PacBio high-fidelity near full-length genome sequencing for HIV-1 quasispecies: methodological framework and validation

This study aimed to develop an optimized sequencing workflow for HIV-1 near full-length genome (NFLG) quasispecies, integrating the PacBio Revio platform with a streamlined bioinformatics pipeline to enable precise characterization of the genetic heterogeneity and dynamic evolution of HIV-1 quasispecies. To this end, the HIV-1 NFLG was divided into three segments for high-fidelity amplification and PacBio sequencing. This method achieved stable amplification (positivity rate >80%) in samples with a viral load >1,000 copies/mL and demonstrated applicability to China's five major epidemic strains and unique recombinant forms (URFs). The adoption of sample-specific asymmetric barcode sequences improved cost-effectiveness, enabling efficient sequencing of 300 to 1,000 samples per sequencing cell. The entire amplification-sequencing workflow exhibited minimal systematic errors (recombination rate