Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants
Andrew N. Banin,
Michael Tuen,
Jude S. Bimela,
Marcel Tongo,
Paul Zappile,
Alireza Khodadadi-Jamayran,
Aubin J. Nanfack,
Josephine Meli,
Xiaohong Wang,
Dora Mbanya,
Jeanne Ngogang,
Adriana Heguy,
Phillipe N. Nyambi,
Charles Fokunang,
Ralf Duerr
Affiliations
Andrew N. Banin
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
Michael Tuen
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
Jude S. Bimela
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
Marcel Tongo
Center of Research for Emerging and Re-Emerging Diseases (CREMER), Institute of Medical Research and Study of Medicinal Plants, BP 906 Yaoundé, Cameroon
Paul Zappile
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
Alireza Khodadadi-Jamayran
Applied Bioinformatics Laboratories (ABL) and Genome Technology Center (GTC), Division of Advanced Research Technologies (DART), New York University Langone Medical Center, New York, NY 10016, USA
Aubin J. Nanfack
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
Josephine Meli
Medical Diagnostic Center, BP 15810 Yaoundé, Cameroon
Xiaohong Wang
Manhattan Veterans Affairs New York Harbor Healthcare System, New York, NY 10010, USA
Dora Mbanya
Faculty of Medicine and Biomedical Sciences, Department of Microbiology, Parasitology and Infectious Diseases, University of Yaoundé 1, BP 1364 Yaoundé, Cameroon
Jeanne Ngogang
Faculty of Medicine and Biomedical Sciences, Department of Biochemistry, University of Yaoundé 1, BP 1364 Yaoundé, Cameroon
Adriana Heguy
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
Phillipe N. Nyambi
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
Charles Fokunang
Faculty of Medicine and Biomedical Sciences, Department of Pharmacotoxicology & Pharmacokinetics, University of Yaoundé 1, BP 1364 Yaoundé, Cameroon
Ralf Duerr
Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
Near full genome sequencing (NFGS) of HIV-1 is required to assess the genetic composition of HIV-1 strains comprehensively. Population-wide, it enables a determination of the heterogeneity of HIV-1 and the emergence of novel/recombinant strains, while for each individual it constitutes a diagnostic instrument to assist targeted therapeutic measures against viral components. There is still a lack of robust and adaptable techniques for efficient NFGS from miscellaneous HIV-1 subtypes. Using rational primer design, a broad primer set was developed for the amplification and sequencing of diverse HIV-1 group M variants from plasma. Using pure subtypes as well as diverse, unique recombinant forms (URF), variable amplicon approaches were developed for NFGS comprising all functional genes. Twenty-three different genomes composed of subtypes A (A1), B, F (F2), G, CRF01_AE, CRF02_AG, and CRF22_01A1 were successfully determined. The NFGS approach was robust irrespective of viral loads (≥306 copies/mL) and amplification method. Third-generation sequencing (TGS), single genome amplification (SGA), cloning, and bulk sequencing yielded similar outcomes concerning subtype composition and recombinant breakpoint patterns. The introduction of a simple and versatile near full genome amplification, sequencing, and cloning method enables broad application in phylogenetic studies of diverse HIV-1 subtypes and can contribute to personalized HIV therapy and diagnosis.
