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Workup of Human Blood Samples for Deep Sequencing of HIV-1 Genomes

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Viral Metagenomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1746))

Abstract

We describe a detailed protocol for the manual workup of blood (plasma/serum) samples from individuals infected with the human immunodeficiency virus type 1 (HIV-1) for deep sequence analysis of the viral genome. The study optimizing the assay was performed in the context of the BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project, which analyzes complete viral genomes from more than 3000 HIV-1-infected Europeans with high-throughput deep sequencing techniques. The goal of the BEEHIVE project is to determine the contribution of viral genetics to virulence. Recently we performed a pilot experiment with 125 patient plasma samples to identify the method that is most suitable for isolation of HIV-1 viral RNA for subsequent long-amplicon deep sequencing. We reported that manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) provides superior results over robotically extracted RNA. The latter approach used the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics), the QIAcube robotic system (Qiagen), or the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular). Here we present a detailed protocol for the labor-intensive manual extraction method that yielded the best results.

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Acknowledgments

The authors want to thank Suzanne Jurriaans, Matthew Hall, Margreet Bakker, Ard van Sighem, Daniela Bezemer, Swee Hoe Ong, Luuk Gras, Peter Reiss, Paul Kellam, and the BEEHIVE collaboration.

Funding

This work was supported by a European Research Council (ERC) grant 339251 to CF.

Author information

Authors and Affiliations

  1. Laboratory of Experimental Virology, Department of Medical Microbiology, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands

    Marion Cornelissen & Antoinette van der Kuyl

  2. Department of Veterinary Medicine, University of Cambridge, Cambridge, UK

    Astrid Gall

  3. Nuffield Department of Medicine, Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, UK

    Chris Wymant & Christophe Fraser

  4. Department of Infectious Disease Epidemiology, Imperial College London, London, UK

    François Blanquart & Christophe Fraser

  5. Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands

    Ben Berkhout

Authors
  1. Marion Cornelissen
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  2. Astrid Gall
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  3. Antoinette van der Kuyl
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  4. Chris Wymant
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  5. François Blanquart
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  6. Christophe Fraser
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  7. Ben Berkhout
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Corresponding author

Correspondence to Marion Cornelissen .

Editor information

Editors and Affiliations

  1. Institute Sustainable Plant Protection of the CNR, Research Unit of Bari, Bari, Italy

    Vitantonio Pantaleo

  2. Institute Sustainable Plant Protection of the CNR, Research Unit of Bari, Bari, Italy

    Michela Chiumenti

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Cornelissen, M. et al. (2018). Workup of Human Blood Samples for Deep Sequencing of HIV-1 Genomes. In: Pantaleo, V., Chiumenti, M. (eds) Viral Metagenomics. Methods in Molecular Biology, vol 1746. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7683-6_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7683-6_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7682-9

  • Online ISBN: 978-1-4939-7683-6

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