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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:BackgroundLower respiratory tract infections (LRTI)are known to be diagnosed late or inaccurately. This has fueled the unscrupulous use of antibiotics, as they are often used empirically and clinically, leading to antibiotic abuse and multidrug resistance in patients. Metagenomic next-generation sequencing (mNGS), now widely used in clinical studies, could be a potential intervention to revolutionize microbiology by rapidly identifying unknown species.MethodsThis review and meta-analysis were conducted on eligible studies with respect to metagenomic sequencing on clinical LRTI diagnostics up to May 01, 2022. QUADAS-2 was employed to assess the methodological bias as well as applicability. After that, a meta-analysis was conducted to analyze the accuracy of mNGS, compared with the composite reference standard (CRS), among the enrolled studies.ResultsThis work collected 1248 samples in 13/21 qualified articles to factor in the accuracy of the diagnostic test. Typically, methods like molecular testing, culture, composite measures, and clinical decision-making were adopted as the reference criteria. With regard to Bronchoalveolar Lavage Samples, their sensitivity was 89% (82-93%) while their specificity was 90% (66-98%), with obvious heterogeneities in these two factors as demonstrated by different studies. The summary receiver operating characteristic (SROC) curve was plotted for mNGS as a function of LRTI, and the area under the curve (AUC) was 0.94. A Funnel plot with a p-value greater than 0.05 indicated the absence of publication bias. Positive and negative likelihood ratios (PLR and NLR) were >10 and > 0.1, respectively. In this pre-test probability-post-probability-likelihood ratio relationship graph, the values were Prior prob (%) = 20, Post-prob-Pos (%) = 77 and Post-prob-Neg (%) = 4.ConclusionThe AUC value of SROC suggested a high accuracy of mNGS in diagnosis, with no publication bias and high reliability. The application of mNGS exhibits notable diagnostic efficacy in discerning pathogens present in bronchoalveolar lavage fluid (BALF) among patients afflicted with LRTI. However, mNGS is more meaningful for the definitive diagnosis of the disease rather than the exclusion of the disease. This post-test probability is significantly higher than the pre-test probability.

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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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