Please see the following for a suggested list of applications PacBio RS II can support. However, the PacBio RSII is still a novel platform and we encourage you to contact us so we can provide you with the most current information and help you plan your project to meet your sequencing needs.
De Novo Assembly: Generate finished genome assemblies with the PacBio® RS II
Faster and more cost-effective DNA sequencing technologies are accelerating investigations of a broad range of genomes – from model organisms to pathogens that may have a direct impact on human health. However, only a limited number of species have been completely sequenced due to the inherent limitations of second-generation sequencing technologies, including GC-bias and the inability to resolve large structural variations. A complete genome is needed to fully understand the biological relevance of an organism.
The complexity of genome assembly is driven by a variety of factors, including the length and number of total fragments and the presence of repeat regions. The PacBio RS II single-molecule, real-time sequencing system provides extra-long read lengths that simplify and improve genome assembly. By reducing the number of contigs and producing superior consensus accuracy, researchers can finish genomes at a lower cost and with higher quality data.
PacBio RS II benefits:
- Complete genome assemblies – long read lengths combine with high accuracy to produce high quality, finished genomes
- Accurate characterization of large structural variations – long read lengths uniquely provide the ability to sequence large repeat regions and resolve complex structure
- Unbiased genome coverage – balanced coverage and minimal GC-bias for high-quality assembly of high or low GC organisms or regions
- Cost-effective and fast – 10x reduction in finishing costs, results in less than a day, and no need for DNA amplification
Target Sequencing: Comprehensively characterize genomic variation with the PacBio® RS II
Targeted sequencing allows researchers to focus on specific areas of interest within the genome, increasing the cost-effectiveness of studies and the depth of coverage. One common use of targeted sequencing is single nucleotide polymorphism (SNP) detection and validation, where the ability to accurately identify true SNPs and distinguish them from false positives is extremely important.
The PacBio RS II directly measures individual molecules, using long reads to fully characterize genetic complexity, including rare SNPs, indels, structural variants, haplotypes and phasing. Single molecule resolution allows comprehensive characterization of heterogeneous samples and identification of variation invisible to multi-molecule sequencing technologies.
PacBio RS II benefits:
- Reduced false positives – no systematic bias provides confidence in results, higher positive predictive value
- Observation of structural variants – long read lengths provide characterization of variants
- Ability to resolve phasing of mutations – observation of haplotypes and correlation to phenotypes or drug response
- Access to the entire genome – flexibility to sequence through repetitive and GC-rich regions
Base Modification Detection: Revealing new insights into biological processes
Base modifications, such as DNA methylation, are key components of biological processes such as gene expression, host-pathogen interactions, DNA damage and DNA repair. Epigenetic processes are a major area of interest to researchers due to their links to major human diseases like cancer. Historically, it has been challenging to study the broad range of base modifications because very few are amenable to molecular biology techniques, especially sequencing.
The PacBio RS II detects single nucleotide additions in real time, directly measuring the kinetic properties of base additions during the sequencing process. These kinetic measurements present characteristic patterns in response to a variety of base modifications such as 5-methylcytosine, 5-hydroxymethylcytosine, 6-methyladenine, 8-oxoguanine, and more. Pacific Biosciences expects that researchers will use this capability to study a broad range of base modifications at single base resolution.
For example, SMRT® sequencing of whole bacterial genomes including positions of 6-methyladenine is currently in development. We believe that once in the hands of researchers this will allow studies that correlate adenine methylation patterns in various bacterial strains to biological processes at single base resolution. This information has the potential to revolutionize our understanding of microbial genomes. As the technology progresses, we expect to be able to address larger genomes in a comprehensive manner.