Introduction: Metagenomic sequencing offers the potential to explore the dynamics of complex microbial communities in wastewater systems for a wide variety of uses, including pathogen surveillance, monitoring the efficacy of the wastewater treatment chain in reducing risk of pathogen transmission, and exploring how toxic and nuisance microorganisms can interrupt wastewater treatment or the supply of recycled water products. However, the robust application of metagenomics in these complex and often poorly characterised communities presents technical and analytical challenges. Furthermore, translating metagenomic detection into a measure of public health or wastewater management risk is both essential and challenging.

Aims: We will discuss the experience and efforts in applying metagenomic sequencing and related methods to the surveillance of pathogens and other microorganisms in complex microbial communities, particularly in wastewater. These observations include insight into method sensitivity and specificity considerations, optimisation for use in complex samples, and a range of analytical considerations and quality control steps to support routine use and translation.

Methods: As a case example, we will present a metagenomic analysis examining seasonal and temporal changes in the composition of seasonal cyanobacterial blooms in wastewater treatment plants, as well as an analysis of how cyanobacterial blooms impact on the composition of markers of antimicrobial resistant pathogens. Additionally, we will draw on specific experiences and observations from our genomic surveillance for SARS-CoV-2 during the COVID-19 pandemic.

Outcomes: Our work highlights key optimisation steps in sample processing and sequencing that can significantly improve the sensitivity of environmental metagenomics or targeted genomic sequencing. We also identified important quality control thresholds to ensure high-quality data output. Finally, we identify critical points in analysing these data that can significantly impact the outcome.

Introduction: Targeted metagenomic sequencing using multi-pathogen enrichment offers a robust and cost-effective approach with clearly defined limits of detection for a very wide range of pathogens, including both DNA and RNA viruses, bacteria, and host markers. The power of the technique lies in co-detection of multiple pathogens simultaneously with no need for additional assay optimisation. In recent years, several targeted protocols have been made available by manufacturers, which provide both pre-designed and custom enrichment panels for targeted metagenomic sequencing.

Aims: In this presentation, we discuss applications of targeted mNGS to a range of clinical specimens, focusing particularly on clinical respiratory swabs. We would also present unique challenges and opportunities for understanding the dynamics of co-infecting and co-circulating pathogens.

Methods: We will be using the targeted mNGS approaches on respiratory swabs containing very large range of bacterial and viral pathogens.

Results: Using two case studies, we compare the application of an off-the-shelf protocol for respiratory viral panel for sequencing seasonal influenza A, and the design and analysis of a custom, powerful multi-pathogen method to examine the impact of both viral and bacterial co-infections on the severity of respiratory symptoms in infants with RSV.

Outcomes: We will address some approaches for effective and robust design of custom enrichment panels and for analysis of targeted enrichment data.​

Introduction: Neisseria gonorrhoeae (Ng) is concerning for sexual and public health professionals due to its systematic development of resistance to most classes of antibiotic. More information on the genomic epidemiology of Ng in Australia is vital in understanding how these drug-resistant strains are entering and circulating in the community. However, due to their high prevalence and culture availability, where only 20% of all Ng cases in Australia have a corresponding isolate, not all Ng cases can have antimicrobial susceptibility testing and whole genome sequencing performed. While PCR screening can be utilised, constant evolution of resistance mechanisms makes this difficult to implement and maintain for public health laboratories. It is possible that pathogen-agnostic metagenomics-based approaches might be able to address this surveillance gap in a cost-effective and efficient manner.

Aims: To develop a cost-effective method that would enable large-scale sequencing of Ng isolates and potentially nucleic acid positive clinical specimens in order to collect AMR gene and typing information.

Methods: We utilised targeted mNGS (amplicon and bait-based sequencing techniques) developed by our laboratory to perform epidemiological typing of Queensland Ng samples. These methods were used to examine population genetics such as long-term epidemiology (MLST), mid-term epidemiology (NG-MAST) and resistance typing (NG-STAR).

Outcomes: The ability to perform large scale sequence-based analysis on Ng isolates and clinical specimens from across Queensland allowed the identification of specific clones circulating in the community and was able to provide important information on AMR resistance genes present. This approach has the potential to expand the molecular tools available for public health laboratories to manage AMR surveillance, potentially across the majority of cases regardless of diagnostic approach.​

Introduction: Metagenomics is a rapidly evolving innovation with the need for continuous discussion around new techniques, best practice methods and future applications. To keep up with the dynamic and differing nature of this work, open discussions and building an understanding of jurisdictional methods are imperative to the long-term use of metagenomics.

Aims: To understand the different methods and challenges faced by laboratories across Australia, as well as the opportunities in metagenomics.

Methods: A panel discussion consisting of key stakeholders with expertise in bacterial, viral and environmental metagenomics will explore several topics: 1. The various techniques and methods used by different labs and their set-up; 2. How to navigate short falls and incidental findings from these technologies; 3. The utility of mNGS in the state-level or national level, and a foreseeable impact to the integration of mNGS into surveillance system. The panel will be interactive, with opportunities for the audience to engage with, and provide feedback to the panel to support an active discussion.

Outcomes: At the conclusion of this session, the audience will have developed a better understanding of the capability and application of metagenomic sequencing for public health purposes, and its caveats.

​