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Distinguish a virus in the crowd

Viruses exist in complex mixtures within each individual, and the complete resolution of each viral variant within that population is critical to understanding evolution, the development of drug-resistant mutations, and immune escape. Given that co-existing isolates may only differ by a handful of changes scattered across their genomes, the distinction of closely related variants within mixtures is particularly challenging.

Confident and complete resolution of viral diversity

Single Molecule, Real-Time (SMRT) Sequencing produces sequencing reads that average > 10 kb in length, providing an unprecedented view of complete viral genes and genomes. By generating single-molecule sequencing reads that allow for the direct detection of phased variants, SMRT Sequencing gives you the ability to:

  • Deconvolute complex mixtures of unique haplotypes
  • Track evolution and phylogeny of viral populations
  • Identify and quantify minor variants
  • Generate complete de novo assemblies of large viral genomes

spotlight-microbiology-icon

Workflow: from DNA to resolved viral populations

  • SMRT Sequencing with PacBio Systems
    • Take advantage of the Sequel System to reduce project costs and generate 7X more reads compared with the PacBio RS II
    • Achieve ~10 kb average read lengths, with some reads as long as 60 kb
    • Obtain consensus accuracies > 99.999% by avoiding mapping and systematic errors
    • Produce high-intra-molecular consensus accuracies of single circularized templates, recommended when examining inserts < 5 kb within a variant population
    • Scalable throughput based on project needs
      • 100X coverage recommended per variant in a mixed population
      • 50X coverage per genome for de novo assembly
  • Data analysis tools available through SMRT Analysis or PacBio DevNet
    • Long Amplicon Analysis (LAA): generate reference-free, phased consensus assemblies from pooled amplicons
    • Minor Variant Analysis: detect and quantify SNPs in a heterogenous data set using a reference
    • ClusterConsensus: reference-free deconvolution of viral genomes in a complex mixture
    • HGAP: create high-quality de novo assembly of large viral genomes

Featured research: development of protocols for full-length HIV envelope sequencing and analysis

Resolve_Viral_Populations.Spotlight2.HIV_envelope_sequencing

The long reads and single-molecule resolution of SMRT Sequencing produced a comprehensive view of the evolution and diversification of HIV env from a single subject’s viral infection through time. The phylogeny of HIV env SMRT sequences agreed with historical Sanger sequences. As HIV env is the main target for neutralizing antibodies, this information will offer unprecedented insights into mechanisms of viral pathogenesis and immune escape1.

Explore this research further.

To learn more about how to resolve your viral populations, contact us.

 

Reference

  1. Smith, M.L. (2015, May) Full-length HIV env Deep Sequencing in a Donor with Broadly Neutralizing V1/V2 Antibodies. Presentation at American Society of Microbiology General Meeting. New Orleans, LA.

 

Selected Resources