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Retrospective Validation of a Metagenomic Sequencing Protocol for Combined Detection of RNA and DNA Viruses Using Respiratory Samples from Pediatric Patients.

ABSTRACT: Viruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables unbiased detection of all potential pathogens. To apply mNGS in viral diagnostics, sensitive and simultaneous detection of RNA and DNA viruses is needed. Herein, were studied the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. The sequencing protocol and bioinformatics analysis were designed and optimized, including exogenous internal controls. Subsequently, the protocol was retrospectively validated using 25 clinical respiratory samples. The developed protocol using Illumina NextSeq 500 sequencing showed high repeatability. Use of the National Center for Biotechnology Information's RefSeq database as opposed to the National Center for Biotechnology Information's nucleotide database led to enhanced specificity of classification of viral pathogens. A correlation was established between read counts and PCR cycle threshold value. Sensitivity of mNGS, compared with PCR, varied up to 83%, with specificity of 94%, dependent on the cutoff for defining positive mNGS results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow, were influenza C, KI polyomavirus, cytomegalovirus, and enterovirus. Sensitivity and analytical specificity of this mNGS protocol were comparable to PCR and higher when considering off-PCR target viral pathogens. One single test detected all potential viral pathogens and simultaneously obtained detailed information on detected viruses.

SUBMITTER: van Boheemen S

PROVIDER: S-EPMC7106021 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Retrospective Validation of a Metagenomic Sequencing Protocol for Combined Detection of RNA and DNA Viruses Using Respiratory Samples from Pediatric Patients.

van Boheemen Sander S van Rijn Anneloes L AL Pappas Nikos N Carbo Ellen C EC Vorderman Ruben H P RHP Sidorov Igor I van T Hof Peter J PJ Mei Hailiang H Claas Eric C J ECJ Kroes Aloys C M ACM de Vries Jutte J C JJC

The Journal of molecular diagnostics : JMD 20191216 2

Viruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables unbiased detection of all potential pathogens. To apply mNGS in viral diagnostics, sensitive and simultaneous detection of RNA and DNA viruses is needed. Herein, were studied the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. The sequencing protocol and bioinformatics analysis were ...[more]

PMID: 31837435

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Project description:BackgroundIn recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking.MethodsA total of 3 QCs were implemented and processed through the whole mNGS workflow: a no-template-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).ResultsThe optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6 to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).ConclusionsAlthough the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

Dataset Information

Retrospective Validation of a Metagenomic Sequencing Protocol for Combined Detection of RNA and DNA Viruses Using Respiratory Samples from Pediatric Patients.

Publications

Retrospective Validation of a Metagenomic Sequencing Protocol for Combined Detection of RNA and DNA Viruses Using Respiratory Samples from Pediatric Patients.

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