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)
Workflow: from targeted region to accurate variant or haplotype
- Library preparation
- 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
- Data analysis with SMRT Analysis or PacBio DevNet
- Long Amplicon Analysis (LAA) for reference-free generation of de novo haplotype sequences from pooled amplicons
- Minor Variant Analysis for detection and quantitation of single nucleotide polymorphisms
- ClusterConsensus for reference-free deconvolution of genomes in a complex mixture
- HGAP for comprehensive de novo assembly of BACs or fosmids
Featured research: mapping viral integration in diploid human cells
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.
- 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.
- Sheppard, Anna E et al. (2016) Nested Russian doll-like genetic mobility drives rapid dissemination of the Carbapenem resistance gene blaKPC Antimicrobial Agents and Chemotherapy
- Badran, Ahmed H et al. (2016) Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance. Nature
- Witek, Kamil et al. (2016) Accelerated cloning of a potato late blight-resistance gene using RenSeq and SMRT sequencing. Nature Biotechnology
- Zhang, Huibin et al. (2016) Comprehensive mutagenesis of the fimS promoter regulatory switch reveals novel regulation of type 1 pili in uropathogenic Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America
- Hall, Andrew Brantley et al. (2016) Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes. Proceedings of the National Academy of Sciences of the United States of America
- De Ravin, Suk See et al. (2016) Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease. Nature Biotechnology
- Neafsey, Daniel E et al. (2015) Genetic diversity and protective efficacy of the RTS,S/AS01 malaria vaccine. The New England Journal of Medicine
- Hargreaves, Chantal E et al. (2015) Fc? receptors: genetic variation, function, and disease. Immunological Reviews
- Burke, Gaelen R et al. (2015) Microplitis demolitor bracovirus proviral loci and clustered replication genes exhibit distinct DNA amplification patterns during replication. Journal of Virology
- Li, Jun-Jie et al. (2015) Complete nucleotide sequences of bla(CTX-M)-harboring IncF plasmids from community-associated Escherichia coli strains in the United States. Antimicrobial Agents and Chemotherapy
- Agarwal, Prasoon et al. (2015) CGGBP1 mitigates cytosine methylation at repetitive DNA sequences. BMC Genomics
- Hüser, Daniela 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
- Zurfluh, Katrin et al. (2014) Vertical transmission of highly similar bla CTX-M-1-harboring IncI1 plasmids in Escherichia coli with different MLST types in the poultry production pyramid. Frontiers in Microbiology
- Lu, Shuguang et al. (2014) Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity. BMC Genomics
- Chen, Xiao et al. (2014) The architecture of a scrambled genome reveals massivel levels of genomic rearrangement during development. Cell
- Parobek, Christian M et al. (2014) Differing patterns of selection and geospatial genetic diversity within two leading Plasmodium vivax candidate vaccine antigens. PLoS Neglected Tropical Diseases
- Fine, Eli J et al. (2014) An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage. Nucleic Acids Research
- Huddleston, John et al. (2014) Reconstructing complex regions of genomes using long-read sequencing technology. Genome Research
- Poster: Lee, Walter et al. (2015) Barcoding strategies for multiplexing of samples using a long-read sequencing technology.