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Penn Center for AIDS Research

Viral / Molecular Core Single Genome Amplification

Core Services Main Page | Virology | Molecular | Sequencing | SGA Services | Intergrated vDNA | Other

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
Single Genome Amplification

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).

Services offered:

  1. Consultation on experimental design
  2. Hands-on SGA training
  3. Complete SGA service
  4. Pricing

Core Service Contact:

Frederic Bibollet-Ruche, PhD
Research Assistant Professor of Medicine
Phone: 215-573-6462
Email: bibof@mail.med.upenn.edu

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Pricing

Service

Cost

Assistance Provided

Initial consultation

No charge

Advice on project feasibility, suitability of SGA approach, assistance with cost estimation.

Assistance with experimental design & set-up

$300

Experimental design, primer design and selection, sample size calculations, technical advice, other detailed experimental set-up based on customized needs.

Hands-on SGA training

$514

Individual instruction in SGA methodology; includes sequencing for up to 30 SGA amplicons (1kb target) for 1 sample.

Complete SGA service

$505

Up to 30 SGA amplicons (1kb target) per sample.  Other amplicon sizes and sample quantities available, with pricing customized based on service.

Other services or combinations of services are available with customized pricing schedule.