Recently, JieYi Biotech and the Department of Clinical Laboratory of Sun Yat-sen Memorial Hospital have made important progress in the research of clinical metagenomic next-generation sequencing (mNGS) automation solutions. The study verifies the performance of the mNGS automation process for the first time, and proposes that the automated process has great advantages for improving the timeliness of mNGS detection and controlling exogenous microbial contamination, and is of landmark significance for realizing the localization of mNGS in medical institutions. The research results were published in the Journal of Applied Microbiology, and the corresponding authors are Professor Duan Zhaohui, Director of the Department of Laboratory, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, and Dr. Wang Jun, Founder and CEO of JieYi Biology.
mNGS is a culture-based, hypothesis-free, pathogen nucleic acid detection technique that has sufficient evidence to improve clinical etiology diagnostics. However, the cumbersome wet experiment process leads to a long mNGS detection cycle and a greater demand for laboratory space, equipment and operators, which limits the localization of mNGS in medical institutions. In addition, the "net-to-net" characteristics of mNGS will also make it possible to detect not only microbial nucleic acids in samples, but also microbial nucleic acids in containers, environments and reagents, increasing the difficulty of interpreting results. Therefore, improving the timeliness of detection and reducing background microbial interference are crucial for the clinical application of mNGS and the localization of medical institutions.
NGSmaster (Patent No. CN106404508A, CN106754339A) is an mNGS automated detection equipment independently developed by Jieyi Bio, which can complete the whole process of nucleic acid extraction and library construction in one stop. Compared with other mNGS automation processes, the NGSmaster-based mNGS automation process has the advantages of short detection cycle (16-18.5 hours), high safety (small size of the instrument, can be placed in a biological safety cabinet), high flexibility (the instrument 4 channels do not interfere with each other, on-the-go inspection), strong anti-pollution ability (single sample independent space), and adopts the PCR-free library construction method, which avoids the PCR preference and aerosol pollution risks, and ensures the authenticity of the mNGS results. Shorten inspection cycles.
The paper validates the automated process of NGSmaster based on the self-developed development of Jieyi Biotech. The results showed that after nucleic acid extraction and library construction of the culture strains by NGSmaster, they could be accurately identified under the average data volume of 15.5 M reads, and the whole genome coverage after sequence assembly reached 95.25±0.46%, and the coefficient of variation (CV) of the pathogen RPM detected on the simulated sample was within 10%.
The paper compares the detection performance of PCR-free library building method and the traditional PCR library building method in mNGS, and finds that the two library construction methods are consistent with the microbial RPM detected by Listeria monocytogenes and Enterobacter vaginalum with diluted concentration gradients, indicating that PCR-free will not affect the performance of mNGS detection, and can shorten the detection cycle and avoid the risk of aerosol pollution.
The paper also assessed the advantages of the NGSmaster automated process over manual process in reducing exogenous microbial contamination, with a total of 161 plasma samples (manual group) and 767 plasma samples (automated group) included. The results showed that a smaller proportion of epidermal symbiotics such as Staphylococcus epidermidis (autogroup vs. manual group = 34.2% vs. 24%), Propionibacterium acid-producing bacteria (autogroup vs. manual group = 0.62% vs. 0.26%), Marassezia spp. spp. (autogroup vs. manual group = 13.0% vs. 6.78%), Acinetobacter adrenherol (auto group vs. manual group = 20.5% vs. 12.3%), It shows that the automated process can greatly reduce the possibility of contamination by exogenous microorganisms compared with manual operation.
The study verified the detection performance of the mNGS automated process based on NGSmaster and PCR-free, and showed that the NGSmaster automated process of "sample in, library out" realized that mNGS detection was faster, more accurate and more convenient, making it easier for mNGS testing projects to be localized in medical institutions, and realizing the greater value of mNGS for infection diagnosis and treatment in critically ill and immunocompromised patients.