TY - JOUR
T1 - From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing
AU - Cornelissen, Marion
AU - Gall, Astrid
AU - Vink, Monique
AU - Zorgdrager, Fokla
AU - Binter, Špela
AU - Edwards, Stephanie
AU - Jurriaans, Suzanne
AU - Bakker, Margreet
AU - Ong, Swee Hoe
AU - Gras, Luuk
AU - van Sighem, Ard
AU - Bezemer, Daniela
AU - de Wolf, Frank
AU - Reiss, Peter
AU - Kellam, Paul
AU - Berkhout, Ben
AU - Fraser, Christophe
AU - van der Kuyl, Antoinette C.
PY - 2017
Y1 - 2017
N2 - 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 similar to 1.9, 3.6, 3.0 and 3.5 kb, 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 >3 kb. 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. (C) 2016 The Authors. Published by Elsevier B.V
AB - 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 similar to 1.9, 3.6, 3.0 and 3.5 kb, 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 >3 kb. 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. (C) 2016 The Authors. Published by Elsevier B.V
U2 - https://doi.org/10.1016/j.virusres.2016.08.004
DO - https://doi.org/10.1016/j.virusres.2016.08.004
M3 - Article
C2 - 27497916
SN - 0168-1702
VL - 239
SP - 10
EP - 16
JO - Virus Research
JF - Virus Research
ER -