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The challenge of missing information

Targeted sequencing allows you to focus on specific areas of interest within microbial genomes, increasing both the cost-effectiveness of studies and the depth of coverage. These regions may include repeat expansions or plasmids, which are critical for the comprehensive characterization of microbes, as they can contribute to pathogenicity, drug resistance, or the assessment of evolutionary relationships among taxa. While many sequencing platforms are capable of resolving differences in SNPs, short-read sequencing platforms struggle to characterize genetic variation across the larger regions needed for mapping complete microbial variation.

Confident identifications with a complete view of variation

Polymorphisms can take the form of structural variation within a genome or its mobile elements, and must be clearly defined for the most confident and accurate conclusions. Single Molecule, Real-Time (SMRT) Sequencing provides the read length needed to clearly resolve structural variants within specific regions of interest in their genomic context. Additionally, SMRT Sequencing allows for the identification of individual members of complex microbial populations — including 16S rRNA species-level identification — and the resolution of complete plasmids or viral quasispecies.

This technology advances your microbial and viral research through the ability to:

  • Resolve complex populations in viral or metagenomic samples
  • Map order and orientation of multiply-inserted phage elements
  • Target regions in host organisms to study viral integration and integration sites
  • Phase variations across targeted regions
  • Perform multiplexed multi-locus sequence typing (MLST)

spotlight-microbiology-icon

Workflow: from targeted region to accurate variant or haplotype

  • 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
    • Adjust run times (0.5 to 6 hours) to maximize sample throughput and turn-around time
    • Obtain consensus accuracies > 99.999% by avoiding mapping and systematic errors
    • Produce high single-molecule consensus accuracies through multiple observations of single circularized templates for complex population resolution

Featured research: mapping viral integration in diploid human cells

Fig3ab0805
SMRT Sequencing identifies the chromosomal distribution of adeno-associated virus (AAV) integration for the first time. The authors required PacBio long reads to unambiguously assign exact AAV-chromosome junctions1.

Explore this research further.

To learn more about obtaining a complete view of microbial variation, contact us.


Reference

  1. Hüser, D. et al., (2014) Adeno-associated virus type 2 wild-type and vector-mediated genomic integration profiles of human diploid fibroblasts analyzed by third-generation PacBio DNA sequencing. Journal of Virology. 88 (19), 11253–11263.

Selected Resources