Application Of High-Throughput Sequencing In The Study On HIV Transmission Network Among Men Who Have Sex With Men

BackgroundThe deep understanding and research of the characteristics of the Human immunodeficiency virus type 1 ?HIV-1? transmission network and the dynamics of the communication network will contribute to the monitoring, prevention and treatment of HIV-1.At present, a large number of studies described HIV-1 transmission network characteristics by analyzing HIV-1 pol gene. All of these sequences were obtained form directing sequencing of nest PCR products or from existing sequence databases. For the newly HIV-1 infected people,env gene and gag gene may be more suitable for analyzing the transimission network because of their evolution rate higher than the pol sequence.The information obtained by these methods was small, it can only detect the sequence of dominant quasispecy and can not infer the direction of transmission at the level of HIV-1 quasispecy. High-throughput sequencing technology allowed the detection of low levels of variation in viruses with a higher sensitivity than normal sequencing. It can also indicate the direction of transmission. In this study, we conducted a transmission sub-network analysis of the pol, gag, and env sequences of HIV-1 in generation and Hiseq high-throughput sequencing ?referred to asHiseq sequencing?, and to explore the application value.ObjectiveExplore the appropriate parameters of HIV gag sequence and env sequence to construct transmission network. Explore the applicate in the dynamic transmission subnetwork analysis of HIV pol sequence, gag sequence and env sequence. Establish a high-throughput sequencing method for Hiseq based on population analysis of HIV pol, gag and env sequences was established. Explore the applicate in the dynamic transmission subnetwork analysis of HIV pol sequence, gag sequence and env sequence by the Hiseq high-throughput sequencing technology.Subjects and Methods1. SubjectsStudy subjects consisted of HIV-1-infected individuals were recruited from the Beijing PRIMO Cohort, a prospective cohort of HIV-negative MSM who were screened for HIV every 8-12 weeks at the Beijing You'an Hospital, Beijing, China, between 2010 and 2012. We chose 100 cases from the cohort using a simple random sampling method, then separated their peripheral blood mononuclear cells ?PBMC? and a plasma ?1.5ml / support? and stored at -80?,respectively, spare. The HIV-1-negative MSM subjects were followed up at two-month intervals. EDTA blood specimens were collected within two months after seroconversion and subjects did not receive antiviral therapy until they were sampled.2. Experiment procedure?1? DNA were extracted from PBMC.?2? For DNA, the specific gene regions were amplified by nest-PCR.?3? Direct sequencing for PCR products and confirm HIV subtype.?4? The dynamic analysis of the transmission sub-network of pol, gag and env sequences was carried out.?5? Primers of pol, gag and env gene was designed for Hiseq sequencing and the reaction conditions were optimized. Then nested PCR was used to amplify the target gene regions.?6? After the PCR product was purified, the DNA library was constructed and then subjected to Hiseq sequencing.?7? The Hiseq sequencing data were preliminarily processed, and then gene distance and phylogenetic analysis of HIV quasispecies and its application value in the dynamic analysis of HIV transmission sub-network were explored.Results1. HIV-1 subtype and epidemic statusIn this study, the subtypes of HIV-1 contained CRF01_AE, CRF07_BC, B, B',CRF55₀1B,CRF65_cpx and unknown subtypes,the respective proportions as follows: 42.7%?41/96?, 25.0% ?24/96?, 15.6% ?15/96?, 1.0% ?1/96?, 1.0% ?1/96?, 3.1% ?3/96? and 11.6%?11/96?. CRF55₀1B and CRF65_cpx subtypes were first found in Beijing.2. Explore of pol, gag and env three sequences for HIV-1 transmission network analysisThe pol sequences of 70 specimens were subjected to transmission network analysis consists of six clusters, consisting of 11 nodes and 7 edges, with clustering rates of 15.71%?11/70?. The results of Fisher test showed that there was no significant difference in the relationship among HIV-1 subtypes, age, education, marital status and CD4+ T cell count in the five influencing factors?Psub=0.2058, Page=0.8652, Pedu=1.000, Pmar=1.0000, PCD4=0.7568?.Fixed bootstrap?90% to investigate the genetic distance used to transmission network analysis for gag and env sequences. Fisher test showed that there were no significant differences between gag sequences with GD?).5%, 1.5%, 2.5% or 3.5%, and Bootstrap?90%?Pgag0.5=0.0257, Pgagi.s=0.7083, Pgag2.5=0.0876, Pgag3.5=0.0156, ?g=0.01? and env sequences with GD?0.5%, 1.5%, 2.5%, 3.5% or 4.5% and Bootstrap?90% with pol sequences GD?1.5% and Bootstrap?90%?Penv0.5=0.0257,Penv1.5=0.0811,Penv2.5=0.3824,Penv3.5=0.3749, Penv3.5=0.0160, ? e=0.0083? in the sample correlation analysis. The comparative analysis of the correlations of different GD values among the pol, gag and env sequences. Fisher test results showed that gag with env sequences, for GD?1.5% and Bootstrap> 90%, were statistically significant ?Pgei s=0.0125?. Pol with gag sequences and pol with env sequences, for GD?3.0% and Bootstrap?90%, were statistically significant ?Ppg3.0 = 0.0008,Ppe3.0<0.0001?; pol with env sequences and gag with env sequences, for GD?4.5% with Bootstrap?90%, were statistically significant ?Ppe4.5<0.0001, Pge4.5 = 0.0005?.The results of the HIV-1 transmission subnetwork analysis of the pol, gag and env sequences indicate 2 specimens?16014, 16035?, 3 specimens?16014, 16017 and 16035? and 4 specimens?16014,16017, 16064 and 16035? that play an important role in HIV-1 transmission. And, while, it suggested an possible transmission path.3. Methodological establishment of Hiseq sequencing for HIV-1 transmission networkThreshold of quasispecies to conduct the transmission network was explored based on env sequence. The first 5, 10, and 20 quasispecies ?labeled A, B, and C groups? in each specimen was selected to analysis the gene distance. It is showed that the results of group A, B, C were not statistically significant ?P=0.5889?. The same with phylogenetic tree analysis' result.Therefore, after considering this study, we decided to select the first five quasi-population sequences for transmission network analysis.4. Explore of pol, gag and env sequences by the Hiseq sequencing for HIV-1 transmission network analysisThe pol, gag and env sequences by the Hiseq sequencing of 40, 42 and 42 specimens of CRF01_AE subtype were subjected to transmission subnetwork analysis. For pol sequences, it was found that 7 specimens ?16001,16003, 16014, 16029, 16082, 16088 and 16097? that play an important role in HIV-1 transmissionand 3 main paths of 10 subnetworks. For gag sequences,it was found that 10 specimens ?16004, 16011,16014, 16032, 16052, 16056, 16061,16082,16089 and 16097? and 3 main paths of 13 subnetworks. For env sequences, it was found that 13 specimens?16007, 16011,16018, 16032, 16056, 16060, 16061,16064, 16070, 16082,16085, 16088 and 16092?and 3 main paths of 34 subnetworks. The pol, gag and env sequences by the Hiseq sequencing of 25, 29 and 29 specimens of CRF07_BC subtype were subjected to transmission subnetwork analysis.,It was found that 2 specimens ?16016 and 16023? for pol sequences,6 specimens?16024,16038, 16054, 16065,16075 and 16090? for gag sequences and 10 specimens?16019, 16028, 16031,16038, 16050, 16065, 16067, 16069, 16090 and 16098?for env sequences. And, while, it suggested an possible transmission path.Conclusions1. Fixed bootstrap?90% and then contained the GD value of transmission network from the HIV-1 gag and env sequences for MSMHIV-1 acute infection, GD?3.5% and GD?4.5%.2. For generation sequencing, the sample association information of the dynamic analysis of the transmission sub-network was in the order of env, gag and pol from high to low.3. The method of quasi-population analysis of HIV-1 pol, gag and env gene sequences based on Hiseq sequencing technology was successfully established.4. It was found that the diversity of HIV-1 quasi-species by Hiseq sequencing helps to make the results of transmission sub-network analysis more accurate and more clear, and is helpful to understand the dynamic process of HIV-1 network.5. Hiseq sequencing technology is simple to operate, low detection cost, and has certain application value in HIV-1 transmission network analysis.