Methodological Study On Rapid Diagnosis Of Pathogens By Metagenomic Nanopore Sequencing

Infectious disease is a local or systemic disease caused by pathogenic microorganisms（viruses,bacteria,fungi,parasites,etc.）,such as bloodstream infection,central nervous system infection,urinary tract infection,lower respiratory tract infection,abdominal infection and so on.There are high morbidity and mortality all over the world.In the treatment of infectious diseases,timely and accurate antimicrobial treatment is very important,but the rapid identification of pathogenic pathogens has always been a key problem for clinicians.Genome sequencing is an unbiased detection method,which can theoretically cover all pathogens.Nanopore sequencing is a long-reading and real-time sequencing technology.Compared with the sanger sequencing and next generation sequencing,it can accurately identify pathogens without too high sequencing depth and too long sequencing time.There is no doubt that it has great potential to apply in clinical pathogen diagnosis.Based on the above understanding,this study explored the clinical feasibility of nanopore sequencing using a large chorht of clinical samples in the etiological diagnosis of various infectious diseases,such as bloodstream infection,meningitis,urinary tract infection,eye infection and so on.The platform of pathogen sequencing and automatic data analysis based on metagenomic nanopore sequencing was initially built,and the potential of POCT in pathogen detection based on m NGS was further explored.Metagenomic sequencing is a powerful tool to speed up the diagnosis of bloodstream infection.However,difficult to deal with blood samples and a large amount of host nucleic acid background seriously affect the detection of pathogens,and put forward higher requirements for the depth of sequencing,resulting in a great waste of sequencing resources.Therefore,the removal of specific host DNA from samples is necessary and important in the study of pathogenic microflora based on metagenomics.Based on the above understanding,we established and evaluated a method based on PMA photolysis to remove host DNA,which can specifically remove host DNA without tedious washing steps.This method is suitable for blood,and has great potential for urine,bronchoalveolar lavage fluid,saliva and other types of samples,especially for low biomass samples,without the loss of pathogen nucleic acid to remove the host DNA to the maximum extent.In addition,we have also established a metagenomic nanopore sequencing process for the detection of bloodstream infection pathogens,and the lowest detection limit is 10CFU/m L,which provides new ideas and powerful tools for the rapid diagnosis of pathogens in clinical bloodstream infections.In this study,a metagenomic nanopore sequencing（m NPS）workflow was developed to detect and identify pathogens and drug resistance genes in urine samples of 76 patients with acute UTI.First,we used 20 samples to compare three different library construction methods from many aspects（construction time,accuracy,etc.）.It was proved that the positive results of the library preparation by PCR barcoding kit（SQK-PBK004）had the highest concordance rate with clinical urine culture,and made it possible to detect drug resistance genes in downstream analysis.The results showed that the sensitivity and specificity of m NPS method were 86.7%（95% CI 73.5%～94.1%）and 93.5%（95% CI78.3%～99.2%）,respectively.It is an effective method for rapid and accurate detection of bacterial pathogens of UTI and can successfully predict drug resistance phenotype.We further optimized the workflow of this method.By combining reliable organism lysis,unbiased sequencing,comprehensive reference databases and automatic data processing software,we reduced the turnaround time from the receipt of the sample to the output result to less than 2 hours,and had high sensitivity（78.9%）and specificity（100%）for the detection of clinical urinary tract infection,especially multi-species infection,which solved the problem of slow characterization of fungal and bacterial infections and difficult detection of parasites and viruses in clinic.Through the automated bioinformatics analysis process,we greatly demonstrated the potential of nanopore sequencing in clinical POCT.To sum up,based on the metagenomic nanopore sequencing technology,we explored,established,and optimized the diagnosis workflow for pathogens of common clinical infectious diseases,evaluated its diagnostic performance,and verified its great potential in clinical application.