Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow

• DOI: 10.1186/s12879-018-3446-5
• Publication Date: 2018-10-29T09:48:59Z
• Authors: A. Bal; M. Pichon; C. Picard; J. S. Casalegno; M. Valette; I. Schuffenecker; L. Billard; S. Vallet; G. Vilchez; V. Cheynet; G. Oriol; S. Trouillet-Assant; Y. Gillet; B. Lina; K. Brengel-Pesce; F. Morfin; L. Josset
• Keywords: Quality Control; 0301 basic medicine; 570; 610; [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry; Infectious and parasitic diseases; RC109-216; Real-Time Polymerase Chain Reaction; 03 medical and health sciences; [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN]; Viral metagenomics; Humans; RNA Viruses; [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology; Respiratory Tract Infections; Molecular Biology/Genomics [q-bio.GN]; Respiratory viruses; 0303 health sciences; Clinical virology; DNA Viruses; Quality control; High-Throughput Nucleotide Sequencing; 3. Good health; [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology; Technical Advance; DNA, Viral; Next-generation sequencing; [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN]; RNA, Viral; Metagenomics

AbstractBackgroundIn 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 notemplate-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.