Integrating DNA and RNA sequencing for enhanced pathogen detection in respiratory infections
Abstract Background The clinical value of shotgun metagenomic next-generation sequencing (mNGS) in improving the detection rates of respiratory pathogens is well-established. However, mNGS is complex and expensive. This study designed and evaluated the performance of targeted NGS (tNGS) in diagnosin...
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BMC
2025-03-01
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| Series: | Journal of Translational Medicine |
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| Online Access: | https://doi.org/10.1186/s12967-025-06342-4 |
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| author | Dejian Gu Jie Liu Jiaping Wang Yuting Yi Yuxing Chu Rui Gao Hao Liu Jun She Binghuai Lu |
| author_facet | Dejian Gu Jie Liu Jiaping Wang Yuting Yi Yuxing Chu Rui Gao Hao Liu Jun She Binghuai Lu |
| author_sort | Dejian Gu |
| collection | DOAJ |
| description | Abstract Background The clinical value of shotgun metagenomic next-generation sequencing (mNGS) in improving the detection rates of respiratory pathogens is well-established. However, mNGS is complex and expensive. This study designed and evaluated the performance of targeted NGS (tNGS) in diagnosing respiratory infections. Methods We retrospectively included samples from 281 patients with lower respiratory tract infections to establish thresholds of pathogens. Subsequently, target pathogens were selected and a probe hybridization system was established. The performance and clinical manifestations of tNGS for 306 pathogens were evaluated using clinical and simulated samples. Results The tNGS method took 16 h with sequencing data sizes of 5 M reads. The limit-of-detection of tNGS was 100–200 CFU/mL, respectively. Bioinformatics simulation confirmed the method’s high specificity and robustness. In 281 patients of clinical validation cohort, tNGS exhibited a sensitivity of 97.73% and specificity of 75.41% compared to the composite reference standard, which notably surpasses those of culture-based and conventional microbiological methods (CMT). In detecting bacterial and viral infection, tNGS demonstrated superior sensitivity relative to CMT. Notably, 61.40% of target viruses were subtype-resolved with the initial establishment of reliable typing cutoffs, with the subtyping results being completely consistent with the PCR results. tNGS allowed for concurrent identification of antimicrobial resistance (AMR) markers and viral subtyping. 80.56% of AMR markers identified by tNGS were consistent with antimicrobial susceptibility testing. Conclusion This research established the robust performance of our tailored tNGS assay in the simultaneous detection of DNA and RNA pathogens, underscoring its prospective suitability for widespread use in clinical diagnostics. |
| format | Article |
| id | doaj-art-c100f4b3d2364dd8a4e695cc0db30022 |
| institution | DOAJ |
| issn | 1479-5876 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Translational Medicine |
| spelling | doaj-art-c100f4b3d2364dd8a4e695cc0db300222025-08-20T03:06:52ZengBMCJournal of Translational Medicine1479-58762025-03-0123111310.1186/s12967-025-06342-4Integrating DNA and RNA sequencing for enhanced pathogen detection in respiratory infectionsDejian Gu0Jie Liu1Jiaping Wang2Yuting Yi3Yuxing Chu4Rui Gao5Hao Liu6Jun She7Binghuai Lu8Geneplus-Beijing Co., Ltd.Shanghai Key Laboratory of Lung Inflammation and Injury, Department of Pulmonary and Critical Care Medicine, Zhongshan Hospital, Fudan UniversitySuzhou GenePlus Clinical Laboratory Co., LtdSuzhou GenePlus Clinical Laboratory Co., LtdSuzhou GenePlus Clinical Laboratory Co., LtdGeneplus-Beijing Co., Ltd.Geneplus-Beijing Co., Ltd.Shanghai Key Laboratory of Lung Inflammation and Injury, Department of Pulmonary and Critical Care Medicine, Zhongshan Hospital, Fudan UniversityLaboratory of Clinical Microbiology and Infectious Diseases, Department of Pulmonary and Critical Care Medicine, Center of Respiratory Medicine, Beijing Key Laboratory of Surveillance, Early Warning and Pathogen Research on Emerging Infectious Diseases, National Center for Respiratory Medicine, China-Japan Friendship HospitalAbstract Background The clinical value of shotgun metagenomic next-generation sequencing (mNGS) in improving the detection rates of respiratory pathogens is well-established. However, mNGS is complex and expensive. This study designed and evaluated the performance of targeted NGS (tNGS) in diagnosing respiratory infections. Methods We retrospectively included samples from 281 patients with lower respiratory tract infections to establish thresholds of pathogens. Subsequently, target pathogens were selected and a probe hybridization system was established. The performance and clinical manifestations of tNGS for 306 pathogens were evaluated using clinical and simulated samples. Results The tNGS method took 16 h with sequencing data sizes of 5 M reads. The limit-of-detection of tNGS was 100–200 CFU/mL, respectively. Bioinformatics simulation confirmed the method’s high specificity and robustness. In 281 patients of clinical validation cohort, tNGS exhibited a sensitivity of 97.73% and specificity of 75.41% compared to the composite reference standard, which notably surpasses those of culture-based and conventional microbiological methods (CMT). In detecting bacterial and viral infection, tNGS demonstrated superior sensitivity relative to CMT. Notably, 61.40% of target viruses were subtype-resolved with the initial establishment of reliable typing cutoffs, with the subtyping results being completely consistent with the PCR results. tNGS allowed for concurrent identification of antimicrobial resistance (AMR) markers and viral subtyping. 80.56% of AMR markers identified by tNGS were consistent with antimicrobial susceptibility testing. Conclusion This research established the robust performance of our tailored tNGS assay in the simultaneous detection of DNA and RNA pathogens, underscoring its prospective suitability for widespread use in clinical diagnostics.https://doi.org/10.1186/s12967-025-06342-4Target NGS testCapture probe enrichmentRespiratory tract infectionmNGStNGSDNA and RNA sequencing |
| spellingShingle | Dejian Gu Jie Liu Jiaping Wang Yuting Yi Yuxing Chu Rui Gao Hao Liu Jun She Binghuai Lu Integrating DNA and RNA sequencing for enhanced pathogen detection in respiratory infections Journal of Translational Medicine Target NGS test Capture probe enrichment Respiratory tract infection mNGS tNGS DNA and RNA sequencing |
| title | Integrating DNA and RNA sequencing for enhanced pathogen detection in respiratory infections |
| title_full | Integrating DNA and RNA sequencing for enhanced pathogen detection in respiratory infections |
| title_fullStr | Integrating DNA and RNA sequencing for enhanced pathogen detection in respiratory infections |
| title_full_unstemmed | Integrating DNA and RNA sequencing for enhanced pathogen detection in respiratory infections |
| title_short | Integrating DNA and RNA sequencing for enhanced pathogen detection in respiratory infections |
| title_sort | integrating dna and rna sequencing for enhanced pathogen detection in respiratory infections |
| topic | Target NGS test Capture probe enrichment Respiratory tract infection mNGS tNGS DNA and RNA sequencing |
| url | https://doi.org/10.1186/s12967-025-06342-4 |
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