LITERATURE UPDATE
of known pathogens and could improve sensitivity.
The authors evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Bioscience Comprehensive Viral Research Panel (CVRP), which targets 3153 viruses. They tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. They further compared the performance of commonly used taxonomic classifiers. Capture with the Twist CVRP
Diagram of the principle of Nanopore DNA sequencing.
In the present chapter, the authors describe the advances of functional metagenomics to decipher food-microbiota and host-microbiota interactions. This powerful high-throughput approach allows for the assessment of the microbiota as a whole (including non-cultured bacteria) and enabled the discovery of new signaling pathways and functions involved in the crosstalk between food, the gut microbiota and its host. The authors present the pipeline and highlight the most important studies that helped to develop the field. To conclude, they emphasise the most recent developments and hot topics in functional metagenomics.
Clinical Metagenomics for Infectious Diseases: Progress toward Operational Value Gaston DC. J Clin Microbiol. 2023 Feb 22;61(2):e0126722. doi: 10.1128/jcm.01267-22.
The field of clinical metagenomics for infectious disease diagnostics has advanced to combining questions of technical methodologies with best-use practices due to lowering barriers of implementation.
This commentary identifies challenges facing further development of the field and proposes methods for advancement by highlighting a recent prospective pilot study evaluating a targeted metagenomic approach for infectious endocarditis. It introduces the concept of operational value as a method for standardizing results generated
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by differing clinical metagenomic approaches.
Operational value includes assessments of result quality, utility, and cost through incorporating methodological aspects of metagenomics as applied to various infectious syndromes, patient populations, and specimen types. Focus is placed on standardising outcome- based metrics using an operational value matrix.
As ambitions of clinical metagenomics
are increasingly realised, new models of study design and collaboration could promote progress toward routine use and positive benefits for patients with infectious diseases.
Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses Buddle S, Forrest L, Akinsuyi N et al. Genome Med. 2024 Sep 9;16(1):111. doi: 10.1186/s13073-024-01380-x.
Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, high sequencing depth requirements, long turnaround times, and limited sensitivity hinder broader adoption. Here, the authors investigated whether they could overcome these limitations using protocols based on untargeted sequencing with Oxford Nanopore technologies (ONT), which offers real-time data acquisition and analysis, or a targeted panel approach, which allows the selective sequencing
increased sensitivity by at least 10- 100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per mL [gc/mL]), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/mL), at lower viral loads (600-6000 gc/mL), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardised results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT.
Metagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist CVRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context.
Sequence-based Functional Metagenomics Reveals Novel Natural Diversity of Functional CopA in Environmental Microbiomes Li W, Wang L, Li X, Zheng X, Cohen MF, Liu YX. Genomics Proteomics Bioinformatics. 2023 Dec;21(6):1182- 1194. doi: 10.1016/
j.gpb.2022.08.006.
Exploring the natural diversity of functional genes/proteins from environmental DNA in high throughput
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