title: Multiplexed next-generation sequencing and de novo assembly to obtain near full-length HIV-1 genome from plasma virus.
abstract: Analysing the HIV-1 near full-length genome (HIV-NFLG) facilitates new understanding into the diversity of virus population dynamics at individual or population level. In this study we developed a simple but high-throughput next generation sequencing (NGS) protocol for HIV-NFLG using clinical specimens and validated the method against an external quality control (EQC) panel. Clinical specimens (n=105) were obtained from three cohorts from two highly conserved HIV-1C epidemics (India and Ethiopia) and one diverse epidemic (Sweden). Additionally an EQC panel (n=10) was used to validate the protocol. HIV-NFLG was performed amplifying the HIV-genome (Gag-to-nef) in two fragments. NGS was performed using the Illumina HiSeq2500 after multiplexing 24 samples, followed by de novo assembly in Iterative Virus Assembler or VICUNA. Subtyping was carried out using several bioinformatics tools. Amplification of HIV-NFLG has 90% (95/105) success-rate in clinical specimens. NGS was successful in all clinical specimens (n=45) and EQA samples (n=10) attempted. The mean error for mutations for the EQC panel viruses were <1%. Subtyping identified two as A1C recombinant. Our results demonstrate the feasibility of a simple NGS-based HIV-NFLG that can potentially be used in the molecular surveillance for effective identification of subtypes and transmission clusters for operational public health intervention.
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden.
authors.forename: Shambhu G
authors.initials: SG
authors.lastname: Aralaguppe
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden.
authors.forename: Abu Bakar
authors.initials: AB
authors.lastname: Siddik
authors.affiliation: HIV/AIDS Division, Department of Clinical Research, National Institute for Research in Tuberculosis, Indian Council of Medical Research, Chennai, India.
authors.forename: Ashokkumar
authors.initials: A
authors.lastname: Manickam
authors.affiliation: SciGenom Lab Pvt. Ltd, Cochin, India.
authors.forename: Anoop T
authors.initials: AT
authors.lastname: Ambikan
authors.affiliation: SciGenom Lab Pvt. Ltd, Cochin, India.
authors.forename: Milner M
authors.initials: MM
authors.lastname: Kumar
authors.affiliation: Unit of Computational Medicine, Center for Molecular Medicine, Department of Medicine & Science for Life Laboratories, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Sunjay Jude
authors.initials: SJ
authors.lastname: Fernandes
authors.affiliation: Department of Internal Medicine, School of Medicine, Addis Ababa University, Addis Ababa, Ethiopia.
authors.forename: Wondwossen
authors.initials: W
authors.lastname: Amogne
authors.affiliation: SciGenom Lab Pvt. Ltd, Cochin, India.
authors.forename: Dhinoth K
authors.initials: DK
authors.lastname: Bangaruswamy
authors.affiliation: HIV/AIDS Division, Department of Clinical Research, National Institute for Research in Tuberculosis, Indian Council of Medical Research, Chennai, India.
authors.forename: Luke Elizabeth
authors.initials: LE
authors.lastname: Hanna
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden; Department of Infectious Diseases, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Sonnerborg
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden. Electronic address: ujjwal.neogi@ki.se.
authors.forename: Ujjwal
authors.initials: U
authors.lastname: Neogi
mesh_terms: D005002:Ethiopia; D015894:Genome, Human; D016679:Genome, Viral; D005838:Genotype; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007194:India; D010949:Plasma / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013548:Sweden
chemical_list: D012367:RNA, Viral
keywords: De novo assembly; HIV-NFLG; Plasma