title: From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing.
abstract: The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.
authors: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors: Marion
authors: M
authors: Cornelissen
authors: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom.
authors: Astrid
authors: A
authors: Gall
authors: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors: Monique
authors: M
authors: Vink
authors: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors: Fokla
authors: F
authors: Zorgdrager
authors: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom.
authors: Špela
authors: Š
authors: Binter
authors: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom.
authors: Stephanie
authors: S
authors: Edwards
authors: Laboratory of Clinical Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors: Suzanne
authors: S
authors: Jurriaans
authors: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors: Margreet
authors: M
authors: Bakker
authors: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom.
authors: Swee Hoe
authors: SH
authors: Ong
authors: Stichting HIV Monitoring, 1105 BD Amsterdam, The Netherlands.
authors: Luuk
authors: L
authors: Gras
authors: Stichting HIV Monitoring, 1105 BD Amsterdam, The Netherlands.
authors: Ard
authors: A
authors: van Sighem
authors: Stichting HIV Monitoring, 1105 BD Amsterdam, The Netherlands.
authors: Daniela
authors: D
authors: Bezemer
authors: Department of Infectious Disease Epidemiology, School of Public Health, Imperial College London, London W21PG, UK.
authors: Frank
authors: F
authors: de Wolf
authors: Stichting HIV Monitoring, 1105 BD Amsterdam, The Netherlands; Department of Global Health, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands; Amsterdam Institute of Global Health and Development, 1105 BM Amsterdam, The Netherlands.
authors: Peter
authors: P
authors: Reiss
authors: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom.
authors: Paul
authors: P
authors: Kellam
authors: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors: Ben
authors: B
authors: Berkhout
authors: Department of Infectious Disease Epidemiology, School of Public Health, Imperial College London, London W21PG, UK.
authors: Christophe
authors: C
authors: Fraser
authors: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Electronic address: a.c.vanderkuyl@amc.uva.nl.
authors: Antoinette C
authors: AC
authors: van der Kuyl
mesh_terms: D016679:Genome, Viral; D023281:Genomics / Q000379:methods; D005838:Genotype; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D012367:RNA, Viral / Q000302:isolation & purification; D019562:Viral Load; D000073336:Whole Genome Sequencing
chemical_list: D012367:RNA, Viral
keywords: Complete genome; HIV-1; High-throughput deep sequencing; QIAamp viral isolation kit; RNA isolation