Metagenomic Sequencing for Combined Detection of Rna and Dna Viruses in Respiratory Samples From Paediatric Patients

S, van Boheemen; Al, van Rijn-Klink; Pappas, Nikolaos; Ec, Carbo; Rh, Vorderman; Pj, van 't Hof; H, Mei; Ec, Claas; Ac, Kroes; J, de Vries

doi:10.1101/492835

Cited by 5 publications

(10 citation statements)

References 37 publications

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“…The metagenomics protocol used has been described and optimized for simultaneously RNA and DNA detection previously [14]. In short, internal controls, Equine Arteritis virus (EAV) for RNA and Phocid Herpesvirus-1 (PhHV) for DNA (kindly provided by prof. dr. H.G.M.…”

Section: Methodsmentioning

confidence: 99%

“…Niesters, the Netherlands), were spiked in 200 μl of the virus transport medium in which the nasopharyngeal swab was stored. Nucleic acids were extracted directly from 200 μl clinical sample using the Magnapure 96 DNA and Viral NA Small volume extraction kit on the MagnaPure 96 system (Roche Diagnostics, Almere, The Netherlands) with 100 μL output eluate (an updated version of the isolation method used for qPCR, tested previously [20]). Extraction buffer was used as negative control (for extraction, library preparation, and sequencing).…”

Section: Methodsmentioning

confidence: 99%

“…Metagenomic next generation sequencing (mNGS) is an innovative method, which enables the detection of all genomes in a given sample. Proof of principle studies have shown that mNGS on respiratory samples can confirm and extend PCR results and deliver typing and resistance data at the same time [10–14]. The performance of mNGS in the clinical diagnostic setting, especially the positive and negative predictive value, has not yet been elucidated and is likely to differ per clinical syndrome and sample.…”

Section: Introductionmentioning

confidence: 99%

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The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease

et al. 2019

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IntroductionExacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an important trigger. However, using conventional diagnostic techniques, a causative agent is not always found. Metagenomic next-generation sequencing (mNGS) allows analysis of the complete virome, but has not yet been applied in COPD exacerbations.ObjectivesTo study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS.Study design88 nasopharyngeal swabs from 63 patients from the Bergen COPD Exacerbation Study (2006–2010) were analysed by mNGS and in-house qPCR for respiratory viruses. Both DNA and RNA were sequenced simultaneously using an Illumina library preparation protocol with in-house adaptations.ResultsBy mNGS, 24/88 samples tested positive. Sensitivity and specificity, as compared with PCR, were 96% and 98% for diagnostic targets (23/24 and 1093/1120, respectively). Additional viral pathogens detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral normalized species reads (log value) (p = 0.002). Patients with viral pathogens had lower percentages of bacteriophages (p<0.001). No correlation was found between viral reads and clinical markers.ConclusionsThe mNGS protocol used was highly sensitive and specific for semi-quantitative detection of respiratory viruses. Excellent negative predictive value implicates the power of mNGS to exclude any pathogenic respiratory viral infectious cause in one test, with consequences for clinical decision making. Reduced abundance of bacteriophages in COPD patients with viral pathogens implicates skewing of the virome during infection, with potential consequences for the bacterial populations, during infection.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease

et al. 2019

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“…The metagenomics protocol used has been described previously 14 . In short, internal controls, Equine Arteritisvirus (EAV) for RNA and Phocid Herpesvirus-1 (PhHV) for DNA (kindly provided by prof. dr. H. G.M.…”

Section: Methodsmentioning

confidence: 99%

“…After quality pre-processing, sequencing reads were taxonomically classified with Centrifuge 20 using an index constructed from NCBI’s RefSeq and taxonomy databases (accessed November 2017) with reference nucleotide sequences for the domains of viruses, bacteria, archaea, fungi, parasites, protozoa. Reads with multiple best matches were uniquely assigned to the lowest common ancestor (k=l setting; previously validated 14 ). Horizontal coverage (%) was determined using www.genomedetective.com 21 version 1.111 (accessed 2018, December 30th).…”

Section: Methodsmentioning

confidence: 99%

The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease

Rijn

Boheemen

Carbo

et al. 2019

Preprint

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Introduction: Exacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (COPD), and respiratory bacterial and viral infections are an important trigger for the occurrence of such exacerbations. However, using conventional diagnostic techniques, a causative agent is not always found. Metagenomic next-generation sequencing (mNGS) allows analysis of the complete virome, but has not yet been applied in COPD exacerbations. Objectives: To study the respiratory virome in nasopharyngeal samples during COPD exacerbations using mNGS. Study design: 88 nasopharyngeal swabs from 63 patients from the Bergen COPD Exacerbation Study (2006-2010) were analysed by mNGS and in-house qPCR for respiratory viruses. Both DNA and RNA were sequenced simultaneously using an Illumina library preparation protocol with in-house adaptations. Results: By mNGS, 23/88 samples tested positive. Sensitivity and specificity were both 96% for diagnostic targets (23/24 and 1067/1120, respectively). Viral pathogens only detected by mNGS were herpes simplex virus type 1 and coronavirus OC43. A positive correlation was found between Cq value and mNGS viral species reads (p=0.008). Patients with viral pathogens had lower percentages of bacteriophages (p<0.000). No correlation was found between viral reads (species and genus level) and clinical markers. Conclusions: The mNGS protocol used was highly sensitive and specific for semi-quantitative detection of respiratory viruses. Excellent negative predictive value implicates the power of mNGS to exclude any infectious cause in one test, with consequences for clinical decision making. Reduced abundance of bacteriophages in COPD patients with viral pathogens implicates skewing of the virome, and speculatively the bacterial population, during infection.

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High‐Throughput Metagenomics for Identification of Pathogens in the Clinical Settings

et al. 2020

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The application of sequencing technology is shifting from research to clinical laboratories owing to rapid technological developments and substantially reduced costs. However, although thousands of microorganisms are known to infect humans, identification of the etiological agents for many diseases remains challenging as only a small proportion of pathogens are identifiable by the current diagnostic methods. These challenges are compounded by the emergence of new pathogens. Hence, metagenomic next‐generation sequencing (mNGS), an agnostic, unbiased, and comprehensive method for detection, and taxonomic characterization of microorganisms, has become an attractive strategy. Although many studies, and cases reports, have confirmed the success of mNGS in improving the diagnosis, treatment, and tracking of infectious diseases, several hurdles must still be overcome. It is, therefore, imperative that practitioners and clinicians understand both the benefits and limitations of mNGS when applying it to clinical practice. Interestingly, the emerging third‐generation sequencing technologies may partially offset the disadvantages of mNGS. In this review, mainly: a) the history of sequencing technology; b) various NGS technologies, common platforms, and workflows for clinical applications; c) the application of NGS in pathogen identification; d) the global expert consensus on NGS‐related methods in clinical applications; and e) challenges associated with diagnostic metagenomics are described.

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Metagenomic Sequencing for Combined Detection of Rna and Dna Viruses in Respiratory Samples From Paediatric Patients

Abstract: 22 Introduction 23

Cited by 5 publications

References 37 publications

The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease

The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease

The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease

High‐Throughput Metagenomics for Identification of Pathogens in the Clinical Settings

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