By Diagnostics World Staff
June 22, 2022 | A team of researchers from Guy's and St Thomas' NHS Foundation Trust and Oxford Nanopore have shared a novel protocol for the rapid metagenomic characterization of DNA and RNA viruses. The workflow—using Oxford Nanopore’s GridION platform, the MinKNOW, software, and ONT Guppy for basecalling—identified pathogens in a matter of hours.
In the paper, currently a preprint posted at SSRN (DOI: 10.2139/ssrn.4132526), researchers used the protocol on eight samples: four nasopharyngeal swabs and four swabs of blistering skin rashes.
For the nasopharyngeal swabs, virus was detected within 30 minutes of sequencing and sufficient data were generated to achieve >90% genome coverage of the viral genomes. The viruses detected were SARS-CoV-2, Adenovirus, Parainfluenza 3, and Seasonal coronavirus.
The same sample workflow was applied to swabs of skin lesions suspected to be the novel monkeypox virus (MKPX). Three samples were confirmed to be MKPX within 30 minutes of sequencing; the fourth sample was identified as varicella zoster virus after 30 minutes of sequencing.
“This finding highlights the importance of a rapid, pathogen-agnostic method to give comprehensive results for confirmation of alternative diagnoses in suspected cases,” the authors write.
Hypothesis-Free Pathogen Detection
This “pathogen-agnostic” method is superior to standard approaches for identifying viruses, such as PCR, the authors write, because the metagenomic approach sequences all genetic material in a sample, therefore providing the capability to identify potential pathogens in an unbiased or ‘hypothesis-free’ manner.
The lack of a targeted PCR assay at the start of a pathogen outbreak often leads to delays in case confirmation, as traditional offsite laboratories can take a few days, sometimes up to five, from first patient contact to result, the authors note.
This novel workflow, developed by a team lead by Guy’s and St Thomas’ with support from Oxford Nanopore Technologies, uses nanopore sequencing and takes seven hours from sample receipt, with results for viral identification available on the same day.
The protocol in the preprint includes altering Oxford Nanopore’s Rapid PCR barcoding kit by increasing PCR cycles to 30 and shortening the extension time to 4 minutes. Samples were sequenced using flowcells (R9.4.1) on a GridION platform.
The MinKNOW software (v21.05.25) was used to acquire the raw data for live basecalling by ONT Guppy (v5.016). Sequencing was run for 72 hours with 16-hour sequencing data used for genome assembly by offline analysis. Initially, human reads were discarded via read-based alignments against genome reference (GRCh38.p13) and non-human reads were retained and used for viral identification and characterization, the protocol outlines.
Viral identification was done using the Antimicrobial Resistance pipeline available within the EPI2ME desktop agent from Oxford Nanopore (v2.59.1896509). For genome assembly, minimap2 (v2.18) was first used to map raw sequencing reads against the viral reference genome (e.g. MKPX: NCBI RefSeq GCA_000857045.1), which were further processed using samtools v1.12. Coverage characteristics were assessed using qualimap (v2.2) and flye (v2.9) was used to assemble the viral genome.