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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Published in:Journal of applied microbiology Vol. 131; no. 2; pp. 1007 - 1016
Main Authors: Luan, Y., Hu, H., Liu, C., Chen, B., Liu, X., Xu, Y., Luo, X., Chen, J., Ye, B., Huang, F., Wang, J., Duan, C.
Format: Journal Article
Language:English
Published: England Oxford University Press 01.08.2021
Subjects:
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
diagnosis of infectious diseases
PCR-free library preparation
metagenomic next-generation sequencing
automation
ISSN:1364-5072, 1365-2672, 1365-2672
Online Access:Get full text
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Summary: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.
Bibliography:Yi Luan, Huiling Hu and Chao Liu, contributed equally to this work.
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ISSN:1364-5072
1365-2672
1365-2672
DOI:10.1111/jam.15003