Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics.

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Retrospective Genotyping of Enteroviruses Using a Diagnostic Nanopore Sequencing Workflow.

Enteroviruses are among the most common viruses pathogenic to humans. They are associated with various forms of disease, ranging from mild respiratory illness to severe neurological diseases. In recent years, an increasing number of isolated cases of children developing meningitis or encephalitis as a result of enterovirus infection have been reported, as well as discrete enterovirus D68 outbreaks in North America in 2014 and 2016. We developed an assay to rapidly genotype enteroviruses by sequencing a region within the VP1 gene using nanopore Flongles. We retrospectively analyzed enterovirus-/rhinovirus-positive clinical samples from the Zurich, Switzerland area mainly collected during two seasons in 2019/2020 and 2021/2022. Respiratory, cerebrospinal fluid, and stool samples were analyzed. Whole-genome sequencing was performed on samples with ambiguous genotyping results and enterovirus D68-positive samples. Out of 255 isolates, a total of 95 different genotypes were found. A difference in the prevalence of enterovirus and rhinovirus infections was observed for both sample type and age group. In particular, children aged 0-4 years showed a higher frequency of enterovirus infections. Comparing the respiratory seasons, a higher prevalence was found, especially for enterovirus A and rhinovirus A after the SARS-CoV-2 pandemic. The enterovirus genotyping workflow provides a rapid diagnostic tool for individual analysis and continuous enterovirus surveillance.

Rapid and sensitive single-sample viral metagenomics using Nanopore Flongle sequencing.

The ability of viral metagenomic Next-Generation Sequencing (mNGS) to unbiasedly detect nucleic acids in a clinical sample is a powerful tool for advanced diagnosis of viral infections. When clinical symptoms do not provide a clear differential diagnosis, extensive laboratory testing with virus-specific PCR and serology can be replaced by a single viral mNGS analysis. However, widespread diagnostic use of viral mNGS is thus far limited by long sample-to-result times, as most protocols rely on Illumina sequencing, which provides high and accurate sequencing output but is time-consuming and expensive. Here, we describe the development of an mNGS protocol based on the more cost-effective Nanopore Flongle sequencing with decreased turnaround time and lower, yet sufficient sequencing output to provide sensitive virus detection. Sample preparation (6 h) and sequencing (2 h) times are substantially reduced compared to Illumina mNGS and allow detection of DNA/RNA viruses at low input (up to 33-38 cycle threshold of specific qPCR). Although Flongles yield lower sequencing output, direct comparison with Illumina mNGS on diverse clinical samples showed similar results. Collectively, the novel Nanopore mNGS approach is specifically tailored for use in clinical diagnostics and provides a rapid and cost-effective mNGS strategy for individual testing of severe cases.