The Role Of Variation Characteristic And Genetic Evolution Of HBVX Gene Quasispecies In Hepatocellular Carcinoma Progression

Objective:(1)To discuss the relationship between the dynamic changes of quasispecies and Hepatocellular Carcinoma(HCC),also the relationship between the dynamic changes of mutation sites and HCC.(2)To reveal the genetic evolution of quasispecies.(3)To compare the sensitivity and accuracy of Next Generation Sequencing(NGS)and Clone-based Sequencing(CBS)for characterizing the genetic variation of HBV quasispecies within the X region.Methods:(1)PCR primers suitable for most of HBV DNA X gene were designed through document retrieval and primer design software,then specificity PCR amplification was validated.we chose optimized methods and definite PCRreaction system and reaction conditions.(2)2 individuals(1 was diagnosed as HCC,1was not)were selected from a prospective cohort of high-risk individuals in Guangxi,China.Peripheral blood samples(5 ml)were collected every six months or one year before diagnosis of HCC,giving a total of 12 samples.HBV DNA was extracted from serum,the HBV X gene was amplified using nested PCR,and PCR products were sequenced using NGS or CBS.(3)NGS and CBS were used to analyze the X region's quasispecies.Heterogeneity characterization of quasispecies was conducted using bioinformatics analysis.Then compare the number of qualified strains and complexity were obtained by NGS and CBS.The phylogenetic trees were used to analysis HBV quasispecies along with the course.Results:9348 quasispecies sequences were obtained by NGS,that was much larger than 98 qualified strains by CBS.NGS detected 40 mutation sites and 1insertion,CBS detected 41 mutation sites and 2 insertion.29 mutation sites were detected by both NGS and CBS,11 sites only by NGS and 12 sites only by CBS.Some mutations associated with HCC,such as A1762T/G1764 A double mutation?C1773T?C1653T?T1753G and T1768 A.Complexity derived from NGS was higher than that from CBS(t=4.20,P<0.05).The d derived from NGS was lower than that from CBS(t=-7.33,P<0.0001).Conclusion:(1)Found that some mutations associated with HCC,could be used to verify the HBV related HCC high-risk objects,finally optimizing the scheme of early screening for HCC.(2)This study confirmed that there were HBV quasispecies in the course of HCC,and they could influence the progression of HCC.The change tendency of quasispecies and superiority strain were more significant in 2-3 years,while in1-1.5 years tend to be stable.(3)In terms of bases mutations,NGS and CBS showed similar ability to discover SNPS,though CBS was better at detecting fragment insertions and deletions.In terms of quasispecies reseach,a:The NGS experiments had advantages of high flux and saving both time and labour,It was suitable for large samples.But the cost was expensive.While The CBS experimental steps were tedious and time-consuming,but the cost was low;b:In terms of results analysis,NGS data was big,and needed professional platform and the software for data processing,While the CBS results processing were easy;c: In terms of detection ability,The number of qualified strains obtained by NGS was much larger than that by CBS,because of the high flux.