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A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses.

ABSTRACT: Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.

SUBMITTER: Reteng P

PROVIDER: S-EPMC8463598 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses.

Reteng Patrick P Nguyen Thuy Linh L Tran Thi Minh Tam T Mares-Guia Maria Angélica Monteiro de Mello MAMM Torres Maria Celeste MC de Filippis Ana Maria Bispo AMB Orba Yasuko Y Kobayashi Shintaro S Hayashida Kyoko K Sawa Hirofumi H Hall William W WW Nguyen Thi Lan Anh LA Yamagishi Junya J

Scientific reports 20210924 1

Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore se ...[more]

PMID: 34561471

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Project description:BackgroundFlaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods.MethodsWe developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates.ResultsThe novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates.ConclusionThis PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.

Project description:BackgroundPrecision in detecting pathogens in sepsis patients is crucial for deploying targeted therapeutic strategies. The objective of the present investigation was to assess the efficacy of targeted nanopore sequencing (TNPseq) as a method for detecting sepsis pathogens.MethodsFor this investigation, we stratified 90 patients with sepsis into 'improved' and 'unimproved' groups based on their clinical outcomes. Pathogenic microorganisms were detected in various sample types on the day of sepsis diagnosis, utilizing both conventional culture and TNPseq techniques. A comparative analysis of these methodologies was undertaken to assess their performance and efficacy in identifying pathogens in sepsis infections and discern pathogens with significant differences between the distinct patient groups.ResultsThe TNPseq analysis demonstrated superior performance in detecting pathogens in sepsis patients, achieving a significantly higher positivity rate (94.4%) compared to the culture method (30.0%, p < 0.001). TNPseq offered enhanced detection capabilities across a diverse array of pathogen types and sample types, outperforming culture particularly in the identification of fungi (35 vs. 8), viruses (46 vs. 1), and atypical pathogens. Both TNPseq and culture yielded concurrent positive results in 26 patient samples, yet TNPseq identified additional pathogens in 14 of these samples. A notable discrepancy in the types of infections was observed between the 'improved' and 'unimproved' groups, with a higher prevalence of single infections among the 'improving' cohort (p < 0.05). Significant differences in the incidence of Acinetobacter baumannii and Clostridium striatum were also evident between the two groups, underscoring the importance of focusing on these two pathogens in future clinical diagnostic efforts.ConclusionThis study underscores the versatility of TNPseq in identifying diverse pathogens in sepsis samples, ensuring its application as a diagnostic when pathogens elude conventional isolation or are difficult to culture. It particularly highlights the significant differences of A. baumannii and C. striatum in the two patient groups, pointing to potential research directions. TNPseq's potential in sepsis diagnosis is promising.

Project description:Among molecular-based techniques for fungal identification, Sanger sequencing of the primary universal fungal DNA barcode, the internal transcribed spacer (ITS) region (ITS1, 5.8S, ITS2), is commonly used in clinical routine laboratories due to its simplicity, universality, efficacy, and affordability for fungal species identification. However, Sanger sequencing fails to identify mixed ITS sequences in the case of mixed infections. To overcome this limitation, different high-throughput sequencing technologies have been explored. The nanopore-based technology is now one of the most promising long-read sequencing technologies on the market as it has the potential to sequence the full-length ITS region in a single read. In this study, we established a workflow for species identification using the sequences of the entire ITS region generated by nanopore sequencing of both pure yeast isolates and mocked mixed species reads generated with different scenarios. The species used in this study included Candida albicans (n = 2), Candida tropicalis (n = 1), Nakaseomyces glabratus (formerly Candida glabrata) (n = 1), Trichosporon asahii (n = 2), Pichia kudriavzevii (formerly Candida krusei) (n = 1), and Cryptococcus neoformans (n = 1). Comparing various methods to generate the consensus sequence for fungal species identification, the results from this study indicate that read clustering using a modified version of the NanoCLUST pipeline is more sensitive than Canu or VSEARCH, as it classified species accurately with a lower abundance cluster of reads (3% abundance compared to 10% with VSEARCH). The modified NanoCLUST also reduced the number of classified clusters compared to VSEARCH, making the subsequent BLAST+ analysis faster. Subsampling of the datasets, which reduces the size of the datasets by approximately tenfold, did not significantly affect the identification results in terms of the identified species name, percent identity, query coverage, percentage of reads in the classified cluster, and the number of clusters. The ability of the method to distinguish mixed species within sub-populations of large datasets has the potential to aid computer analysis by reducing the required processing power. The herein presented new sequence analysis pipeline will facilitate better interpretation of fungal sequence data for species identification.

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A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses.

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A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses.

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