What You're Missing Matters: Advancing Infectious Disease/ Microbiology & Genomics Across the Nordics & Baltics

Over the past decade, Oxford Nanopore Technologies (ONT) sequencing has evolved from an emerging technology to a valuable tool for clinical microbiology in Sweden. Through Genomic Medicine Sweden (GMS), clinical microbiology laboratories across the country have collaborated to develop harmonized methods, shared infrastructure, and national bioinformatics solutions supporting large-scale implementation.

This presentation will describe the Swedish GMS Nanopore journey, from early adoption to nationwide implementation. A major milestone was the first national multicentre evaluation of Nanopore-based 16S rRNA gene sequencing, involving laboratories across Sweden. Within only two months, participating laboratories established the protocol, generated sequencing data, and contributed to a common analytical framework, demonstrating the power of coordinated national collaboration.

The talk will further highlight the potential of Nanopore-based methods to be introduced into routine diagnostic workflows, expanding the clinical application of long-read whole-genome sequencing for pathogen surveillance and outbreak detection, with a particular focus on MRSA and ESBL-producing Klebsiella pneumoniae. Shared national analysis pipelines and visualization tools enable harmonized genomic epidemiology, antimicrobial resistance profiling, and outbreak investigations across regions.

Finally, an overview of the national bioinformatics infrastructure developed within GMS will be presented, illustrating how collaborative development and data sharing can accelerate clinical implementation, strengthen precision microbiology, and improve preparedness for future infectious disease threats.

Oxford Nanopore Technologies (ONT) has emerged as a versatile sequencing platform with applications spanning public health, clinical microbiology, and infectious disease research. At Akershus University Hospital, nanopore sequencing is being applied across a range of use cases, from genomic surveillance and outbreak investigations to microbial identification and metagenomics.

In this presentation, we will share our experience implementing and utilizing nanopore sequencing in a hospital microbiology setting. Examples will include whole-genome sequencing (WGS) for outbreak investigations and pathogen surveillance, illustrating how rapid sequencing can support infection prevention and control efforts. We will further highlight the use of metagenomic next-generation sequencing (mNGS) in research projects exploring new approaches to infectious disease diagnostics and precision medicine. In addition, we will briefly touch upon the ongoing implementation of full-length 16S rRNA gene sequencing for microbial identification.

By highlighting diverse applications from a single institution, this presentation aims to demonstrate how a single sequencing platform can address multiple needs across microbiology, infection prevention and control, and translational research.

In our laboratory, 16S rRNA gene Sanger-sequencing has been the standard diagnostic method for culture-negative bacterial infections. However, this method is limited in its ability to detect polymicrobial infections, and may not provide sufficient taxonomic resolution. To address these limitations, we established and evaluated a more cost-effective workflow that leverages sequencing of the nearly complete 16S rRNA gene using nanopore-based technology combined with an in-house reads-to-results pipeline. Comparison of Sanger and nanopore sequencing results from 174 samples showed higher sensitivity and improved detection of polymicrobial infections in a clinical setting.

In this talk, we will describe how we have optimized various parameters to improve the workflow and reduce costs. To aid in identifying environmental contaminants and contamination across samples, negative controls are included for all sample materials during DNA extraction and a negative PCR template control is included in each PCR setup. Sequencing time has been shortened from 16 hours to 4 hours or until 1.2 Gb data has been generated, whichever occur first. Switching to P2i flow cells has further reduced sequencing time. In addition, we have lowered our costs per sample by optimizing flowcell re-use.

We have also implemented a bioinformatic pipeline based on established tools to analyze the results of our optimized sequencing workflow. This pipeline, which is publicly available, can easily be started by non-bioinformaticians. It provides detailed output that can integrate sample metadata for interpretation by a microbiologist and a molecular biologist to accommodate the increased complexity introduced by increased resolution and the ability to differentiate polymicrobial results.

In our laboratory, 16S rRNA gene Sanger-sequencing has been the standard diagnostic method for culture-negative bacterial infections. However, this method is limited in its ability to detect polymicrobial infections, and may not provide sufficient taxonomic resolution. To address these limitations, we established and evaluated a more cost-effective workflow that leverages sequencing of the nearly complete 16S rRNA gene using nanopore-based technology combined with an in-house reads-to-results pipeline. Comparison of Sanger and nanopore sequencing results from 174 samples showed higher sensitivity and improved detection of polymicrobial infections in a clinical setting.

In this talk, we will describe how we have optimized various parameters to improve the workflow and reduce costs. To aid in identifying environmental contaminants and contamination across samples, negative controls are included for all sample materials during DNA extraction and a negative PCR template control is included in each PCR setup. Sequencing time has been shortened from 16 hours to 4 hours or until 1.2 Gb data has been generated, whichever occur first. Switching to P2i flow cells has further reduced sequencing time. In addition, we have lowered our costs per sample by optimizing flowcell re-use.

We have also implemented a bioinformatic pipeline based on established tools to analyze the results of our optimized sequencing workflow. This pipeline, which is publicly available, can easily be started by non-bioinformaticians. It provides detailed output that can integrate sample metadata for interpretation by a microbiologist and a molecular biologist to accommodate the increased complexity introduced by increased resolution and the ability to differentiate polymicrobial results.

Clinical metagenomics has been providing benefit to patients for the last decade, with direct from blood identification of all major types of human pathogens. The ability to identify microbial cell free DNA (mcfDNA) in a vast excess of human DNA background (99.9%) has been mainly facilitated by deep sequencing levels. This has meant a need for larger sequencing instruments and centralized lab-solutions, reducing the positive effect on patient out-comes as sample transfer often results in time to ID well above 24 hrs. However, with the advent of table-top rapid continuous sequencers distributed high yield sequencing has become common place. To facilitate the usage of these instruments for clinical metagenomics we have developed a fully agnostic microbial cfDNA enrichment concept, SepTRACE, that coupled with a fully automated bioinformatic pipeline (TRACER.ID) allows identification of any pathogen in 6-8 hrs from < 1mL of whole blood, directly at the hospital. We have applied this method across a group of high need patient groups, fibril neutropenic cancer, paediatric joint infections, neonates and vascular endograft infections (VEGI) to show case applicability. In general the applied method shows sensitivity gains, enabling identification of chronic low yield polymicrobial infections, detects EBV virus levels prior to cancer development and mcfDNA derived from difficult to sample compartments.

Bloodstream infections (BSIs) and sepsis remain leading causes of death, and survival depends on early, effective antimicrobial therapy. Yet blood culture, the diagnostic gold standard, takes around two days and misses the causative pathogen in more than half of patients with clinical sepsis. We therefore developed an assay using real-time Oxford Nanopore sequencing to detect and quantify microbial cell-free DNA directly in plasma, with a potential turnaround of about 6 hours.

In a prospective study in the emergency ward at Aalborg University Hospital, we analysed plasma from 40 patients with suspected BSI, drawn alongside routine blood cultures. Nanopore sequencing detected all pathogens found in the 11 culture-positive patients and identified clinically relevant pathogens in 11 additional culture-negative patients, doubling the diagnostic yield. Spike-in standards enabled absolute quantification of pathogen DNA, and no pathogens were detected in patients without suspected infection. In a retrospective assessment, results could have altered antimicrobial treatment in 13 of 22 patients, including five changes from ineffective to effective therapy.

I will present the assay, our clinical findings and the challenge of interpreting culture-negative detections, and discuss next steps towards clinical implementation.

In recent years, long-read whole-genome sequencing with Oxford Nanopore Technologies (ONT) has become an increasingly accessible tool for genomic epidemiology. Implementing it across many laboratories, however, requires methods that are simple, robust and reproducible between sites.

This presentation will describe the building of a standard operating procedure (SOP) for sequencing of Staphylococcus aureus and Klebsiella pneumoniae in a national study, covering both wet-lab and bioinformatic optimisation. The SOP was designed for simplicity, with on-site quality control limited to DNA quantification, placing high demands on the robustness of each step from DNA extraction to sequencing.

The talk will further highlight how data analysis became the central quality safeguard. With quality assessable only after sequencing, data-based metrics were used to accept or reject isolates and to trace wet-lab problems back to their source. The impact of basecalling, assembly and typing choices on cgMLST and SNP-based results, compared with short-read data, will also be discussed.

Finally, the struggles encountered along the way will be presented together with tips on how to avoid them, illustrating how careful protocol design and data-driven quality control can turn the challenges of a multicentre SOP into possibilities for reliable nanopore-based surveillance.

Bacterial diversity is usually measured as differences in DNA sequence. Yet bacteria also carry an epigenetic layer: methyl groups added to specific DNA motifs by methyltransferases. Methylation underpins restriction-modification systems, a major defence against bacteriophages, and can also regulate gene expression. Until recently, methylomes were too costly to obtain at scale. Nanopore long-read sequencing has changed this: a single dataset now yields both a complete genome and a methylome (6mA, 4mC, 5mC) from native reads. We applied this approach to traditional Danish dairy starter cultures, simple communities of four to five lactic acid bacterial species that have co-evolved with their phages for decades. From two cultures we isolated ~400 strains and obtained complete genomes and methylomes of ~100 non-redundant strains. Diversity proved even higher than expected. In Pseudolactococcus laudensis, nearly every strain from a single culture carried its own methylation profile, with 43 motifs (25 novel) and 74 orphan methyltransferases located on mobile genetic elements. Strikingly, strains with nearly identical genomes showed different methylation profiles, a layer of diversity invisible to sequence-based analysis. These findings suggest that the epigenome remains largely unexplored, functional dimension of strain diversity that may shape phage susceptibility and community resilience. Long-read sequencing thus opens a view of microbial communities beyond the genome.

Antimicrobial resistance (AMR) represents one of the most urgent global health threats of the 21st century, undermining the effectiveness of life-saving treatments and jeopardizing modern medicine. Misuse and overuse of antibiotics are estimated to contribute to nearly 5 million deaths annually worldwide, accelerating the spread of resistant pathogens. Without effective intervention, AMR could claim up to 10 million lives per year by 2050 and impose a cumulative economic burden exceeding $1 trillion.

A major challenge in combating AMR lies in the time required for accurate diagnosis. Conventional bacterial culture methods typically require 2–4 days to identify pathogens and determine appropriate antibiotic treatment. In critical conditions such as sepsis, this delay necessitates immediate empirical treatment, often involving broad-spectrum or inappropriate antibiotics, further driving resistance.

This project addresses two major clinical burdens, including sepsis and urinary tract infections, by advancing strategies to better understand, clinically manage, and prevent AMR. We aim to develop rapid, point-of-care diagnostic tools that significantly reduce time-to-result while improving diagnostic accuracy. By integrating metagenomics and artificial intelligence, we propose a novel diagnostic prototype that delivers results within 4–5 hours.

This approach represents a transformative step toward precision antimicrobial therapy, enabling timely and targeted treatment decisions, reducing unnecessary antibiotic use, and strengthening antimicrobial stewardship. Ultimately, this work has the potential to mitigate the global impact of AMR, preserve the efficacy of existing antibiotics, and save millions of lives.

National surveillance rarely has a single optimal sequencing strategy. The appropriate resolution depends on the question: Are we tracking transmission between isolates, screening broadly for unexpected organisms, or sensitively detecting antimicrobial resistance (AMR) determinants in complex samples?

Drawing on experience from high-throughput bacterial sequencing and wastewater AMR surveillance at Statens Serum Institut, this presentation compares three complementary Oxford Nanopore approaches. High-throughput whole-genome sequencing of cultured bacterial isolates provides the resolution needed for routine typing, outbreak investigation, resistance characterisation and analysis of mobile genetic elements. Scalable barcoding, automated library preparation and real-time sequencing support large and diverse isolate collections while retaining genomic context for epidemiological interpretation.

Direct, amplification-free metagenomic sequencing broadens surveillance beyond organisms that can be cultured or specifically targeted. In wastewater, it can simultaneously profile microbial communities, potential pathogens and resistance genes. However, low-abundance targets, background DNA and finite sequencing depth may limit sensitivity and complicate interpretation.

Targeted hybridisation capture provides an intermediate strategy. Enriching AMR targets before long-read sequencing increases the proportion of informative reads and often retain the sequence context surrounding resistance determinants. This approach can bridge broad metagenomics and isolate sequencing when culture is impractical or relevant targets are rare.

These methods should therefore be viewed not as competitors but as a tiered surveillance toolbox.

Fast and reproducible pathogen identification is central to infectious disease care and outbreak response. Yet standard diagnostics may be delayed by culture requirements, prior antibiotic treatment, limited infrastructure and cost. Real-time Oxford Nanopore metagenomics might offer faster, culture-independent detection directly from clinical samples.

I will share our experience in settings where we assessed rapid Oxford Nanopore sequencing, and compared it when feasible to conventional methods. During a suspected cholera outbreak in a refugee camp, we used portable Oxford Nanopore sequencing and offline, laptop-based analysis to investigate gastrointestinal infection samples. We detected Vibrio cholerae on-site without prior culture, alongside other enteric bacteria and antimicrobial resistance and virulence genes. This exposed barriers to field deployment.

We also assessed rapid metagenomic sequencing for necrotizing soft tissue infections (NSTI) in trauma centres and hospitals, where early identification may be critical. We tracked bacterial signals as DNA reads accumulate, compared results with routine microbiology, and assessed how quickly clinically interpretable data can be obtained (app. 6 hours from samples to output).

I will introduce our easy-to-use, non-specialist pipelines (LPAP and GPAP) and discuss the workflow from DNA extraction and sample quality to computational analysis and interpretation. Together, these studies show what Oxford Nanopore metagenomics can deliver, its limitations, and what is needed to integrate rapid sequencing into routine diagnostics.