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. 2020 Feb;22(2):196-207.
doi: 10.1016/j.jmoldx.2019.10.007. Epub 2019 Dec 16.

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

Sander van Boheemen  1 , Anneloes L van Rijn  2 , Nikos Pappas  3 , Ellen C Carbo  1 , Ruben H P Vorderman  3 , Igor Sidorov  1 , Peter J van T Hof  3 , Hailiang Mei  3 , Eric C J Claas  1 , Aloys C M Kroes  1 , Jutte J C de Vries  1
Affiliations

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

Sander van Boheemen et al. J Mol Diagn. 2020 Feb.

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.

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Figures

Figure 1
Figure 1
The bioinformatic workflow of the metagenomic next-generation sequencing protocol studied. NCBI, National Center for Biotechnology Information.
Figure 2
Figure 2
Comparison of fragmentation methods on target reads (species level, log scale). Asterisks indicate not tested with Bioruptor setting high intensity. ADV, adenovirus; HBOV, human bocavirus; INFC, influenza C virus; NL63, coronavirus NL63; PIV, parainfluenza virus; RSV, respiratory syncytial virus.
Figure 3
Figure 3
Serial dilutions of an influenza A–positive clinical sample. Cq, quantification cycle.
Figure 4
Figure 4
Analysis of in silico simulated equine arteritis virus (EAV) reads with the different bioinformatic settings of the Centrifuge pipeline.
Figure 5
Figure 5
Spurious Lassa virus reads detected using the National Center for Biotechnology Information’s (NCBI's) nucleotide database (top), versus NCBI's RefSeq database (bottom). Black arrow points to the spurious Lassa virus reads. dsDNA, double-stranded DNA; ssRNA, single-stranded RNA.
Figure 6
Figure 6
Receiver-operating characteristic curve for metagenomic next-generation sequencing detection of PCR target viruses, depending on the cutoff level of the number of mapped sequence reads for defining a positive result.
Figure 7
Figure 7
Semiquantification of the metagenomic next-generation sequencing assay for target virus detection in clinical samples with real-time quantitative PCR confirms human respiratory viruses. Cq, quantification cycle.
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