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

ABSTRACT: BACKGROUND:In 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. METHODS:A 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). RESULTS:The 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). CONCLUSIONS:Although 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.

SUBMITTER: Bal A

PROVIDER: S-EPMC6206636 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Bal A A Pichon M M Picard C C Casalegno J S JS Valette M M Schuffenecker I I Billard L L Vallet S S Vilchez G G Cheynet V V Oriol G G Trouillet-Assant S S Gillet Y Y Lina B B Brengel-Pesce K K Morfin F F Josset L L

BMC infectious diseases 20181029 1

<h4>Background</h4>In 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.<h4>Methods</h4>A 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 externa ...[more]

PMID: 30373528

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Project description:Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

Project description:BackgroundThe usefulness of metagenomic next-generation sequencing (mNGS) in identifying pathogens is being investigated. We aimed to compare the power of microbial identification between mNGS and various methods in patients with acute respiratory failure.MethodsWe reviewed 130 patients with respiratory failure, and 184 specimens including blood, bronchoalveolar lavage fluid (BALF), sputum, pleural effusion, ascitic fluid, and urine were tested by mNGS and conventional methods (culture, PCR). We also enrolled 13 patients to evaluate the power of mNGS and pathogen targets NGS (ptNGS) in microbial identifications. Clinical features and microbes detected were analyzed.ResultsmNGS outperformed the conventional method in the positive detection rate of Mycobacterium tuberculosis (MTB) (OR, ∞; 95% CI, 1-∞; P < 0.05), bacteria (OR, 3.7; 95% CI, 2.4-5.8; P < 0.0001), fungi (OR, 4.37; 95% CI, 2.7-7.2; P < 0.0001), mycoplasma (OR, 10.5; 95% CI, 31.8-115; P = 0.005), and virus (OR, ∞; 95% CI, 180.7-∞; P < 0.0001). We showed that 20 patients (28 samples) were detected with Pneumocystis jirovecii (P. jirovecii) by mNGS, but not by the conventional method, and most of those patients were immunocompromised. Read numbers of Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), P. jirovecii, cytomegalovirus (CMV), and Herpes simplex virus 1 (HSV1) in BALF were higher than those in other sample types, and the read number of Candida albicans (C. albicans) in blood was higher than that in BALF. We found that orotracheal intubation and type 2 diabetes mellitus (T2DM) were associated with a higher detection rate of bacteria and virus by mNGS, immunosuppression was associated with a higher detection rate of fungi and virus by mNGS, and inflammatory markers were associated with mNGS-positive detection rate of bacteria. In addition, we observed preliminary results of ptNGS.ConclusionmNGS outperformed the conventional method in the detection of MTB, bacteria, fungi, mycoplasma, and virus. Orotracheal intubation, T2DM, immunosuppression, and inflammatory markers were associated with a higher detection rate of bacteria, fungi, and virus by mNGS. In addition, ptNGS results were consistent with the detection of abundant bacteria, fungi, and mycoplasma in our specimens.

Dataset Information

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

Publications

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

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