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Introduction to the protocol
This protocol has been developed by Adela Alcolea-Medina, Prof. Jonathan Edgeworth and colleagues, with support from Oxford Nanopore Technologies (Novel, rapid metagenomic method to detect emerging viral pathogens applied to human monkeypox infections by Alcolea-Medina et al., 2022). It outlines a rapid method to perform metagenomic sequencing from extracted DNA and RNA from routine nasopharyngeal swab samples in viral transport media to identify viral pathogens. This method has also been used to sequence the virus currently known as mpox from skin lesion swabs (in viral transport media) of diagnosed patients, demonstrating an alternative sample site is compatible with this protocol. However, further work is required to assess and validate different sample sites and types.
While this protocol is available in the Nanopore Community, we kindly ask users to ensure they are citing the authors of the paper who have been behind the development of these methods.
This method uses random hexamers and oligo-dT primers to synthesis cDNA strands from RNA within the sample. Subsequently, the Rapid PCR Barcoding kit (SQK-RPB004) is used to amplify the DNA present from the sample alongside the cDNA strands. During development, typically three samples per patient were pooled together after the PCR and clean-up step before sequencing the sample on a single flow cell.
We recommend users follow their local health and safety recommendations when handling samples. Confirmed mpox samples were used for the development of this method and were handled in a Containment Level 3 (CL3) facility.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:- Extract your DNA/RNA. An example extraction method is provided in this protocol but other options are available if preferred
- Ensure you have your sequencing kit, the correct equipment and third-party reagents required
- Download the software for acquiring and analysing your data
- Check your flow cell to ensure it has enough pores for a good sequencing run
Library preparation
You will need to:- Prepare full-length cDNAs via reverse transcription and 2nd strand synthesis
- Tagment your DNA using the Fragmentation Mix in the kit
- Amplify the sample by PCR using the barcoded primers supplied in the kit
- Attach the sequencing adapters supplied in the kit to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cell
Sequencing and analysis
You will need to:- Start a sequencing run of 48 hours using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads.
- For samples positive for mpox using the wf-mpx, after removing human/host reads, users should expect a significant part of all their reads to be the mpox virus and the remainder of reads typically being the flora found at the sample sites.