Viral / Molecular Core Single Genome Amplification
Single genome amplification (SGA) followed by direct sequencing of amplicon DNA is a technology that allows an unbiased characterization of the genetic diversity of a population of target sequences in the absence of potentially confounding PCR-induced artifacts, such as recombination and nucleotide misincorporations. The SGA technology is ideal for the genetic characterization of viral quasispecies, including those of SIV, HIV, and HCV, bacteria, and unicellular parasites such as Plasmodium. Depending on the genetic complexity of the sample of interest and the desired depth of analysis, up to 100 templates can be amplified, sequenced and (if necessary) cloned. SGA technology is routinely used to infer and enumerate transmitted founder HIV/SIV/HCV strains.
Identification of transmitted founder env genes using SGA in a subject with acute HIV-1 infection
Left: Full-length HIV-1 envelope genes were PCR-amplified using SGA protocols from viral RNA present in the plasma of subject ZM247F with acute HIV-1 infection. This gel shows 64 SGA replicates, which yielded 10 positive amplifications (15%) as indicated by red arrows.
Center and Right: A total of 44 full-length env sequences from unique SGA-derived amplicons were used to analyze the genetic complexity of the HIV-1 quasi-species present in this subject during acute infection. Phylogenetic reconstruction (center panel) and Highlighter plot (right panel) identify two distinct populations, labeled variant 1 and 2, which are represented by 35 and 9 sequences, respectively. Sequences within each cluster are nearly identical, differing at most by 2 nucleotides (indicated by tick marks in the highlighter plot) across the entire length of the 2500 bp env gene.
This pattern of sequence diversity indicated that subject ZM247F acquired two distinct transmitted/founder viruses, the nucleotide sequence of which was inferred by determining the clade consensus sequences (Keele BF et al., PNAS 105:7552-7557, 2008).
Core Service Contact:
Frederic Bibollet-Ruche, PhD
Research Assistant Professor of Medicine
This service provides hand-on training of investigators using detailed protocols for each aspect of the SGA procedure. The required steps for SGA include (i) nucleic acid extraction (DNA, RNA, viral or eukaryote genomes), (ii) cDNA synthesis for RNA targets, (iii) optimization of the amplification conditions depending of the abundance of the target sequences and the sample composition, and (iv) direct sequencing of the amplification products and analysis. Completion of this training requires five days.