Research On The Application Of Metagenomic Sequencing Technology Based On Illumina Nextseq2000 In Respiratory Pathogen Identification

Objective:Based on Illumina Nextseq2000 platform,a metagomic sequencing scheme was established.The metagenomic sequencing characteristics of Illumina Nextseq2000 were obtained,and the application value of Illumina Nextseq2000 in pathogen identification of respiratory tract infection was discussed.Methods:1.Single and multi-pathogen simulation samples were established by adding four pathogens(Staphylococcus aureus represented gram-positive bacteria;Vibrio parahaemolyticus stands for Gram-negative bacteria;Candida albicans stands for fungus;Influenza B virus stands for RNA virus)in gradient dilution to human healthy pharyngeal swab eluent and non-pathogenic bronchoalveolar lavage solution.Through metagenomic sequencing and fluorescence quantitative PCR detection of simulated samples,the library construction process and nucleic acid extraction method were optimized,and the Illumina Nextseq2000 metagenomic sequencing scheme was established to discuss the sequencing performance and characteristics of Illumina Nextseq2000 metagenomic sequencing.2.Metagenomic sequencing was performed on negative fluorescent quantitative PCR samples from 2 fever outpatients in Huazhong University of Science and Technology Union Shenzhen Hospital in March 2022 by RNA library building method,and the detection results were obtained.To verify the application value of Illumina Nextseq2000based metagomic sequencing method in the identification of unknown pathogens in upper respiratory tract.3.From January 2022 to November 2022,55 pneumonia cases collected in the Hospital of Qianhai Shekou Free Trade Zone of Shenzhen were divided into immunosuppressive group and non-immunosuppressive group,and metagenomic sequencing was performed by DNA library building.The culture results were compared and statistically analyzed,and the application value of Illumina Nextseq2000 based metagenomic sequencing scheme for DNA identification of pathogens in lower respiratory tract was verified.Results:1.In the results of multi-pathogen simulated metagenic sequencing,the number of homo reads in the multi-pathogen simulated sample of bronchoalveolar lavage solution was higher than that in pharyngeal swab eluent.Pathogens with sequencing data volume of 20M and 50M detected the same type of pathogen.The number of pathogens reads with sequencing data volume of 20M accounts for 6.87%～75.71%of that of50M,with an average ratio of 46.86%.The number of homo reads in DNA library was higher than that in RNA library.2.In different concentrations of Staphylococcus aureus and Candida albicans diluents,the Ct values of DNA extracted by QIAamp DNA Blood&Tissue Kit(filtration)were lower than those of the other two methods(P<0.05).In Vibrio parahaemolyticus 10~4～10~5diluent,The Ct values of DNA extracted by TIANLONG Cq Ex-DNA extraction Kit(magnetic bead method),QIAamp DNA Blood&Tissue Kit(filtration method),and DNeasy Power Soil Pro Kit(grinding bead+filtration method)were the same(P>0.05).3.The pathogen dilution ratio of the single pathogen simulated sample was roughly linear with the Ct value of quantitative fluorescence PCR,the Ct value of quantitative fluorescence PCR was roughly reversing linear with the number of log-transformed metagenomic sequencing reads,and the DNA pathogen concentration was roughly linear with the number of log-transformed metagenomic sequencing reads.Illumina Nextseq2000 metagenomic sequencing showed lower detection limits for the four pathogens than fluorescence quantitative PCR.4.Metagenomic sequencing was performed on 57 patients with respiratory tract infection.The potential pathogens of patients 1 with upper respiratory tract infection were Haemophilus parainfluenza,Fusobacterium nucleatum,Actinomyces oris,Burkholderia contaminans,Ralstonia insidiosa,and Comamonas testosteroni.In patients 2 with upper respiratory tract infection,the potential pathogen was Haemophilus influenzae.A total of 64 species of microorganisms were detected in bronchoalveolar lavage fluid of 55 patients with pneumonia,including 14species of Gram-positive bacteria,46 species of Gram-negative bacteria,1 species of fungi,2 species of mycoplasma,and 1 species of DNA virus.The microorganisms with the top five reads detected and the top four cases detected were Gram-negative bacteria.5.Statistical analysis was conducted on the culture results of bronchoalveolar lavage fluid of pneumonia patients and the results of metagenomic sequencing.The results showed that there was a statistically significant difference in the detection reads of Corynebacterium striatum and Stenotrophomonas maltophilia(P<0.05),and the detection rate of metagenomic sequencing was higher than that of culture.The sensitivity,specificity,consistency,and Kappa value of metagenomic sequencing compared with culture for Klebsiella pneumoniae detection were 81.82%,87.50%,85.71%,and 0.677.The results of pathogen detection of bronchoalveolar lavage fluid in immunosuppressive group and non-immunosuppressive group showed that there were statistically significant differences in the detection number of Stenotrophomonas maltophilia,Corynebacterium striatum,Achromobacter xylosoxidans,and Alcaligenes faecalis(P<0.05),and the detection rates in immunosuppressive group were higher than that in non-immunosuppressive group.Conclusion:1.Compared with DNA library building methods,RNA library building method can remove part of r RNA,reduce the number of homo reads.In metagenomic sequencing,the library construction method should be flexibly selected according to the characteristics of samples and the purpose of sequencing.The detected pathogens with 20M were found to be the same as 50M.However,there is a large fluctuation(6.87%～75.71%)between 20M and 50M in the ratio of detection reads.In order to ensure data quality,50M should be used for the experiment.2.Compared Tianlong Cq Ex-DNA extraction Kit(magnetic bead method),QIAamp DNA Blood&Tissue Kit(filtration method),DNeasy Power Soil Pro Kit(grinding bead+filtration method),a total of three kinds of DNA extraction kit,QIAamp DNA Blood&Tissue Kit(filtration method)has the highest efficiency of DNA extraction.3.The detection limit of Illumina Nextseq2000 was lower than that of fluorescence quantitative PCR.There was a linear relationship between log-transformed detection reads,Ct value of fluorescence quantitative PCR and DNA pathogen concentration,suggesting that metagenomic sequencing could assist in determining pathogen load on the basis of detecting unknown pathogens.4.Compared with the results of fluorescence quantitative PCR and culture,metagenomic sequencing has highly sensitivity and accuracy.Based on the established Illumina Nextseq2000 metagenomic sequencing protocol,it can be applied to the unknown respiratory pathogen samples to identify possible pathogens and find the mixed infection of patients.