Next-generation sequencing (NGS) is increasingly being explored in clinical microbiology for applications ranging from pathogen detection to outbreak investigation and antimicrobial resistance surveillance. Among these technologies, nanopore sequencing has drawn interest for its flexibility and potential use in routine laboratory workflows.
Here, Jose Alexander, Executive Director and Medical Microbiologist at AdventHealth, discusses the opportunities and challenges associated with implementing NGS in clinical microbiology and the evolving role of sequencing in diagnostic practice.
Where do you see the biggest gaps in current clinical microbiology workflows that NGS can help address?
One important opportunity is addressing the limitations of traditional culture methods. Conventional cultures may miss fastidious organisms or pathogens that take weeks to grow, delaying diagnosis and treatment. NGS can help close this gap by enabling faster, more comprehensive pathogen detection with high specificity and accurate organism identification.
Beyond detection, NGS can provide additional clinically relevant information, including antimicrobial resistance mechanisms and virulence factors. It may also support molecular epidemiology and outbreak investigations by helping laboratories track pathogen transmission and characterize emerging infectious threats.
However, broader implementation still requires standardized criteria and validated clinical frameworks for routine diagnostic use.
What advantages does nanopore sequencing offer over traditional and other sequencing approaches in routine diagnostics?
Nanopore technology offers flexibility that may make it easier to integrate into routine clinical microbiology workflows. Clinical laboratories require diagnostic platforms that are rapid, practical, and adaptable to day-to-day patient care operations.
One advantage is flexibility across different sample types and testing volumes. Workflows can be optimized to fit existing laboratory infrastructure and operational needs.
Another important feature is long-read sequencing capability, which may improve characterization of pathogens, antimicrobial resistance mechanisms, structural genetic variation, and transmission patterns compared with short-read approaches.
How feasible is it to implement nanopore sequencing in day-to-day clinical microbiology laboratories?
From our experience, implementation is feasible even in a routine clinical microbiology laboratory without dedicated research infrastructure or personnel. We introduced the technology approximately three years ago in a standard clinical diagnostic setting and have continued refining the workflow since then.
A key requirement is expertise in laboratory-developed tests and robust validation processes to ensure clinically reliable results. At the same time, testing must fit routine laboratory workflows and deliver actionable information quickly enough to support patient care.
The field is evolving rapidly, and an important goal is integrating sequencing technologies as closely as possible into traditional clinical microbiology laboratories, where much of the initial clinical value is likely to be realized.
What are the main technical and operational challenges laboratories face when adopting NGS?
NGS workflows are more complex than many conventional laboratory instruments. They typically involve multiple instruments, coordinated workflows, and extensive validation rather than “plug-and-play” testing.
One major challenge is limited awareness and training. Many microbiologists have extensive experience with traditional methods but limited exposure to NGS technologies. Expanding education and accessible training programs will be important for broader adoption.
Infrastructure is another consideration, as NGS often requires dedicated space, multiple instruments, and strict quality-management processes. Bioinformatics has also been a barrier, particularly for smaller laboratories without dedicated computational teams. However, cloud-based databases and online analytical tools may help reduce this burden.
Automation of analytical workflows will also be important. While microbiologists should remain responsible for interpretation and final clinical decisions, increasing automation may simplify data processing and improve routine implementation.
How can sequencing be integrated with existing diagnostic methods to enhance, rather than replace, current workflows?
NGS is unlikely to fully replace traditional culture methods or PCR assays in the near term. Many conventional diagnostics remain cost-effective, clinically validated, and deeply integrated into patient care workflows.
Instead, NGS is more likely to complement existing methods by improving sensitivity, specificity, and potentially turnaround time in selected applications. Culture, PCR, and sequencing technologies will likely continue to coexist as the field evolves.
There are already areas where NGS may offer advantages, including mycobacterial and mold identification, where sequencing can improve species-level characterization. In the future, sequencing may also support faster assessment of antimicrobial resistance markers and other clinically relevant genomic features.
What role can nanopore sequencing play in outbreak detection and public health surveillance?
Microbiology laboratories play an important role beyond organism identification alone. Nanopore sequencing generates detailed genomic data that can support epidemiology, resistance monitoring, and outbreak investigation.
For example, in invasive Streptococcus pneumoniae infections, sequencing can identify serotypes, genotypes, resistance markers, and virulence factors. These findings can be correlated with patient and susceptibility data to better understand regional epidemiology and support vaccination strategies.
Sequencing also allows laboratories to assess how closely related strains are during outbreaks, helping clarify transmission patterns and outbreak dynamics. Monitoring regional resistance profiles may further support laboratory testing strategies, treatment decisions, and public health surveillance efforts.
How do you address concerns around data quality, standardization, and interpretation in routine sequencing-based diagnostics?
There is still limited standardization across the field, and many institutions currently use their own workflows and validation processes. As sequencing moves further into clinical practice, laboratories must build on the established quality and validation frameworks already used for laboratory-developed tests.
Key considerations include data quality, assay variability, platform limitations, and overall clinical performance. Clear validation and quality-control processes are essential to ensure reliable results.
Interpretation is equally important. Raw sequencing data should not be reported directly to clinicians without expert review. Clinical microbiologists play a critical role in interpreting and contextualizing sequencing findings to ensure that results are clinically relevant and actionable.
Looking ahead, do you see sequencing becoming a frontline diagnostic tool in clinical microbiology, and what needs to happen to achieve that?
The field is moving in that direction, and NGS applications in clinical microbiology are expanding rapidly. However, broader adoption will require inclusion in clinical and microbiology guidelines, along with clearer standards for validation, quality control, and performance assessment.
Greater standardization will also be important to improve consistency and comparability of sequencing results across institutions. Similar challenges already exist with some laboratory-developed molecular assays, particularly highly specialized virology PCR tests that can vary between laboratories.
NGS may initially follow a similar path, but continued efforts toward standardization and harmonization could eventually allow sequencing results to become more interoperable between laboratories and more widely integrated into routine patient care.
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