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
Workflow: from DNA to resolved viral populations
- 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
- 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
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.
- 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.
- Betz-Stablein, B D et al. (2016) Single-molecule sequencing reveals complex genomic variation of hepatitis B virus during 15 years of chronic infection following liver transplantation. Journal of Virology
- Kanda, Teru et al. (2016) Highly efficient CRISPR/Cas9-mediated cloning and functional characterization of gastric cancer-derived Epstein-Barr virus strains. Journal of Virology
- Bull, Rowena A et al. (2016) A method for near full-length amplification and sequencing for six hepatitis C virus genotypes. BMC Genomics
- Poon, Leo L M et al. (2016) Quantifying influenza virus diversity and transmission in humans. Nature Genetics
- Huang, Da Wei et al. (2016) Towards better precision medicine: PacBio Single-Molecule long reads resolve the interpretation of HIV drug resistant mutation profiles at explicit quasispecies (haplotype) level. Journal of Data Mining in Genomics & Proteomics
- Dilernia, Dario A et al. (2015) Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing. Nucleic Acids Research
- Deymier, Martin J et al. (2015) Heterosexual transmission of subtype C HIV-1 selects consensus-like variants without increased replicative capacity or interferon-? resistance. PLoS Pathogens
- Kugelman, Jeffrey R et al. (2015) Emergence of ebola virus escape variants in infected nonhuman primates treated with the MB-003 antibody cocktail. Cell Reports
- Erdmann, Nathan et al. (2015) HLA Class-II associated HIV polymorphisms predict escape from CD4+ T Cell responses. PLoS Pathogens
- Peretti, Alberto et al. (2015) Hamburger polyomaviruses. The Journal of General Virology
- Rogers, Matthew B et al. (2015) Intrahost dynamics of antiviral resistance in influenza a virus reflect complex patterns of segment linkage, reassortment, and natural selection. mBio
- McBride, Carolyn S et al. (2014) Evolution of mosquito preference for humans linked to an odorant receptor. Nature
- Flint, Mike et al. (2014) Inhibitors of the tick-borne, hemorrhagic fever-associated flaviviruses. Antimicrobial Agents and Chemotherapy
- Ocwieja, Karen E et al. (2012) Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing. Nucleic Acids Research
- Zhang, Jie et al. (2016) Evolution of coreceptor utilization to escape CCR5 antagonist therapy. Virology
- Xiao, Wenming et al. (2016) Challenges, solutions, and quality metrics of personal genome assembly in advancing precision medicine. Pharmaceutics
- Seifert, David et al. (2016) Estimating fitness of viral quasispecies from next-generation sequencing data. Current Topics in Microbiology and Immunology
- Bergfors, Assar et al. (2016) Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing. Antiviral Research
- Serrao, Erik et al. (2016) Sites of retroviral DNA integration: From basic research to clinical applications. Critical Reviews in Biochemistry and Molecular Biology
- Deng, Kai et al. (2015) Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations. Nature
- Marsic, Damien et al. (2014) Vector design Tour de Force: integrating combinatorial and rational approaches to derive novel adeno-associated virus variants. Molecular Therapy
- Zeng, Feng et al. (2014) ProbAlign: a re-alignment method for long sequencing reads bioRxiv
- Giallonardo, Francesca Di et al. (2014) Full-length haplotype reconstruction to infer the structure of heterogeneous virus populations. Nucleic Acids Research
- Alquezar-Planas, David E et al. (2013) Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing. Scientific Reports
- Archer, John et al. (2012) Use of four next-generation sequencing platforms to determine HIV-1 coreceptor tropism. PLoS One
- Poster: Smith, M.L. et al. (2016) An improved circular consensus algorithm with an application to detect HIV-1 Drug Resistance Associated Mutations (DRAMs)
- Poster: Hepler, N. Lance et al. (2016) An improved circular consensus algorithm with an application to detection of HIV-1 Drug-Resistance Associated Mutations (DRAMs)
- Presentation: Alexander, David et al. (2015) PacBio SMRT Analysis 3.0 preview
- Poster: Brown, Michael et al. (2015) High-accuracy, single-base resolution of near-full-length HIV genomes.
- Poster: Murrell, Ben et al. (2015) Full-length env deep sequencing in a donor with broadly neutralizing V1/V2 antibodies.
- Poster: Brown, Michael et al. (2015) Sequencing complex mixtures of HIV-1 genomes with single-base resolution.
- Poster: Laird, Melissa E. et al. (2014) Next generation sequencing of full-length HIV-1 env during primary infection.
- Poster: Imamichi, Hiromi et al. (2014) High-throughput analysis of full-length proviral HIV-1 genomes from PBMCs.
- Poster: Brown, Michael et al. (2013) Rapid sequencing of HIV-1 genomes as single molecules from simple and complex samples.
- Poster: Sethuraman, Anand et al. (2014) Characterization of NNRTI mutations in HIV-1 RT using Single Molecule, Real-Time SMRT Sequencing.
- Paxinos, Ellen (2016) AGBT Virtual Poster: An improved circular consensus algorithm with an application to detect HIV-1 Drug Resistance Associated Mutations (DRAMs)
- Murrell, Ben (2016) AGBT Roche and PacBio Workshop: Deeply sequencing entire genes from rapidly evolving viruses
- Gyllensten, Ulf (2014) Mendelspod: The progress of clinical genomics in Sweden
- Gyllensten, Ulf (2014) AGBT Conference: High-throughput NGS for screening of microbial pathogens
- Case Study: Scientists advance understanding of microbial genomes, epigenomes and communities with SMRT Sequencing (2016)
- Industrial Biotechnology Brochure: Fuel biotech discovery with confident characterization of microbes and their communities (2016)
- Certified Service Provider Profile: Institute for Genome Sciences expands long-read sequencing services with new Sequel System (2016)
- Microbiology and Infectious Disease Brochure: Novel insights into microbial and viral complexity. (2015)
- Featured Interview: Revolutionize Translational Research – Uppsalas Ulf Gyllensten on how long reads give access to new areas of the genome. (2015)