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

Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

HIV-1 Nearly complete genome RNA isolation QIAamp viral isolation kit High-throughput deep sequencing 

Notes

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.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Laboratory of Experimental Virology, Department of Medical MicrobiologyAcademic Medical Center of the University of AmsterdamAmsterdamThe Netherlands
  2. 2.Department of Veterinary MedicineUniversity of CambridgeCambridgeUK
  3. 3.Nuffield Department of MedicineBig Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of OxfordOxfordUK
  4. 4.Department of Infectious Disease EpidemiologyImperial College LondonLondonUK
  5. 5.Laboratory of Experimental Virology, Department of Medical MicrobiologyCenter for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of AmsterdamAmsterdamThe Netherlands

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