Study On Transmission And Microevolution Of Pathogen Based On Whole Genome Sequencing

Pathogens pose great threats to human health.Tracing the source and law of its transmission as soon as possible,and adopting the timely prevention and control measures can help preventing further spread of the epidemic.Studies on the adaptive mechanism of pathogens that can build long-term infection in the human body can help pathogen detection,drug development and disease treatment.The development of sequencing technology enable researchers sequence large quantities of samples at a lower cost and faster speed,thus promoting the studies on transmission and micro evolution of pathogens.Experimental and analytical methods for pathogen transmission and micro evolution were established,and the technical points were also defined.Then,three pathogens SARS-CoV-2,Pseudomonas aeruginosa and Helicobacter pylori were studied based on their characteristics,scientific problems and research objectives respectively.Sample collection,genome sequencing and data analysis were carried out to achieve the research objectives,and some methodological innovations were made.The specific research work includes the following four aspects:Firstly,complete methodology suitable for most pathogen transmission tracking and microevolution analysis were established.This section first explains the reasons for choosing these three pathogens and their representativeness.After that,based on the characteristics of bacterial or viral pathogens and the goals of pathogen transmission tracking and microevolution analysis,complete methodological methods including sample collection,processing,related information collection,sequencing experiments,and sequencing data analysis were established,which can help researchers quickly and accurately select the appropriate experimental and analysis schemes to track the transmission and conduct microevolution analysis of pathogens rapidly and accurately.Secondly,transmission and microevolution analysis of SARS-CoV-2 during the recurrence of COVID-19 in Guangzhou in March 2020 were performed.In consideration of large amplification bias for SARS-CoV-2 genome based on the ARTIC primmer set,a primer panel with ～1000-bp amplicon lengths for full-genome sequencing of SARS-CoV-2 was developed,which could obtain more uniform genome coverage.At the same time,the primer set also performed well in nanopore sequencing,and it is confirmed that accurate viral genome consistent sequences can be obtained even under low sequencing flux,but the accuracy of nanopore sequencing in detecting intra-host mutations was low.Based on the primmer set for SARS-CoV-2 genome,near-real-time genomic surveillance of SARS-CoV-2 was conducted during the resurgences of COVID-19 in Guangzhou that began in March 2020.The lineages of the virus were then classified and the genome-based phylogenetic analyses was performed.The results showed that the epidemic was caused by two lineages virus from imported cases.In the earlier period of this epidemic,the viruses carried by imported viruses from overseas had been found had high genomic diversity and been assign to multiple lineages,and provided information about the epidemics in countries where research data are insufficient or unavailable.Then,the genomes of the virus that spread locally in Guangzhou are relatively single,local transmission was predominately attributed to two specific variants imported from Africa.Then,further analysis were conducted based on the epidemiological investigation information of local cases and the genome characteristics of the infected virus,and the virus transmission chain centered on the three local places was traced.In general,the transmission chain was reconstructed based on genome data,providing scientific basis and data for the implementation of strict epidemic prevention and control and other public health policies for immigrants,and reflecting the role of virus whole genome sequencing in the prevention and control of the new resurgences of epidemic.Then,the microevolution of longitudinal Pseudomonas aeruginosa in inpatients based on whole genome sequencing were studied.Taking the Pseudomonas aeruginosa samples from seven elderly patients(P1 to P7)with prolonged intensive care unit(ICU)stays of 4 – 19 months duration as the research subjects.Clinical samples,such as sputum specimens and wound secretions,were collected from the patients for routine bacterial cultures and species identification.Pseudomonas aeruginosa was the most common bacteria among all the isolates,so Pseudomonas aeruginosa was selected for whole-genome sequencing,and the Longitudinal Pseudomonas aeruginosa samples were used to study the dynamic and microevolution of Pseudomonas aeruginosa in hospitalized patients.The results of phylogenetic analysis based on SNP of core-genome and multilocus sequence typing(MLST)showed that two of the cases were infected with the same ST type strain,and it was also found that multiple Pseudomonas aeruginosa clone transient occurred in the two patients over time.This is the first report of P.aeruginosa clonal turnover in inpatient patients.Through mutation-based analysis of the genome diversity,hypermutator phenotypes were found in two MLST strains isolated from two patients,and the hypermutators had significantly more microindels in homopolymeric regions.And the two hypermutators both found non-synonymous mutations in DNA mismatch repair(MMR)genes mutL.In addition,this study also conducted a horizontal comparison of Pseudomonas aeruginosa strains of different MLST types from 7 patients,and found that multiple genes were found to have mutations in the independent microevolution process.Many of these genes are related to the adaptive process of Pseudomonas aeruginosa from the in vitro environment to the in vivo environment,such as fptA and pvdS,and some others are related to drug resistance,such as mexZ,lasR,fleQ.Besides,two deletions variations related to drug resistance genes and flagellar genes were identified during the analysis of structural variations.In summary,this study emphasizes the importance of longitudinal surveillance of P.aeruginosa hypermutators based on whole-genome sequencing.The diversity of the bacterial in the host and the vertical and horizontal comparisons showed that the Pseudomonas aeruginosa in ICU patients has a convergent microevolution mode,which deepens the understanding of the microevolution of Pseudomonas aeruginosa in the host under the background of nosocomial infection.Finally,a heterogeneous sub-clonal phasing method was developed,and the diversity and micro-evolution of Helicobacter pylori in the host were analyzed.In this study,cases with samples of Helicobacter pylori isolated and cultured from multiple regions of the stomach were selected as the research object,and they were divided into groups with differences in resistance to levofloxacin and clarithromycin(14 persons,41samples)and groups without differences in resistance(11 persons,31 samples).Combined the NGS and single-molecule sequencing technology,the Helicobacter pylori culture broth(not serially subculture)was used for whole-genome sequencing.The consensus genomes of each Helicobacter pylori sample were obtained,and the genome-based phylogenetic tree was constructed.At the consensus genome level,it was found that there was a case of multiple infections,in which different strains were colonized in different region of the stomach,and they were assigned to the East Asian lineage(hsp EAsia)and the European lineage(hp Europe).After in-depth analysis at the sub-clonal level,another case was found that also had multiple infections.For tne other cases,the diversity within the host is relatively low,and it can be considered that the diversity that comes from microevolution within the host.Through comparative analysis,it was found that,at the level of consensus sequence analysis and sub-clonal analysis,the diversity of strains within the host of the case group with drug resistance differences was statistically significantly higher than that of the case group without drug resistance differences.In order to further analyze the diversity of Helicobacter pylori host in detail,NGS and Pac Bio sequencing data were combined,the mutation sites were phased in the host at the sub-clonal level,the characterization the sub-clonal haplotype at the sub-clonal haplotype level were also described.The results showed that,after excluding samples with multiple infections,mutations imported by recombination were an absolute dominant power to generate the intra-host diversity,rather than spontaneous mutations.Among the 16 samples with more than 400 within-host mutations,the proportion of mutations contributed by recombination was conservatively estimated to be 94.5%(SD,3.38).By tracing the donor of the mutations imported by the recombination,and visualizing the phased region block,the length of the recombination events was obtained,with a median length of 445 bp.Furthermore,this study focused on the levofloxacin resistance-related gyrA gene and the clarithromycin resistance-related23 S rRNA gene.In the two cases,intra-host diversity of the resistance mutations of gyrA were observed,and the resistance mutations in the haplotypes clearly derived from the recombination region block.In addition,in case P4,the drug resistance mutation on23 S rRNA gene was also found to come from recombination.In general,based on the combination of NGS and single molecule sequencing,through the innovation of analytical methodology,this research studied the microevolution of Helicobacter pylori at the sub-clonal haplotype level for the first time,and obtained a high-resolution map of the genome in-host.The results show that recombination is the main force for the diversity of Helicobacter pylori in the hosts and may lead to differences in drug resistance among different clones in the same host,which increases the understanding of Helicobacter pylori microevolution.In general,analysis of transmission and microevolution of three different pathogens were performed based on whole genome sequencing,which not only realized their analysis for pathogen transmission,but also conducted in-depth research on the adaptability and microevolution of pathogens in hosts based on longitudinal samples or at sub-clonal level,which all fully reflected the role of whole genome sequencing in studies on pathogen transmission and micro-evolution.