Multiplexed next-generation sequencing and de novo assembly to obtain near full-length HIV-1 genome from plasma virus
| Autor: | Ashokkumar Manickam, Shambhu G Aralaguppe, Anoop T. Ambikan, Ujjwal Neogi, Milner M. Kumar, Anders Sönnerborg, Abu Bakar Siddik, Dhinoth K. Bangaruswamy, Sunjay Jude Fernandes, Luke Elizabeth Hanna, Wondwossen Amogne |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Genotype Population India Sequence assembly HIV Infections Genome Viral Biology Genome DNA sequencing Virus Plasma 03 medical and health sciences Virology Humans education Sweden Genetics education.field_of_study Genome Human High-Throughput Nucleotide Sequencing Sequence Analysis DNA Subtyping 030104 developmental biology HIV-1 RNA Viral Human genome Ethiopia |
| Zdroj: | Journal of Virological Methods. 236:98-104 |
| ISSN: | 0166-0934 |
| DOI: | 10.1016/j.jviromet.2016.07.010 |
| Popis: | 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 |
| Databáze: | OpenAIRE |
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