Multiplexed next-generation sequencing and de novo assembly to obtain near full-length HIV-1 genome from plasma virus

被引：17

作者：

Aralaguppe, Shambhu G. ^{[1
]}

Siddik, Abu Bakar ^{[1
]}

Manickam, Ashokkumar ^{[2
]}

Ambikan, Anoop T. ^{[3
]}

Kumar, Milner M. ^{[3
]}

Fernandes, Sunjay Jude ^{[4
]}

Amogne, Wondwossen ^{[5
]}

Bangaruswamy, Dhinoth K. ^{[3
]}

Hanna, Luke Elizabeth ^{[2
]}

Sonnerborg, Anders ^{[1
,6
]}

Neogi, Ujjwal ^{[1
]}

机构：

[1] Karolinska Inst, Div Clin Microbiol, Dept Lab Med, Stockholm, Sweden

[2] Indian Council Med Res, Natl Inst Res TB, Div HIV AIDS, Dept Clin Res, Madras, Tamil Nadu, India

[3] SciGenom Lab Pvt Ltd, Cochin, Kerala, India

[4] Karolinska Inst, Dept Med & Sci Life Labs, Unit Computat Med, Ctr Mol Med, Stockholm, Sweden

[5] Univ Addis Ababa, Sch Med, Dept Internal Med, Addis Ababa, Ethiopia

[6] Karolinska Univ Hosp, Karolinska Inst, Dept Infect Dis, Stockholm, Sweden

来源：

JOURNAL OF VIROLOGICAL METHODS | 2016年 / 236卷

基金：

瑞典研究理事会;

关键词：

HIV-NFLG; De novo assembly; Plasma; RECOMBINATION; PERFORMANCE; RNA; AMPLIFICATION; ALIGNMENTS;

D O I：

10.1016/j.jviromet.2016.07.010

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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. (C) 2016 Elsevier B.V. All rights reserved.

引用

页码：98 / 104

页数：7

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