Drug Resistance Mutation Analysis Of Hepatitis B Virus Using Next Generation Sequencing Technology

BackgroundHigh-throughput sequencing(also known as second-generation sequencing)technology has a strong parallel sequencing capability and is widely used in life science.In recent years,reduction in the cost and advancements in technology have made this approach applicable for clinical gene detection,which is widely used in the field of individualized gene therapy for cancer and microbes infections..HBV drug resistance is an important clinical issue.The current clinical treatment for chronic hepatitis B is administration of nucleoside drugs.Long-term treatment by this kind of chemicals causes varying degrees of drug resistance.As anti-hepatitis B drug resistance of nucleoside is increasing monitoring the drug resistance mutation in HBV patients and timely adjustment of treatment have gained more concerns.HBV mutations are currently detected by polymerase chain reaction(PCR)-based direct sequencing,which is only effective when the specific mutation late in samples is higher than 20%.Cloning and sequencing of PCR products can identify mixed strains,but the operation is comlicated and the sensitivity is limited.In addition,real-time quantitative PCR,restriction fragment length polymorphism,and linear probe reverse hybridization have been used clinically.All these methods can only detect specific known resistance mutations,resulting in low efficiency.In this study,we established a NGS method to detect HBV drug resistance mutations for clinical drug guidance.This method can analyze all drug-resistant mutation points within the HBV polymerase gene for HBV patients.MethodFirst,the fusion primer srtategy was used to design primers for the Ion Torrent NGS platform(Waltham,MA,USA)and Miseq sequencing platform(San Diego,CA,USA).Based on the specific primers,we linked adapter and barcode sequences to the primers for high-throughput sequencing.Next,an Ampli Seq DNA library was prepared,and sequencing was conducted to obtain sequence data for the target area.In this study,specific primers were firstly designed to amplify the drug resistance region.Furthermore,the HBV plasmids was used to evaluate the detection efficiency of the NGS method in detecting drug resistance mutation.Type B and type C HBV genomic plasmids were mixed at ratios of 1:99,5:95,10:90 for Ampli Seq DNA library preparation.Sequencing was conducted on the Ion Torrent PGM and Miseq.At the same time,primers containing specific mutations were used to amplify the target regions by PCR amplification.Mutations were then introduced into the HBV mutant plasmid.A commercial HBV DNA standard substance was used for sensitivity experiments.Blood samples were collected from 15 patients with chronic hepatitis B and HBV DNA was extracted using the PureLink? Viral RNA/DNA Mini kit(Thermo Scientific).HBV DNA extracted from patient serum was detected using the established PGM method and also by Sanger sequencing.Result1.In this study,primers covering all known drug resistance mutation were designed for gene amplification.PCR products amplified using specific primers were confirmed by molecular weight detection and sequencing.PCR produced three fragments(165,324,and 135 bp).The drug resistance sites included in the amplicons were as follows: L80V/I,I169 T,V173L,L180 M,A181T/V,T184 A,S202G/I,M204I/V/S,V214 A,Q215S,N236 T,M250V,and G1896 A.2.Amplicon primers based on the Ion Torrent PGM and Miseq platforms were designed.The PCR product were used for molecular weight detection and sequencing to evaluate the performance of the sequencing primers.The constructed plasmids were identified by capillary electrophoresis and Sanger sequencing.The results showed that the HBV mutant plasmid was successfully constructed.3.Library preparation methods for the Ion Torrent? platform and Miseq platform were established.This method can be used for library preparation when serum HBV DNA > 50IU/mL.In simulated samples,mutation rates greater than 5% can be detected(SNR> 10).In the 15 cases of clinical samples,5 drug-resistant mutation were detected,while these sites were not detected by the Sanger method.ConclusionA NGS method for detecting HBV resistance mutations was established.This method can be used for dynamic clinical monitoring of hepatitis B virus drug resistance regional mutation.