Streamlined Subpopulation, Subtype, and Recombination Analysis of HIV-1 Half-Genome Sequences Generated by High-Throughput Sequencing

mSphere

Mars Stone¹  Manzoor Ahmed¹  Chang Su¹  Yue Chen¹  Feng Gao²  Thomas N. Denny³  Myron S. Cohen³  Fangping Cai⁴  Sharaf Ali Shah⁴  Naila Gulzar⁴  Ana M. Sanchez⁴  Krista Smith⁵  Vahan Simonyan⁵  Konstantinos Karagiannis⁵  Bhavna Hora⁵  Raja Mazumder⁶ 

[1] Molecular Medicine, The George Washington School of Medicine and Health Sciences, Washington, DC, USA;Blood Systems Research Institute, San Francisco, California, USA;Bridge Consultants Foundation, Karachi, Pakistan;;Department of Biochemistry &Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, USA;Locus Biosciences, Morrisville, North Carolina, USA;

关键词: HIV-1;    genetic recombination;    quasispecies;    sequencing;   

DOI  :  10.1128/mSphere.00551-20

来源: DOAJ

【 摘 要 】

ABSTRACT High-throughput sequencing (HTS) has been widely used to characterize HIV-1 genome sequences. There are no algorithms currently that can directly determine genotype and quasispecies population using short HTS reads generated from long genome sequences without additional software. To establish a robust subpopulation, subtype, and recombination analysis workflow, we amplified the HIV-1 3′-half genome from plasma samples of 65 HIV-1-infected individuals and sequenced the entire amplicon (∼4,500 bp) by HTS. With direct analysis of raw reads using HIVE-hexahedron, we showed that 48% of samples harbored 2 to 13 subpopulations. We identified various subtypes (17 A1s, 4 Bs, 27 Cs, 6 CRF02_AGs, and 11 unique recombinant forms) and defined recombinant breakpoints of 10 recombinants. These results were validated with viral genome sequences generated by single genome sequencing (SGS) or the analysis of consensus sequence of the HTS reads. The HIVE-hexahedron workflow is more sensitive and accurate than just evaluating the consensus sequence and also more cost-effective than SGS. IMPORTANCE The highly recombinogenic nature of human immunodeficiency virus type 1 (HIV-1) leads to recombination and emergence of quasispecies. It is important to reliably identify subpopulations to understand the complexity of a viral population for drug resistance surveillance and vaccine development. High-throughput sequencing (HTS) provides improved resolution over Sanger sequencing for the analysis of heterogeneous viral subpopulations. However, current methods of analysis of HTS reads are unable to fully address accurate population reconstruction. Hence, there is a dire need for a more sensitive, accurate, user-friendly, and cost-effective method to analyze viral quasispecies. For this purpose, we have improved the HIVE-hexahedron algorithm that we previously developed with in silico short sequences to analyze raw HTS short reads. The significance of this study is that our standalone algorithm enables a streamlined analysis of quasispecies, subtype, and recombination patterns from long HIV-1 genome regions without the need of additional sequence analysis tools. Distinct viral populations and recombination patterns identified by HIVE-hexahedron are further validated by comparison with sequences obtained by single genome sequencing (SGS).

【 授权许可】

Unknown