A proof‐of‐concept study of an automated solution for clinical metagenomic next‐generation sequencing
Aims Metagenomic next‐generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture‐free and hypothesis‐free nucleic acid test for diagnosing all pathogens with known genomic sequences, including bacteria, fungi, viruses and parasites. While this technique greatl...
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| Vydáno v: | Journal of applied microbiology Ročník 131; číslo 2; s. 1007 - 1016 |
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| Hlavní autoři: | , , , , , , , , , , , |
| Médium: | Journal Article |
| Jazyk: | angličtina |
| Vydáno: |
England
Oxford University Press
01.08.2021
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| Témata: | |
| ISSN: | 1364-5072, 1365-2672, 1365-2672 |
| On-line přístup: | Získat plný text |
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| Abstract | Aims
Metagenomic next‐generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture‐free and hypothesis‐free nucleic acid test for diagnosing all pathogens with known genomic sequences, including bacteria, fungi, viruses and parasites. While this technique greatly expands the clinical capacity of pathogen detection, it is a second‐line choice due to lengthy procedures and microbial contaminations introduced from wet‐lab processes. As a result, we aimed to reduce the hands‐on time and exogenous contaminations in mNGS.
Methods and Results
We developed a device (NGSmaster) that automates the wet‐lab workflow, including nucleic acid extraction, PCR‐free library preparation and purification. It shortens the sample‐to‐results time to 16 and 18·5 h for DNA and RNA sequencing respectively. We used it to test cultured bacteria for validation of the workflow and bioinformatic pipeline. We also compared PCR‐free with PCR‐based library prep and discovered no differences in microbial reads. Moreover we analysed results by automation and manual testing and found that automation can significantly reduce microbial contaminations. Finally, we tested artificial and clinical samples and showed mNGS results were concordant with traditional culture.
Conclusion
NGSmaster can fulfil the microbiological diagnostic needs in a variety of sample types.
Significance and Impact of the Study
This study opens up an opportunity of performing in‐house mNGS to reduce turnaround time and workload, instead of transferring potentially contagious specimen to a third‐party laboratory. |
|---|---|
| AbstractList | Aims
Metagenomic next‐generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture‐free and hypothesis‐free nucleic acid test for diagnosing all pathogens with known genomic sequences, including bacteria, fungi, viruses and parasites. While this technique greatly expands the clinical capacity of pathogen detection, it is a second‐line choice due to lengthy procedures and microbial contaminations introduced from wet‐lab processes. As a result, we aimed to reduce the hands‐on time and exogenous contaminations in mNGS.
Methods and Results
We developed a device (NGSmaster) that automates the wet‐lab workflow, including nucleic acid extraction, PCR‐free library preparation and purification. It shortens the sample‐to‐results time to 16 and 18·5 h for DNA and RNA sequencing respectively. We used it to test cultured bacteria for validation of the workflow and bioinformatic pipeline. We also compared PCR‐free with PCR‐based library prep and discovered no differences in microbial reads. Moreover we analysed results by automation and manual testing and found that automation can significantly reduce microbial contaminations. Finally, we tested artificial and clinical samples and showed mNGS results were concordant with traditional culture.
Conclusion
NGSmaster can fulfil the microbiological diagnostic needs in a variety of sample types.
Significance and Impact of the Study
This study opens up an opportunity of performing in‐house mNGS to reduce turnaround time and workload, instead of transferring potentially contagious specimen to a third‐party laboratory. Metagenomic next-generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture-free and hypothesis-free nucleic acid test for diagnosing all pathogens with known genomic sequences, including bacteria, fungi, viruses and parasites. While this technique greatly expands the clinical capacity of pathogen detection, it is a second-line choice due to lengthy procedures and microbial contaminations introduced from wet-lab processes. As a result, we aimed to reduce the hands-on time and exogenous contaminations in mNGS.AIMSMetagenomic next-generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture-free and hypothesis-free nucleic acid test for diagnosing all pathogens with known genomic sequences, including bacteria, fungi, viruses and parasites. While this technique greatly expands the clinical capacity of pathogen detection, it is a second-line choice due to lengthy procedures and microbial contaminations introduced from wet-lab processes. As a result, we aimed to reduce the hands-on time and exogenous contaminations in mNGS.We developed a device (NGSmaster) that automates the wet-lab workflow, including nucleic acid extraction, PCR-free library preparation and purification. It shortens the sample-to-results time to 16 and 18·5 h for DNA and RNA sequencing respectively. We used it to test cultured bacteria for validation of the workflow and bioinformatic pipeline. We also compared PCR-free with PCR-based library prep and discovered no differences in microbial reads. Moreover we analysed results by automation and manual testing and found that automation can significantly reduce microbial contaminations. Finally, we tested artificial and clinical samples and showed mNGS results were concordant with traditional culture.METHODS AND RESULTSWe developed a device (NGSmaster) that automates the wet-lab workflow, including nucleic acid extraction, PCR-free library preparation and purification. It shortens the sample-to-results time to 16 and 18·5 h for DNA and RNA sequencing respectively. We used it to test cultured bacteria for validation of the workflow and bioinformatic pipeline. We also compared PCR-free with PCR-based library prep and discovered no differences in microbial reads. Moreover we analysed results by automation and manual testing and found that automation can significantly reduce microbial contaminations. Finally, we tested artificial and clinical samples and showed mNGS results were concordant with traditional culture.NGSmaster can fulfil the microbiological diagnostic needs in a variety of sample types.CONCLUSIONNGSmaster can fulfil the microbiological diagnostic needs in a variety of sample types.This study opens up an opportunity of performing in-house mNGS to reduce turnaround time and workload, instead of transferring potentially contagious specimen to a third-party laboratory.SIGNIFICANCE AND IMPACT OF THE STUDYThis study opens up an opportunity of performing in-house mNGS to reduce turnaround time and workload, instead of transferring potentially contagious specimen to a third-party laboratory. Metagenomic next-generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture-free and hypothesis-free nucleic acid test for diagnosing all pathogens with known genomic sequences, including bacteria, fungi, viruses and parasites. While this technique greatly expands the clinical capacity of pathogen detection, it is a second-line choice due to lengthy procedures and microbial contaminations introduced from wet-lab processes. As a result, we aimed to reduce the hands-on time and exogenous contaminations in mNGS. We developed a device (NGSmaster) that automates the wet-lab workflow, including nucleic acid extraction, PCR-free library preparation and purification. It shortens the sample-to-results time to 16 and 18·5 h for DNA and RNA sequencing respectively. We used it to test cultured bacteria for validation of the workflow and bioinformatic pipeline. We also compared PCR-free with PCR-based library prep and discovered no differences in microbial reads. Moreover we analysed results by automation and manual testing and found that automation can significantly reduce microbial contaminations. Finally, we tested artificial and clinical samples and showed mNGS results were concordant with traditional culture. NGSmaster can fulfil the microbiological diagnostic needs in a variety of sample types. This study opens up an opportunity of performing in-house mNGS to reduce turnaround time and workload, instead of transferring potentially contagious specimen to a third-party laboratory. AIMS: Metagenomic next‐generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture‐free and hypothesis‐free nucleic acid test for diagnosing all pathogens with known genomic sequences, including bacteria, fungi, viruses and parasites. While this technique greatly expands the clinical capacity of pathogen detection, it is a second‐line choice due to lengthy procedures and microbial contaminations introduced from wet‐lab processes. As a result, we aimed to reduce the hands‐on time and exogenous contaminations in mNGS. METHODS AND RESULTS: We developed a device (NGSmaster) that automates the wet‐lab workflow, including nucleic acid extraction, PCR‐free library preparation and purification. It shortens the sample‐to‐results time to 16 and 18·5 h for DNA and RNA sequencing respectively. We used it to test cultured bacteria for validation of the workflow and bioinformatic pipeline. We also compared PCR‐free with PCR‐based library prep and discovered no differences in microbial reads. Moreover we analysed results by automation and manual testing and found that automation can significantly reduce microbial contaminations. Finally, we tested artificial and clinical samples and showed mNGS results were concordant with traditional culture. CONCLUSION: NGSmaster can fulfil the microbiological diagnostic needs in a variety of sample types. SIGNIFICANCE AND IMPACT OF THE STUDY: This study opens up an opportunity of performing in‐house mNGS to reduce turnaround time and workload, instead of transferring potentially contagious specimen to a third‐party laboratory. AimsMetagenomic next‐generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture‐free and hypothesis‐free nucleic acid test for diagnosing all pathogens with known genomic sequences, including bacteria, fungi, viruses and parasites. While this technique greatly expands the clinical capacity of pathogen detection, it is a second‐line choice due to lengthy procedures and microbial contaminations introduced from wet‐lab processes. As a result, we aimed to reduce the hands‐on time and exogenous contaminations in mNGS.Methods and ResultsWe developed a device (NGSmaster) that automates the wet‐lab workflow, including nucleic acid extraction, PCR‐free library preparation and purification. It shortens the sample‐to‐results time to 16 and 18·5 h for DNA and RNA sequencing respectively. We used it to test cultured bacteria for validation of the workflow and bioinformatic pipeline. We also compared PCR‐free with PCR‐based library prep and discovered no differences in microbial reads. Moreover we analysed results by automation and manual testing and found that automation can significantly reduce microbial contaminations. Finally, we tested artificial and clinical samples and showed mNGS results were concordant with traditional culture.ConclusionNGSmaster can fulfil the microbiological diagnostic needs in a variety of sample types.Significance and Impact of the StudyThis study opens up an opportunity of performing in‐house mNGS to reduce turnaround time and workload, instead of transferring potentially contagious specimen to a third‐party laboratory. |
| Author | Luo, X. Huang, F. Luan, Y. Chen, J. Hu, H. Liu, X. Xu, Y. Liu, C. Chen, B. Wang, J. Duan, C. Ye, B. |
| Author_xml | – sequence: 1 givenname: Y. surname: Luan fullname: Luan, Y. organization: Sun Yat‐Sen University – sequence: 2 givenname: H. surname: Hu fullname: Hu, H. organization: Sun Yat‐Sen University – sequence: 3 givenname: C. surname: Liu fullname: Liu, C. organization: Matridx Biotechnology Co., Ltd – sequence: 4 givenname: B. surname: Chen fullname: Chen, B. organization: Matridx Biotechnology Co., Ltd – sequence: 5 givenname: X. surname: Liu fullname: Liu, X. organization: Sun Yat‐Sen University – sequence: 6 givenname: Y. surname: Xu fullname: Xu, Y. organization: Sun Yat‐Sen University – sequence: 7 givenname: X. surname: Luo fullname: Luo, X. organization: Sun Yat‐Sen University – sequence: 8 givenname: J. surname: Chen fullname: Chen, J. organization: Matridx Biotechnology Co., Ltd – sequence: 9 givenname: B. surname: Ye fullname: Ye, B. organization: Matridx Biotechnology Co., Ltd – sequence: 10 givenname: F. surname: Huang fullname: Huang, F. organization: Matridx Biotechnology Co., Ltd – sequence: 11 givenname: J. orcidid: 0000-0001-9308-663X surname: Wang fullname: Wang, J. email: wangjun@matridx.com organization: Matridx Biotechnology Co., Ltd – sequence: 12 givenname: C. surname: Duan fullname: Duan, C. email: 1725012289@qq.com, wangjun@matridx.com organization: Sun Yat‐Sen University |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/33440055$$D View this record in MEDLINE/PubMed |
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| Copyright | 2021 The Society for Applied Microbiology 2021 The Society for Applied Microbiology. Copyright © 2021 The Society for Applied Microbiology |
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| Keywords | diagnosis of infectious diseases PCR-free library preparation metagenomic next-generation sequencing automation |
| Language | English |
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Metagenomic next‐generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture‐free and hypothesis‐free nucleic acid... Metagenomic next-generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture-free and hypothesis-free nucleic acid test... AimsMetagenomic next‐generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture‐free and hypothesis‐free nucleic acid... AIMS: Metagenomic next‐generation sequencing (mNGS) has been utilized for diagnosing infectious diseases. It is a culture‐free and hypothesis‐free nucleic acid... |
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| SubjectTerms | Automation Bacteria bioinformatics Contamination Deoxyribonucleic acid diagnosis of infectious diseases DNA DNA sequencing Infectious diseases Libraries metagenomic next‐generation sequencing Metagenomics Microbial contamination microbial detection Microorganisms Nucleic acids Parasites Pathogens PCR‐free library preparation Polymerase chain reaction RNA Workflow |
| Title | A proof‐of‐concept study of an automated solution for clinical metagenomic next‐generation sequencing |
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