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A metagenomics study for the identification of respiratory viruses in mixed clinical specimens: an application of the iterative mapping approach.

ABSTRACT: Metagenomic approaches to detect viral genomes and variants in clinical samples have various challenges, including low viral titers and bacterial and human genome contamination. To address these limitations, we examined a next-generation sequencing (NGS) and iterative mapping approach for virus detection in clinical samples. We analyzed 40 clinical specimens from hospitalized children diagnosed with acute bronchiolitis, croup, or respiratory tract infections in which virus identification by viral culture or polymerase chain reaction (PCR) was unsuccessful. For our NGS data analysis pipeline, clinical samples were pooled into two NGS groups to reduce sequencing costs, and the depth and coverage of assembled contigs were effectively increased using an iterative mapping approach. PCR was individually performed for each specimen according to the NGS-predicted viral type. We successfully detected previously unidentified respiratory viruses in 26 of 40 specimens using our proposed NGS pipeline. Two dominant populations within the detected viruses were human rhinoviruses (HRVs; n = 14) and human coronavirus NL63 (n = 8), followed by human parainfluenza virus (HPIV), human parechovirus, influenza A virus, respiratory syncytial virus (RSV), and human metapneumovirus. This is the first study reporting the complete genome sequences of HRV-A101, HRV-C3, HPIV-4a, and RSV, as well as an analysis of their genetic variants, in Taiwan. These results demonstrate that this NGS pipeline allows to detect viruses which were not identified by routine diagnostic assays, directly from clinical samples.

SUBMITTER: Gong YN

PROVIDER: S-EPMC7087367 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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A metagenomics study for the identification of respiratory viruses in mixed clinical specimens: an application of the iterative mapping approach.

Gong Yu-Nong YN Yang Shu-Li SL Chen Guang-Wu GW Chen Yu-Wen YW Huang Yhu-Chering YC Ning Hsiao-Chen HC Tsao Kuo-Chien KC

Archives of virology 20170419 7

Metagenomic approaches to detect viral genomes and variants in clinical samples have various challenges, including low viral titers and bacterial and human genome contamination. To address these limitations, we examined a next-generation sequencing (NGS) and iterative mapping approach for virus detection in clinical samples. We analyzed 40 clinical specimens from hospitalized children diagnosed with acute bronchiolitis, croup, or respiratory tract infections in which virus identification by vira ...[more]

PMID: 28424887

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Project description:Background: Metagenomic sequencing has the potential to identify a wide range of pathogens in human tissue samples. Sarcoidosis is a complex disorder whose etiology remains unknown and for which a variety of infectious causes have been hypothesized. We sought to conduct metagenomic sequencing on cases of ocular and periocular sarcoidosis, none of them with previously identified infectious causes. Methods: Archival tissue specimens of 16 subjects with biopsies of ocular and periocular tissues that were positive for non-caseating granulomas were used as cases. Four archival tissue specimens that did not demonstrate non-caseating granulomas were also included as controls. Genomic DNA was extracted from tissue sections. DNA libraries were generated from the extracted genomic DNA and the libraries underwent next-generation sequencing. Results: We generated between 4.8 and 20.7 million reads for each of the 16 cases plus four control samples. For eight of the cases, we identified microbial pathogens that were present well above the background, with one potential pathogen identified for seven of the cases and two possible pathogens for one of the cases. Five of the eight cases were associated with bacteria ( Campylobacter concisus, Neisseria elongata, Streptococcus salivarius, Pseudopropionibacterium propionicum, and Paracoccus yeei), two cases with fungi ( Exophiala oligosperma, Lomentospora prolificans and Aspergillus versicolor) and one case with a virus (Mupapillomavirus 1). Interestingly, four of the five bacterial species are also part of the human oral microbiome. Conclusions: Using a metagenomic sequencing we identified possible infectious causes in half of the ocular and periocular sarcoidosis cases analyzed. Our findings support the proposition that sarcoidosis could be an etiologically heterogenous disease. Because these are previously banked samples, direct follow-up in the respective patients is impossible, but these results suggest that sequencing may be a valuable tool in better understanding the etiopathogenesis of sarcoidosis and in diagnosing and treating this disease.

Project description:UnlabelledBovine respiratory disease (BRD) is a common health problem for both dairy and beef cattle, resulting in significant economic loses. In order to identify viruses associated with BRD, we used a metagenomics approach to enrich and sequence viral nucleic acids in the nasal swabs of 50 young dairy cattle with symptoms of BRD. Following deep sequencing, de novo assembly, and translated protein sequence similarity searches, numerous known and previously uncharacterized viruses were identified. Bovine adenovirus 3, bovine adeno-associated virus, bovine influenza D virus, bovine parvovirus 2, bovine herpesvirus 6, bovine rhinitis A virus, and multiple genotypes of bovine rhinitis B virus were identified. The genomes of a previously uncharacterized astrovirus and picobirnaviruses were also partially or fully sequenced. Using real-time PCR, the rates of detection of the eight viruses that generated the most reads were compared for the nasal secretions of 50 animals with BRD versus 50 location-matched healthy control animals. Viruses were detected in 68% of BRD-affected animals versus 16% of healthy control animals. Thirty-eight percent of sick animals versus 8% of controls were infected with multiple respiratory viruses. Significantly associated with BRD were bovine adenovirus 3 (P < 0.0001), bovine rhinitis A virus (P = 0.005), and the recently described bovine influenza D virus (P = 0.006), which were detected either alone or in combination in 62% of animals with BRD. A metagenomics and real-time PCR detection approach in carefully matched cases and controls can provide a rapid means to identify viruses associated with a complex disease, paving the way for further confirmatory tests and ultimately to effective intervention strategies.ImportanceBovine respiratory disease is the most economically important disease affecting the cattle industry, whose complex root causes include environmental, genetics, and infectious factors. Using an unbiased metagenomics approach, we characterized the viruses in respiratory secretions from BRD cases and identified known and previously uncharacterized viruses belonging to seven viral families. Using a case-control format with location-matched animals, we compared the rates of viral detection and identified 3 viruses associated with severe BRD signs. Combining a metagenomics and case-control format can provide candidate pathogens associated with complex infectious diseases and inform further studies aimed at reducing their impact.

Dataset Information

A metagenomics study for the identification of respiratory viruses in mixed clinical specimens: an application of the iterative mapping approach.

Publications

A metagenomics study for the identification of respiratory viruses in mixed clinical specimens: an application of the iterative mapping approach.

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