A Comparative Study On The Characterization Of Hepatitis B Virus Quasispecies By Clone Based Sequencing And Third Generation Sequencing

BackgroundHepatitis B virus(HBV)infects more than 350 million people worldwide,and more than 1 million people die each year from liver cirrhosis,hepatic failure and hepatocellular carcinoma(HCC),HBV infection has become one of the threats to global public health security.The extremely high replication rate and the proofreading deficiency during reverse transcription may result in a high mutation rate in the HBV life cycle.The generated variants with genetic heterogeneity are defined as the viral quasispecies(QS).QS variation of HBV was demonstrated to have an impact on the response of antiviral treatment,so more comprehensive understandings toward mutations and QS of HBV full-length genome will help to guide the clinical antiviral treatment.By so far HBV QS analysis depends on direct polymerase chain reaction(PCR)sequencing,clone-based sequencing(CBS)and next generation sequencing(NGS).Direct PCR sequencing only detected mutations when mutations reach approximately 20%of the total HBV QS population.CBS is considered as the "gold standard",but this method is time-consuming,laborious,with relative low sensitivity.NGS has obvious advantages over the previous two methods,but there are limitations,such as the restriction of read length(100-300bp)and the need to combine the results of molecular cloning.Presenting as the third generation sequencing(TGS)technology,the innovation and development of single-molecule real-time(SMRT)sequencing make giant increasing application in a variety of genome research in recent years.Along with the significant increase in reading length,the new research opportunities have been brought forward to HBV QS.MethodsA total of 13 examples were enrolled,containing control group and experiment group.The control samples consisted of HBV full gene genotype B and genotype C plasmids,named C01,C02 and C03.The recruited patients of experiment group were admitted to the Department of Infections Disease of the Second Hospital of Shan Dong University from February 2015 to December 2015 and met the clinical diagnostic criteria for chronic hepatitis B(CHB).In this study,all the samples were taken by HBV DNA extraction,PCR amplification,CBS and TGS,and then from the analysis of HBV QS complexity,diversity,and HBV mutation detection to investigate the utility of TGS DNA sequencing to characterize genetic heterogeneity of HBV QS and assessed the possible contribution of TGS technology in HBV QS studies.ResultsIn terms of the quantity of HBV QS sequences,an average of 21.77±3.63 clones per sample was obtained by CBS and 560.31±233.42 sequences of HBV full-length genome per sample was obtained by TGS.The number of sequences generated by TGS was significantly larger than those in CBS(P<0.01).The result of comparing the heterogeneity determination capacities of CBS and TGS from control group showed that the proportion derived from both CBS and TGS were consistent with the expected standard value,and the proportion derived from TGS was more approximate to the true value.Furthermore,complexity values derived from TGS of control group were highly correlated with those from CBS.Both CBS and TGS showed high consistence with their theoretical values.In patient samples,at nucleotide level except PreS2 region(P=0.021)and at amino acid level except PreSl region(P=0.025)and PreS2 region(P=0.019),the QS complexity value derived with TGS showed no difference with that derived with CBS within HBV genome and BCP region,C region,P region,PreC region,RT region,S region and X region(P value>0.05,Student's t test or Mann-Whitney test).The correlation of the two methods showed that the QS complexity values at nucleotide level and amino acid level derived with the two methods were highly correlated except for whole genome(P= 0.13).As indicated by Bland-Altman approach,a high level of agreement was discovered between CBS and TGS except for HBV genome.Both at nucleotide level and amino acid level,diversity values and dS,dN derived with the two methods had a high consistency,however,at the nucleotide level,the QS distance value derived with the TGS within BCP region and X region(P value =0.044 and 0.021,respectively)and dS at S region(P=0.030)had a significant difference with the genetic distance derived with the CBS.The correlation of the two methods showed that the diversity value at nucleotide and amino acid level,and dS,dN calculated from CBS was highly correlated with that calculated from TGS method.Bland-Altman analysis demonstrated that there was a high consistency by comparing the two methods for each region.According to reference D00330 for genotype B and reference AB033556 for genotype C,for CBS sequencing detected a total of 1101 variations with an average of 110.1±26.98 variations per patient.TGS sequencing detected 3059 variations at an average of 305.9±162.45 variations per patient.For common single allele mutation which had described by several reports RT resign,S region,C region,X region,PreC region,BCP region for the two methods in each patient and totally 50 mutual mutations were detected both by TGS and CBS,reaching a positive rate equaled 15.15%and no mutation was detected only by CBS(positive rate =0%),40 mutations were detected only by TGS and the positive rate was 12.12%,which was significantly higher than CBS(X2 =157.14,P<0.01).The result showed that TGS had a much higher sensitivity for variation detection than CBS.Furthermore,Kappa consistency test(0.645)demonstrated that the consistency of the two methods was substantial.For combination mutations,A1762T/G1764A(5/10 patients)and M204[?/?]/A181T(4/10 patients)were detected only by TGS.Phylogenetic trees were generated using the full-length HBV genomes and two trees were very similar.Besides,phylogenetic tree constructed from TGS data was more complex than that constructed by CBS data.ConclusionsThe present study indicated the utility of TGS to characterize genetic heterogeneity of HBV QS matched perfectly with CBS which was considered as "gold standard".In terms of quantity and the sensitivity of mutation detection,TGS was superior to CBS.However,TGS for the study of HBV QS was still in its infancy stage,so larger sample size was needed to have a further verification.With decreases in costs and improvements in sequencing quality,TGS may be used to be convenient for clinicians to diagnose and treat the patients with different kinds of HBV.