Evaluation on the accuracy of consensus sequence of Next-generation sequencing of blood DNA for human immunodeficiency virus dominant quasispecies

doi:10.3877/cma.j.issn.1674-1358.2021.01.005

Abstract

Abstract:

Objective

To assess the representativeness and accuracy of consensus sequence generated from Next-generation sequencing (NGS) for human immunodeficiency virus (HIV) quasispecies.

Methods

Total of 32 blood samples from 29 patients with HIV infection were collected. Among them, 3 blood samples were collected from 3 patients when virologic failure occurred. HIV DNA pol gene was amplified and sequenced by Sanger sequencing and NGS sequencing, respectively. The sequencing data of Sanger and NGS were processed by Sequencher (v4.10.1) and Bowtie2 (v2.2.5) separately. Furtherly, the consensus sequence from NGS was defined by the homemade PERL scripts. Finally, cluster analysis was processed by Mega software and the different sites between the consensus sequence of NGS and Sanger sequencing were identified and analyzed, respectively.

Results

Cluster analysis showed that 90.6% (29/32) sequences derived from the same sample were clustered in the same branch, the average credibility was 95.5%. The accuracy of each nucleotide site in consensus sequences was 99.6% compared to Sanger sequencing. Furthermore, the 81.6% (155/190) nucleotide type of inconsistent sites was transition, and the transition between Guanine (G) and Adenine (A) was the most common with the occurrence ratio of 39% (74/190).

Conclusions

The consensus sequence of NGS sequencing could be used in the study of HIV dominant quasispecies, which could present the high accurate results with Sanger sequencing.

Key words: Sanger sequencing, Next-generation sequencing, Consensus sequence, Human immunodeficiency virus, Dominant quasispecies

Yuanyuan Zhang, Qianqian Yin, Pengcheng Du, Yibo Ding, Junyan Han, Qianru Lin, Liying Ma. Evaluation on the accuracy of consensus sequence of Next-generation sequencing of blood DNA for human immunodeficiency virus dominant quasispecies[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2021, 15(01): 22-28.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhsyhlcgrbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-1358.2021.01.005

https://zhsyhlcgrbzz.cma-cmc.com.cn/EN/Y2021/V15/I01/22

Figures/Tables 5

References 26

[1]	Coffin JM. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy[J]. Science,1995,267(5197):483-489.
[2]	冯凯迪，张志敏，张秋月, 等. 深度测序用于分析抗病毒治疗急性期感染者的HIV-1准种群变化[J]. 中国艾滋病性病,2017,23(7):582-586,591.
[3]	Rouzioux C, Trémeaux P, Avettand-Fenoël V. HIV DNA: a clinical marker of HIV reservoirs[J]. Curr Opin HIV AIDS,2018,13(5):389-394.
[4]	Avettand-Fènoël V, Hocqueloux L, Ghosn J, et al. Total HIV-1 DNA, a marker of viral reservoir dynamics with clinical implications[J]. Clin Microbiol Rev,2016, 29(4):859-880.
[5]	刘志英，袁霖，张欣, 等. 总HIV-1 DNA定量检测技术的建立[J]. 中国艾滋病性病,2019,25(3):222-226,235.
[6]	张淳，张媛媛. 基因测序技术在细菌性传染病监测中的应用[J]. 中国医学装备,2017,14(7):134-138.
[7]	Eriksson N, Pachter L, Mitsuya Y, et al. Viral population estimation using pyrosequencing [J]. PLoS Comput Biol,2008,4(4):e1000074.
[8]	贺健梅，邹潇白，陈曦, 等. 超深度焦磷酸测序技术用于HIV-1 RT基因区原发性耐药突变的研究[J]. 中华流行病学杂志,2014,35(10):1142-1145.
[9]	Schneider TD. Consensus sequence Zen[J]. Appl Bioinformatics, 2002,1(3):111-119.
[10]	Andino R, Domingo E. Viral quasispecies[J]. Virology,2015,479-480:46-51.
[11]	中国疾病预防控制中心. 全国艾滋病检测技术规范(2015年修订版)[EB/OL]. 2015. URL
[12]	殷倩倩，徐维四，焦洋, 等. HIV-1感染者血浆病毒RNA与全血前病毒DNA的比较[J]. 中国艾滋病性病,2016,22(3):155-159.
[13]	Ho DD. Perspectives series: host/pathogen interactions. Dynamics of HIV-1 replication in vivo[J]. J Clin Invest,1997,99(11):2565-2567.
[14]	孔德生，马丽英. HIV的清除和艾滋病功能性治愈的研究进展[J]. 中国热带医学,2016,16(11):1145-1150.
[15]	Song H, Giorgi EE, Ganusov VV, et al. Tracking HIV-1 recombination to resolve its contribution to HIV-1 evolution in natural infection[J]. Nat Commun,2018,9(1):1928.
[16]	周佳佳，邢辉. 我国HIV重组株流行情况的研究进展[J]. 国际病毒学杂志,2019,26(1):66-68.
[17]	Ghimire D, Rai M, Gaur R.Novel host restriction factors implicated in HIV-1 replication[J]. J Gen Virol,2018,99(4):435-446.
[18]	张昱，李敬云. APOBEC3G与Vif的相互作用及其对HIV复制的影响[J]. 中华实验和临床病毒学杂志,2019,33(2):221-224.
[19]	欧阳雅博，王琛，张岱, 等. HIV-1中枢特征性突变的中和敏感性和免疫逃逸分析[J]. 国际病毒学杂志,2019,26(4):272-276.
[20]	Pollpeter D, Parsons M, Sobala AE, et al. Deep sequencing of HIV-1 reverse transcripts reveals the multifaceted antiviral functions of APOBEC3G[J]. Nat Microbiol,2018,3(2):220-233.
[21]	莫婷，胡志亮，魏洪霞, 等. HIV传播耐药的研究进展[J]. 现代医学,2016,44(4):584-590.
[22]	宋霆，粟斌，刘志英, 等. 总HIV-DNA作为病毒储存库动力学标志物的研究进展[J]. 北京医学,2019,41(6):494-497.
[23]	Chabria SB, Gupta S, Kozal MJ. Deep sequencing of HIV: clinical and research applications[J]. Annu Rev Genomics Hum Genet,2014,15:295-325.
[24]	Collins DW, Jukes TH. Rates of transition and transversion in coding sequences since the human-rodent divergence[J]. Genomics,1994,20(3):386-396.
[25]	苏齐鉴，闭志友，周平, 等. 123株人类免疫缺陷病毒-1重组亚型主要耐药性突变的基因屏障[J]. 中华传染病杂志,2010,28(8):449-454.
[26]	Piantadosi A, Freije CA, Gosmann C, et al. Metagenomic sequencing of HIV-1 in the blood and female genital tract reveals little quasispecies diversity during acute infection[J]. J Virol,2019,3(2):1-16.

引物名称	序列（5′→3′）	位置^a	片段长度（bp）	退火温度（℃）
F1a	TGAARGAITGYACTGARAGRCAGGCTAAT	2 057～2 085	1 291	50
F1b	ACTGARAGRCAGGCTAATTTTTTAG	2 068～2 092
RT-R1	ATCCCTGCATAAATCTGACTTGC	3 370～3 348
PRT-F2	CTTTARCTTCCCTCARATCACTCT	2 243～2 266	1 061	55
RT-R2	CTTCTGTATGTCATTGACAGTCC	3 326～3 304	1 061	55
F1	xxx^bGCCAGGAATGGATGGCCCAAAAG	2 588～2 610	431	60
R1	xxx^bTGGTGATCCTTTCCATCCCTGTGG	3 020～2 997	431	60
F2	xxx^bAGTATACTGCATTTACCATACC	2 926～2 947	400	60
R2	xxx^bCTTCTGTATGTCATTGACAGTCC	3 326～3 304	400	60

引物名称	序列（5′→3′）	位置^a	片段长度（bp）	退火温度（℃）
F1a	TGAARGAITGYACTGARAGRCAGGCTAAT	2 057～2 085	1 291	50
F1b	ACTGARAGRCAGGCTAATTTTTTAG	2 068～2 092
RT-R1	ATCCCTGCATAAATCTGACTTGC	3 370～3 348
PRT-F2	CTTTARCTTCCCTCARATCACTCT	2 243～2 266	1 061	55
RT-R2	CTTCTGTATGTCATTGACAGTCC	3 326～3 304	1 061	55
F1	xxx^bGCCAGGAATGGATGGCCCAAAAG	2 588～2 610	431	60
R1	xxx^bTGGTGATCCTTTCCATCCCTGTGG	3 020～2 997	431	60
F2	xxx^bAGTATACTGCATTTACCATACC	2 926～2 947	400	60
R2	xxx^bCTTCTGTATGTCATTGACAGTCC	3 326～3 304	400	60

Please choose a citation manager

Content to export