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Pandemic Preparedness Capabilities

Rapid diagnostic metagenomic sequencing


PI(s)/Head responsible for the resource:

Tobias Allander

Host organisation(s):

Clinical Microbiology at Karolinska University Hospital

Resource description:

Diagnostic metagenomic next-generation sequencing (mNGS) is a rapidly developing cutting-edge method for diagnosis of infectious diseases. It has the potential for unbiased, hypothesis-independent detection of all infectious agents and can in addition provide genotyping and susceptibility prediction as well as information about the patient’s immunological response to the infection. It also has the advantage of detecting new infectious agents. Clinical Microbiology at Karolinska University Hospital has established a diagnostic mNGS service with the support of PLP funding. The current workflow is built on Illumina NovaSeq sequencing performed at GMCK/Clinical Genomics. The time from sampling to result is about 10-14 days. Considering the acute nature of many infectious diseases, the long analysis time limits the clinical benefit of the results. There is therefore a great need to shorten the time from sampling to result.

Nanopore sequencing technology offers a rapid alternative where results can be collected in real time during ongoing analysis. Nanopore sequencing has until recently been limited by a high error rate and a low number of reads not sufficient for sensitive diagnostic mNGS. With recent technical development these obstacles are now largely overcome enabling mNGS analysis in 24-48h with maintained sensitivity.

This project aims to establish and validate an mNGS service with results within 48h based on Nanopore sequencing. The project involves optimization of sample preparation and library construction, adaption of the bioinformatic pipeline and validation with clinical samples.

Research findings:
  • We have developed a sample preparation protocol that allows deep sequencing on Nanopore, giving over 40 M reads per Promethion flow cell. This allows for a more sensitive assay, capable of detecting low concentrations of pathogens in clinical samples.
  • We have also developed a protocol that selectively depletes human sequences. Since high levels of human background DNA severely reduces the sensitivity of the assay, this achievement further improves the ability to detect low concentrations of pathogens in clinical samples.
  • We have modified our current bioinformatic analysis pipeline for Nanopore sequence data. This part of the project is ongoing and will be finalized during the spring of 2025.
  • We have analyzed 27 clinical samples (respiratory, CSF and serum) in parallel using the “old” and optimized protocols, and also on both the Illumina and Nanopore platforms. The results confirm that the optimized sample prep significantly improves sensitivity, especially for DNA viruses. We also find that Nanopore sequencing gives comparable sensitivity to Illumina sequencing.
  • Compared to current routine assay, the Nanopore assay will reduce turn-around times from 10 days to 48 hours.
Impact on prepardness for future pandemics:

The developed nanopore sequencing assay allow for reduced turn-around times and lower costs. This allows for a wider use of the assay, including respiratory samples from ICU patients. As pandemic viruses often infect the respiratory system, this will improve our preparedness for future pandemics.

Implementing fast Nanopore sequencing in routine use will lower the threshold for implementing fast sequencing also in other areas such as cancer and rare disease diagnostics. Furthermore, the anticipated broader use of the assay will benefit the development of NGS based detection of antibiotic resistance and precision medicine in the form of analysis of the patient immunological response to the infection

Contact information:

Tobias Allander
Dept. of Clinical Microbiology, Karolinska University Hospital and Dept. of Microbiology, Tumor, and Cell Biology, Karolinska Institutet
Email: tobias.allander@regionstockholm.se