Auto Tag: DNA isolation

Background: HIV-1 proviruses in peripheral blood mononuclear cells (PBMCs) are felt to be an important reservoir of HIV-1 infection. Given that this pool represents an archival library, it can be used to study virus evolution and CD4+ T cell survival. Accurate study of this pool is burdened by difficulties encountered in sequencing a full-length proviral genome, typically accomplished by assembling overlapping pieces and imputing the full genome. Methodology: Cryopreserved PBMCs collected from a total of 8 HIV+ patients from 1997-2001 were used for genomic DNA extraction. Patients had been receiving cART for 2-8 years at the time samples were obtained. 7 patients had pVL >50 copies/mL (mean: 312,282, range: 18,372-683,400) and 1 had pVL <50. Genomic DNA was subjected to limiting dilution prior to amplification of near-full-length genomes by a newly developed nested PCR. The predicted size of the PCR product was 9.0 kb, spanning from the 5’ LTR through the 3’ LTR. Single molecules were sequenced as near-full-length amplicons directly from PCR products without shearing using commercially available P4-C2 reagents and standard protocols on a PacBio RS II instrument. Quality of the genomes was validated by clonal positive controls and synthetic mixtures. Results: Near-full-length provirus genome sequences were successfully obtained from all 8 patients as continuous long reads from single molecules. PacBio sequencing required approximately 10% of the PCR product needed for Sanger sequencing and generated 325 MB per 3-hour run including 1,800 full-length intact genome reads on average. One patient’s sample was not at a limiting dilution and analysis revealed multiple subspecies. For 8 near-fulllength provirus genomes derived from the other 7 patients, large internal deletions were noted in 2 proviruses; APOBEC-mediated hypermutations were seen in 2 proviruses; and 4 proviruses appeared to be intact genomes. All of the defective proviruses showed a complete absence of resistance mutations in either RT or protease, even after 2-8 years of cART. On the contrary, all of the intact proviruses contained evidence of ART-resistance associated mutations suggesting that they represented relatively recent variants. Conclusions: Combining a novel protocol for full-length limiting dilution amplification of proviruses with PacBio SMRT sequencing allowed for the generation of near-full-length genomes with good quality and an ability to detect minor variants at the 1-10% level. Preliminary data analyses suggest that defective proviruses may represent archival variants that persist long-term in host cells, while intact proviruses within the PBMC pool showing evidence of active virus replication may represent more recent variants.