Single-molecule Complete HIV-1 Genome Sequencing from 2 Linked Transmission Pairs
Malinda Schaefer*1, M Brown2, W Kilembe3, S Allen1, Y Guo2, E Hunter1, and E Paxinos2
1Emory Univ, Atlanta, GA, US; 2Pacific Biosci, Menlo Park, CA, US; and 3Zambia-Emory HIV Res Project, Lusaka
Background: In order to better understand the relationships between the specific sequences of transmitted viruses and disease progression, we explored the feasibility of using a novel sequencing technology to sequence full-length HIV-1 genomes.
Methods: Four samples, representing 2 transmission pairs, consisting of a donor (D) and an epidemiologically linked recipient (LR) were selected from the Zambia Emory HIV Research Project (Lusaka, Zambia) for sequencing. Pairs Z190 and Z1076 were identified soon after transmission had occurred, with the newly infected individual in pair Z190 being p24+. Pair Z190 was chosen because of the LR’s p24 status, low early set point viral load, and lack of HLA-I allele sharing with D. Pair Z1076 was chosen because the D and LR share 4 HLA-I alleles and the high early set point viral load in the recipient. Patient viruses were amplified as a single whole-genome amplicon in 8-15 replicate reactions, pooled, and sequenced. No amplicon fragmentation or specific sequencing primers are required, and amplicons were sequenced using Pacific biosciences commercially available reagents and protocols.
Results: In less than 4 hours, we obtained >50Mbp of sequence data for each sample. Importantly, the data contained multiple full-length (>9Kbp) continuous reads for each sample. Whole genome consensus sequences were constructed for each patient virus population by simple plurality for verification of sequencing results.
Conclusions: Transmission pairs were easily identified by relative sequence divergence. The single molecule nature of Pacific Biosciences RS sequencing further allows us to characterize the diversity of the individual quasi-species comprising the patient viral populations and opens up the potential for reference-agnostic and cost-effective full genome sequencing of HIV-1.