets users adjust these weights to compare pipelines according to their own priorities.

Linezolid is a critical last-resort antimicrobial for multidrug-resistant Enterococcus faecium , particularly against vancomycin-resistant lineages where therapeutic options are severely limited. While resistance has historically arisen through de novo chromosomal mutations, the global emergence of transferable resistance mechanisms threatens to render more infections untreatable. Here, we characterize a recent (2023–2024) hospital-associated outbreak of linezolid-resistant E. faecium in Queensland, Australia. Although the cohort comprised a variety of sequence types, the outbreak was primarily driven by the clonal expansion of an ST80 lineage carrying the plasmid-borne poxtA gene. Standard short-read genomic surveillance failed to resolve the genetic context of the resistance determinant. However, long-read sequencing revealed that poxtA was carried within a novel transposon, Tn 8026 , situated on a linear plasmid. Structural analysis defined Tn 8026 as a unique element flanked by IS 1678 and the novel insertion sequence IS Efa26 . Furthermore, we identified an instance of Tn 8026 integration into the chromosome, providing functional evidence of its mobility and capacity for stabilization within the genome. Global genomic screening demonstrated that Tn 8026 significantly predates the local outbreak, identified in a historical Norwegian isolate from 2012, indicating a long-standing yet unrecognized global reservoir. Phylogenomic analysis provided strong evidence that the linear plasmid was imported from the Indian subcontinent, initiating a chain of silent dissemination in eastern Australia where the lineage circulated undetected prior to clinical recognition. Crucially, we also confirmed the presence of the linear plasmid in Enterococcus gallinarum , demonstrating its capacity to mobilize transmissible linezolid resistance across enterococcal species boundaries. These findings emphasize the need for detailed long-read-based surveillance of mobile genetic elements, with a particular focus on identifying linear plasmids that are often overlooked.

Background Microbial colonisation of the human body begins immediately after birth, with each body site forming a distinct ecological niche. While early gut microbial dynamics and their links to paediatric health have been considerably studied, the bacterial colonisation of the early infant respiratory system remains poorly understood, particularly at the species and strain level. Results Here, we generated and analysed whole genome sequencing of 925 isolates from six dominant genera in nasopharyngeal samples from 58 healthy infants enrolled in the Childhood Asthma Study (CAS) in Western Australia, collected longitudinally from birth (2, 6, and 12 months old). Our results expanded genomic reference catalogues and uncovered substantial strain-level diversity in dominant infant airway taxa. Plate-sweep metagenomics identified four microbiome profile groups (MPGs) with age-dependent membership, confirming prior observations while enabling high-resolution species and strain analyses. Community maturation was characterised by a shift from early Staphylococcus aureus dominance to increased Moraxella catarrhalis dominance by 12 months of age, alongside marked temporal changes in prevalence and cohort-level strain diversity. Conclusions These findings resolve infant nasopharyngeal microbiota composition at the species and strain level, revealing taxon-specific colonisation patterns. By substantially expanding publicly available reference genomes for underrepresented airway taxa, this work also provides a foundation for functional follow-up studies of the early respiratory microbiota and respiratory outcomes. Keywords : airway microbiome; metagenomics; paediatric; Moraxella ; Staphylococcus ; Streptococcus ; Corynebacterium ; Rothia

Background: Oxford Nanopore Technologies (ONT) sequencing is increasingly used for whole-genome sequencing (WGS) across a wide range of applications. However, the platform has evolved rapidly through updates to flow cell chemistry and basecalling algorithms, altering the characteristics of the resulting sequencing data. Read simulators provide synthetic datasets with known ground truth, enabling controlled development and evaluation of methods. However, many existing simulators were developed for earlier versions of ONT sequencing or use generic long-read assumptions, and their realism for contemporary ONT data is unclear. Results: We benchmarked six ONT-compatible read simulators (Badread, LongISLND, lrsim, NanoSim, PBSIM3 and SimLoRD) using a microbial genome reference and ONT R10.4.1 reads as the empirical standard. Each tool was configured to maximise realism, including training on empirical reads when supported. We compared simulated and real datasets with respect to read length, read accuracy, FASTQ quality scores and sequence error profiles. No simulator reproduced all metrics of the real data well. PBSIM3 most closely reproduced read length, read accuracy and FASTQ quality scores, making it a strong simulator for broad read-level realism. However, it did not capture important features of the real error profile, including context-dependent substitution rates and homopolymer-length errors. Badread and LongISLND better reproduced some aspects of the error profile, but showed other departures from the real data. Conclusion: PBSIM3 is a good general-purpose choice for many ONT WGS simulation tasks because it reproduced several key read-level properties well. However, Badread or LongISLND may be preferable for applications where error structure is more important. No evaluated tool was realistic across all tested metrics, highlighting a gap for improved long-read simulators.

Shigella flexneri is the leading causative agent of shigellosis globally. The public health threat posed by S. flexneri is compounded by its emergence as a sexually transmissible infection, importance of international travel in driving dissemination, and the increasing prevalence of antimicrobial resistance (AMR). A rapid and robust computational method is needed to enhance genomic surveillance and systematically explore features of the population structure of this WHO priority pathogen, which is scalable and readily implementable across jurisdictions, particularly as vaccine development efforts are underway. Here, we present Flex-It, a genomic framework and genotyping scheme implemented in Mykrobe for S. flexneri serotypes 1-5, X & Y, compatible with previous approaches used to describe S. flexneri's population structure. To develop Flex-It, we curated a retrospective dataset of 5,819 publicly available S. flexneri genomes. We characterised the global population structure for S. flexneri, exploring geographical and temporal traits, and showed the granular diversity of AMR and serotype profiles. We applied Flex-It to >13,000 genomes routinely generated by public health laboratories from Australia, the UK and the USA across a ten-year period. We found significant genotype diversity in all three locations, with the emergence of genotypes with converged resistance to all major drugs currently used for treatment. Flex-It provides an open-source, novel genotyping method that rapidly characterises S. flexneri and its ciprofloxacin resistance determinants in <1 minute from both short and long whole-genome sequencing reads. Flex-It provides the community with a standardised nomenclature to monitor the emergence and spread of S. flexneri lineages.

Linezolid is a critical last-resort antimicrobial for multidrug-resistant Enterococcus faecium , particularly against vancomycin-resistant lineages where therapeutic options are severely limited. While resistance has historically arisen through de novo chromosomal mutations, the global emergence of transferable resistance mechanisms threatens to render more infections untreatable. Here, we characterise a recent (2023–2024) hospital-associated outbreak of linezolid-resistant E. faecium in Queensland, Australia. Although the cohort comprised a variety of sequence types, the outbreak was primarily driven by the clonal expansion of an ST80 lineage carrying the plasmid-borne poxtA gene. Standard short-read genomic surveillance failed to resolve the genetic context of the resistance determinant. However, long-read sequencing revealed that poxtA was carried within a novel transposon, Tn 8026 , situated on a linear plasmid. Structural analysis defined Tn 8026 as a unique element flanked by IS 1678 and the novel insertion sequence IS Efa26 . Furthermore, we identified an instance of Tn 8026 integration into the chromosome, providing functional evidence of its mobility and capacity for stabilisation within the genome. Global genomic screening demonstrated that Tn 8026 significantly predates the local outbreak, identified in a historical Norwegian isolate from 2012, indicating a long-standing yet unrecognised global reservoir. Phylogenomic analysis provided strong evidence that the linear plasmid was imported from the Indian subcontinent, initiating a chain of silent dissemination in eastern Australia where the lineage circulated undetected prior to clinical recognition. Crucially, we also confirmed the presence of the linear plasmid in Enterococcus gallinarum , demonstrating its capacity to mobilise transmissible linezolid resistance across enterococcal species boundaries. These findings emphasise the need for detailed long-read-based surveillance of mobile genetic elements, with a particular focus on identifying linear plasmids that are often overlooked.

Motivation: Assembly graphs are a fundamental data structure used by genome and metagenome assemblers to represent sequences and their overlap information, facilitating the assembler in constructing longer genomic fragments. Apart from their core use in assemblers, assembly graphs have become increasingly important in a range of downstream applications such as metagenomic binning, plasmid detection, viral genome resolution, and haplotype phasing. However, there is a need for a comprehensive tool that allows programmatic access to manipulate assembly graphs (e.g. parse, convert, filter, and analyze) across different assembly graph formats. Results: Here we present agtools, an open-source Python framework to manipulate assembly graphs produced by commonly used assemblers. agtools provides a command-line interface for tasks such as assembly graph format conversion, segment filtering, and component extraction. It also exposes a Python package interface to load, query, and analyze assembly graphs from popular genome and metagenome assemblers. This enables streamlined assembly-graph-based analyses that can be integrated into other bioinformatics software and workflows. Availability and implementation: The source code of agtools is hosted on GitHub at https://github.com/Vini2/agtools and the documentation is available at https://agtools.readthedocs.io/. agtools can also be installed from Bioconda (https://anaconda.org/bioconda/agtools) and PyPI (https://pypi.org/project/agtools/).

Members of the family Rhizobiaceae typically have multipartite genomes that are split between two or more replicons, including the chromosome and a variable number of extrachromosomal replicons (chromids and plasmids). Nearly all Rhizobiaceae replicons sequenced and described to date have a circular topology, except the linear chromid found in the genomes of most Agrobacterium spp. In this study, genomes of five non-pathogenic Agrobacterium strains and one plant tumourigenic Allorhizobium strain were fully sequenced. Surprisingly, genome analysis revealed that these six strains each carry an 80-kbp linear plasmid. Linear plasmids have so far not been identified in this bacterial family or other bacteria within the class Alphaproteobacteria . The ends of all six plasmids identified in this study have a hairpin structure with covalently closed ends. The plasmid sequences showed a high degree of homology, clearly indicating their common ancestry. Database searches led to the identification of additional linear plasmids in previously published Rhizobiaceae genome assemblies that were not previously recognized to have linear plasmids, suggesting that these replicons may be more widespread. Most likely, linear plasmids may be even more widely distributed than anticipated. Although the biological functions of the linear plasmids identified in this study remain unknown, they are associated with both non-pathogenic and plant tumourigenic Rhizobiaceae strains.

Motivation: Long-read sequencing enables complete bacterial genome assemblies, but individual assemblers are imperfect and often produce sequence-level and structural errors. Consensus assembly using Trycycler can improve accuracy, but its lack of automation limits scalability. There is a need for an automated method to generate high-quality consensus bacterial genome assemblies from long-read data. Results: We present Autocycler, a command-line tool for generating accurate bacterial genome assemblies by combining multiple alternative long-read assemblies of the same genome. Without requiring user input, Autocycler builds a compacted De Bruijn graph from the input assemblies, clusters and filters contigs, trims overlaps and resolves consensus sequences by selecting the most common variant at each locus. It also supports manual curation when desired, allowing users to refine assemblies in challenging or important cases. In our evaluation using Oxford Nanopore Technologies reads from five bacterial isolates, Autocycler outperformed individual assemblers, automated pipelines and other consensus tools, producing assemblies with lower error rates and improved structural accuracy. Availability and implementation: Autocycler is implemented in Rust, open-source and freely available at github.com/rrwick/Autocycler. It runs on Linux and macOS and is extensively documented. Supplementary information: Supplementary figures, tables and methods are available at github.com/rrwick/Autocycler-paper and archived on Zenodo (doi: 10.5281/zenodo.16916187). Assemblies, reference genomes and read sets used in the analysis are available at figshare.unimelb.edu.au/projects/Autocycler/247142.

Linezolid is an oxazolidinone antibiotic and is a last-line agent for treating infections caused by vancomycin-resistant Enterococcus (VRE). Limited work has been done to study the genomic epidemiology and population structure of linezolid-resistant Enterococcus (LRE) spp. in Victoria, Australia, and globally. We aimed to elucidate the genomic epidemiology and population structure of LRE in Victoria, Australia. We screened our local collection of Enterococcus faecalis and Enterococcus faecium for LRE from 2015 to 2023 and identified 15/349 (4.3 %) and 26/9,207 (0.28 %) E. faecalis and E. faecium, respectively, which had either or both phenotypic and genotypic resistance to linezolid. We next contextualized the Victorian LRE isolates with a global set of 684 E. faecalis and 324 E. faecium genomes from public databases. Integrated phylogenetic and epidemiologic data suggested that the LRE isolates in Victoria were likely introduced through multiple independent events from international travellers, with evidence of putative linear plasmids co-carrying vanA and linezolid resistance genes. We observed a predominance of the linezolid resistance-conferring optrA gene in E. faecalis irrespective of source origin in both the global and Victorian collections, whereas in E. faecium, cfr and cfr(B) occurred more frequently among clinical isolates, with no apparent geographical signatures. Our study provides a genomic snapshot of a large global collection of LRE isolates and establishes the epidemiological context for LRE circulating in Victoria, Australia.