Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses
Abstract Background 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...
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2024-09-01
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| Online Access: | https://doi.org/10.1186/s13073-024-01380-x |
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| author | Sarah Buddle Leysa Forrest Naomi Akinsuyi Luz Marina Martin Bernal Tony Brooks Cristina Venturini Charles Miller Julianne R. Brown Nathaniel Storey Laura Atkinson Timothy Best Sunando Roy Sian Goldsworthy Sergi Castellano Peter Simmonds Heli Harvala Tanya Golubchik Rachel Williams Judith Breuer Sofia Morfopoulou Oscar Enrique Torres Montaguth |
| author_facet | Sarah Buddle Leysa Forrest Naomi Akinsuyi Luz Marina Martin Bernal Tony Brooks Cristina Venturini Charles Miller Julianne R. Brown Nathaniel Storey Laura Atkinson Timothy Best Sunando Roy Sian Goldsworthy Sergi Castellano Peter Simmonds Heli Harvala Tanya Golubchik Rachel Williams Judith Breuer Sofia Morfopoulou Oscar Enrique Torres Montaguth |
| author_sort | Sarah Buddle |
| collection | DOAJ |
| description | Abstract Background 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. We investigated whether we 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 of known pathogens and could improve sensitivity. Methods We 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. We 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. We further compared the performance of commonly used taxonomic classifiers. Results Capture with the Twist CVRP 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 standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT. Conclusions 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. |
| format | Article |
| id | doaj-art-e2af225a703e4fe19e0e320c7515d0b3 |
| institution | Kabale University |
| issn | 1756-994X |
| language | English |
| publishDate | 2024-09-01 |
| publisher | BMC |
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| series | Genome Medicine |
| spelling | doaj-art-e2af225a703e4fe19e0e320c7515d0b32025-01-05T12:42:18ZengBMCGenome Medicine1756-994X2024-09-0116112010.1186/s13073-024-01380-xEvaluating metagenomics and targeted approaches for diagnosis and surveillance of virusesSarah Buddle0Leysa Forrest1Naomi Akinsuyi2Luz Marina Martin Bernal3Tony Brooks4Cristina Venturini5Charles Miller6Julianne R. Brown7Nathaniel Storey8Laura Atkinson9Timothy Best10Sunando Roy11Sian Goldsworthy12Sergi Castellano13Peter Simmonds14Heli Harvala15Tanya Golubchik16Rachel Williams17Judith Breuer18Sofia Morfopoulou19Oscar Enrique Torres Montaguth20Infection, Immunity and Inflammation Department, Great Ormond Street Institute of Child Health, University College LondonGenetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College LondonInfection, Immunity and Inflammation Department, Great Ormond Street Institute of Child Health, University College LondonGenetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College LondonGenetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College LondonInfection, Immunity and Inflammation Department, Great Ormond Street Institute of Child Health, University College LondonDepartment of Microbiology, Virology and Infection Prevention & Control, Great Ormond Street Hospital for Children NHS Foundation TrustDepartment of Microbiology, Virology and Infection Prevention & Control, Great Ormond Street Hospital for Children NHS Foundation TrustDepartment of Microbiology, Virology and Infection Prevention & Control, Great Ormond Street Hospital for Children NHS Foundation TrustDepartment of Microbiology, Virology and Infection Prevention & Control, Great Ormond Street Hospital for Children NHS Foundation TrustDepartment of Microbiology, Virology and Infection Prevention & Control, Great Ormond Street Hospital for Children NHS Foundation TrustGenetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College LondonGenetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College LondonGenetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College LondonNuffield Department of Medicine, University of OxfordRadcliffe Department of Medicine, University of OxfordNuffield Department of Medicine, University of OxfordGenetics and Genomic Medicine Department, Great Ormond Street Institute of Child Health, University College LondonInfection, Immunity and Inflammation Department, Great Ormond Street Institute of Child Health, University College LondonInfection, Immunity and Inflammation Department, Great Ormond Street Institute of Child Health, University College LondonInfection, Immunity and Inflammation Department, Great Ormond Street Institute of Child Health, University College LondonAbstract Background 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. We investigated whether we 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 of known pathogens and could improve sensitivity. Methods We 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. We 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. We further compared the performance of commonly used taxonomic classifiers. Results Capture with the Twist CVRP 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 standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT. Conclusions 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.https://doi.org/10.1186/s13073-024-01380-xClinical metagenomicsViral diagnosticsPathogen detectionEpidemiological surveillanceNext-generation sequencing |
| spellingShingle | Sarah Buddle Leysa Forrest Naomi Akinsuyi Luz Marina Martin Bernal Tony Brooks Cristina Venturini Charles Miller Julianne R. Brown Nathaniel Storey Laura Atkinson Timothy Best Sunando Roy Sian Goldsworthy Sergi Castellano Peter Simmonds Heli Harvala Tanya Golubchik Rachel Williams Judith Breuer Sofia Morfopoulou Oscar Enrique Torres Montaguth Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses Genome Medicine Clinical metagenomics Viral diagnostics Pathogen detection Epidemiological surveillance Next-generation sequencing |
| title | Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses |
| title_full | Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses |
| title_fullStr | Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses |
| title_full_unstemmed | Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses |
| title_short | Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses |
| title_sort | evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses |
| topic | Clinical metagenomics Viral diagnostics Pathogen detection Epidemiological surveillance Next-generation sequencing |
| url | https://doi.org/10.1186/s13073-024-01380-x |
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