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Towards Better Precision Medicine: PacBio Single-Molecule Long Reads Resolve the Interpretation of HIV Drug Resistant Mutation Profiles at Explicit Quasispecies (Haplotype) Level

Da Wei Huang, Castle Raley, Min Kang Jiang, Xin Zheng, Dun Liang, M Tauseef Rehman, Helene C Highbarger, Xiaoli Jiao, Brad Sherman, Liang Ma, Xiaofeng Chen, Thomas Skelly, Jennifer Troyer, Robert Stephens, Tomozumi Imamichi, Alice Pau, Richard A Lempicki, Bao Tran, Dwight Nissley, H Clifford Lane and Robin L Dewar

Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient’s HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only ≥ 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger scope (hundreds of samples per run). However, NGS technologies produce reads that are too short to enable the detection of the physical linkages of individual SNVs across the haplotype of each HIV strain present. In this article, we demonstrate that the single-molecule long reads generated using the Third Generation Sequencer (TGS), PacBio RS II, along with the appropriate bioinformatics analysis method, can resolve the HDRM profile at a more advanced quasispecies level. The case studies on patients’ HIV samples showed that the quasispecies view produced using the PacBio method offered greater detection sensitivity and was more comprehensive for understanding HDRM situations, which is complement to both Sanger and NGS technologies. In conclusion, the PacBio method, providing a promising new quasispecies level of HDRM profiling, may effect an important change in the field of HIV drug resistance research.