Performance verification and results analysis of DNA workflow for metagenomic next-generation sequencing
- VernacularTitle:病原宏基因组测序DNA检测流程性能验证及结果分析
- Author:
Shangdong YANG
1
;
Yang XIAO
1
;
Wen XI
1
;
Zhe LIU
1
;
Fang WANG
1
;
Xiaoqin WANG
1
Author Information
- Publication Type:Journal Article
- Keywords: metagenomic next-generation sequencing(mNGS); DNA detection; performance verification; reference material
- From: Journal of Xi'an Jiaotong University(Medical Sciences) 2025;46(1):162-168
- CountryChina
- Language:Chinese
- Abstract: Objective To establish a performance verification scheme for the metagenomic next-generation sequencing(mNGS)DNA workflow.Methods Reference materials and clinical samples were used for conducting experiments.The mNGS detection results were evaluated in terms of limit of detection(LOD),repeatability,robustness,anti-interference ability,specificity and accuracy,as well as the patterns of library construction and the performance of sequencers.Results All species in the reference materials were stably detected,and the LOD of mNGS was 5.0E+02 CFU/mL(copies/mL).The repeatability was 100％and the within-batch(coefficient of variation)CV ranged from 8.53％to 38.73％.The linear correlation coefficient|r|＞0.9 was found between the input pathogenic microorganism concentration and the read count.Meanwhile,the experimental robustness was found to be good.The results of the anti-interference test showed that the higher concentration of human DNA inputted,the fewer pathogenic microorganism read counts detected by mNGS.Meanwhile,the read counts of related species presented a proportional relationship with the corresponding pathogenic microorganisms concentration inputted,which meant the validation of the cross-interference test had been passed.Furthermore,the detection result of D0 was negative.The accuracy of clinical samples testing was 90.9％(10/11).In addition,the library quality control results obtained by the automatic liquid handling workstation and manually operation were all acceptable.The performance of the three Illumina sequencers met or were better than the factory standards.Conclusion The clinical laboratory performance verification scheme for mNGS detection was established,which included the design for reference materials,comparison of different patterns for library construction,and performance evaluation of the sequencers.More importantly,the performance verification scheme can be used to evaluate and ensure the quality of mNGS DNA workflow detection process.
