Research And Application Of Nanopore Sequencing Technology Based On Pathogen Cell-free DNA And Rolling Circle Amplification

Infectious diseases pose a major threat to human health and global public health security,and pathogen identification is the prerequisite for accurate prevention and control.The detection cycle of pathogen isolation and culture is long,which takes 3-5days,and most pathogens cannot be cultured in vitro.Molecular biological detection such as PCR requires known pathogen information,and can only detect a single or limited pathogen.Metagenomic sequencing does not require prior knowledge,and can detect all microorganisms in a sample at one time,and can simultaneously detect pathogenic variation,drug resistance genes,virulence genes,etc.At present,the next generation sequencing platform and third generation sequencing platform have their own advantages in metagenomic sequencing.The next generation sequencing has high throughput and high accuracy,while the third generation sequencing has real-time and long read length,which can obtain more complete gene sequence information.In metagenomic sequencing,the high background level of host nucleic acid is the key to the sensitivity of pathogen detection.Cell-free DNA(cf DNA)is the free and partially degraded DNA outside the cell,and the level of host nucleic acid is significantly lower than that of tissue samples or other body fluid samples.Therefore,pathogen detection based on cf DNA has attracted much attention.In this study,we explored the technology of metagenomic sequencing based on cf DNA for pathogen detection.Rolling circle amplification(RCA)was combined with cf DNA to construct simulated samples containing DNA fragments of eight microorganisms(80bp)and human genomic DNA fragments(170bp).Short cf DNA sequence extension was achieved,and Nanopore sequencing was used to achieve 100%detection rate of target microorganisms in simulated samples,indicating that cf DNA sequencing after RCA amplification can achieve effective detection of pathogens.The blood sample of a patient with respiratory tract infection was simultaneously subjected to next-generation sequencing of cf DNA and the third generation real-time Nanopore sequencing after RCA amplification,and blood microbial isolation and culture.The results showed that both next-generation sequencing and Nanopore sequencing could effectively detect Klebsiella pneumoniae sequences,accounting for 83.5% and 85.6% of the total microbial sequences,respectively.Sequence number ranked first among all detected microorganisms,which was consistent with the results of pathogenic culture.The detection time required for next-generation sequencing,Nanopore sequencing and isolation and culture was about 54 h,17.5 h and 50 h,respectively.This indicates that third-generation Nanopore sequencing combined with cf DNA and RCA amplification can effectively detect pathogens,and has a shorter detection cycle than the other two schemes.In order to further evaluate the application value of Nanopore sequencing technology based on cf DNA and RCA amplification in clinical infectious diseases,137 different types of clinical body fluid samples were collected for microbial isolation,culture and sequencing.All the 137 samples were positive for isolation and culture.A total of 33 kinds of microorganisms were cultured,including common clinical contaminating microorganisms,but no virus isolation and culture were performed.cf DNA-based next-generation sequencing detected 24 microorganisms in 137 samples,with a positive rate of 75.2%,and 3 different types of herpesviruses were detected in 6samples.Among them,detected 9 pathogens in at least three samples,including Klebsiella pneumoniae,Pseudomonas aeruginosa,Acinetobacter baumannii,etc.The consistency between the results of isolation culture and the next generation sequencing was 41%.Considering that blood sample collection is relatively minimally invasive and can reduce the harm caused by sampling the infection site of critically ill patients,we selected the blood samples for RCA amplification and Nanopore sequencing.It was found that the consistency between the isolation and culture results of 28 blood samples and the next generation sequencing was 89.3%,and the consistency between the isolation and culture results of 28 blood samples and the RCA-based Nanopore sequencing was 78.6%.The consistency of next-generation sequencing and RCA-based Nanopore sequencing in pathogen detection was 81.5%,suggesting that the three methods had good complementarity in pathogen detectionIn summary,this study established a Nanopore sequencing technology based on pathogenic cf DNA and RCA amplification,and evaluated its efficiency in the identification of pathogens in infectious diseases.It provides a direction for the better application of short cf DNA sequences in Nanopore sequencing.Compared with cf DNAbased next-generation sequencing,the detection cycle is further shortened.This study confirmed that cf DNA based on blood samples can be used to identify pathogens of infectious diseases,explored the possibility of rapid non-invasive diagnosis of infectious diseases,and provided an important basis for the rapid determination of infectious diseases in the future.