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September 03, 2025
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Metagenomic sequencing for pathogen surveillance
The Oxford Nanopore Technologies’ dual-arm rapid metagenomic sequencing method can be used for surveillance of bacterial, fungal and viral pathogens. This post describes a new metagenomics dataset release following the rapid metagenomic surveillance protocol for two qualitative control panels (Zeptometrix Respiratory Panel 2.1) covering DNA viruses, RNA viruses, bacteria and atypical bacteria (intracellular or lacking a cell wall) spiked into human sputum sample from healthy donors purchased from Cambridge BioScience.
Sample
| Detail | Description |
|---|---|
| Sample Name | Inclusivity test |
| Organism | Human, bacteria, virus |
| Molecule Type | gDNA, RNA |
| Sample Type | Human sputum, qualitative control panel |
| Technical replicates | 3 per panel |
Preparation
Sample preparation was performed according to the Rapid metagenomic sequencing for pathogen surveillance protocol (v1) published on the Oxford Nanopore Technologies website.
| Detail | Description |
|---|---|
| Extraction | Host depletion carried out per arm followed by total nucleic acid extraction using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit |
| Reverse transcription | The DNA/RNA viral sample is prepared following an optimised version of a method established by Josh Quick and Ingra M. Claro which utilises a shotgun approach using 9N primers to randomly reverse transcribe RNA and subsequently PCR-amplify DNA/RNA present in a sample. (https://wellcomeopenresearch.org/articles/6-241/v1) |
| Library Prep | Rapid metagenomic sequencing for pathogen surveillance |
| Kit | SQK-RPB114-24 |
Further preparation information such as sample storage suggestions can be found on the Oxford Nanopore Website.
Sequencing
Sequence data was generated using the following configuration:
| Detail | Description |
|---|---|
| Flow Cell | FLO-MIN114 |
| Device | GridION |
| Chemistry | R10.4.1 |
| Basecall Model | HAC v4.3.0 |
| MinKNOW Version | 6.0.14 (24.06.15) |
| Dorado Version (rebasecalled) | v0.9.1 |
Data Download
The dataset is available for anonymous download, without login, from a public Amazon Web Services S3 bucket. The bucket is part of the Open Data on AWS project enabling sharing and analysis of a wide range of data. The data can be downloaded with the AWS CLI command:
aws s3 sync --no-sign-request s3://ont-open-data/pathogen_surveillance_2025.09 pathogen_surveillance_2025.09
See the tutorials page for information on downloading the dataset.
You can also browse and download the files in your web browser courtesy of 42basepairs.
| Folder name | Size | Description |
|---|---|---|
| raw | 255 GB | POD5 files |
| basecalls | 37 GB | BAM files |
| analysis | 13 MB | Workflow outputs |
Analysis
Analysis outputs are available. The analysis results are located in the S3 bucket under the prefix:
s3://ont-open-data/pathogen_surveillance_2025.09/analysis
EPI2ME workflows used to generate analysis outputs:
Related Materials
- Protocol: https://nanoporetech.com/document/rapid-sequencing-metagenomics-sqk-rpb114-24
- Know-how: https://nanoporetech.com/document/requirements/rapid-metagenomic-sequencing-for-surveillance-of-bacterial-fungal-and-viral-pathogens-using-sqk-rpb114-24-know-how-document
- Knowledge exchange: https://nanoporetech.com/resource-centre/metagenomics-for-pathogen-surveillance
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