Nabsys is pioneering the development of semiconductor-based tools for genomic analysis. The Nabsys positional sequencing and mapping platform simultaneously generates information about both the DNA sequence and its location - in the genome and with respect to other segments of DNA sequence. Classical sequencing typically generates only hundreds of contiguous bases from each read that then must be assembled with no knowledge of sequence location. In contrast, positional sequencing generates many short stretches of non-contiguous sequence spread over extremely long distances, simultaneously revealing the relative position of each sequence during reads of single DNA molecules that can be as large as 100s of kilobases. Knowledge of the location of each of many sequences spread over large regions greatly facilitates assembly, thereby enabling rapid, accurate and complete de novo assembly of whole genome sequences without the need to rely on reference databases.
Similarly, whole genome maps can be readily generated from this long-range information even when no short-read data is available.
How it works:
Sample DNA is first isolated by established protocols, tagged with probes specific to particular locations, incubated with a reaction mixture that stiffens the double-stranded DNA, and then loaded into the instrument. Single molecules of DNA with bound tags pass through solid-state nanodetectors that can distinguish DNA from background and identify the locations of bound tags, by direct electrical detection. Long distance maps are thus generated from each single-molecule read, revealing the locations of tag binding. With just a single tag type, this technology platform can be used to assemble whole genomes, to query genome structural variation, to create genome maps, and to target specific genes for analysis. When used for sequencing DNA, independent maps are generated from hybridization with different pools of probes, the pools having been designed to generate overlapping sequence information. The independent maps are then combined and the underlying sequence reconstructed. Each individual base is queried many times by overlapping probes of different sequence, hybridized in different pools, so that many independent events confirm the presence of the correct base. The general workflow is described further both in schematic drawings and video.
Positional sequencing features and benefits:
• Speed: DNA passes through solid-state detectors at a rate of approximately 1 million bases per second. A single chip would generate data at rates surpassing existing next-gen sequencing systems. Data from only a few minutes of collection are sufficient for many applications.
• Scalability and throughput: The current detectors contain only a single detector per module but future generations will contain tens to thousands of individual detectors on a single chip, and many chips can be assembled in a single instrument, providing the opportunity for unprecedented levels of throughput.
• Precision: Electronic measurements enable the location of binding events to be determined at well below the diffraction limit of light, enabling creation of maps with greater precision than has ever been possible with optical techniques.
In contrast to approaches that rely on optical analysis, Nabsys positional sequencing is able to electronically measure the location of DNA probes with sub-diffraction limit resolution, enabling creation of the most precise maps ever produced. There is no requirement for expensive cameras or lasers. Position is simply measured by the time between direct electrical detection of tagging events.
• Information content: Each read provides information over as many as 10s to 100s of kilobases on single DNA molecules, revealing long range relationships and facilitating data assembly. Single-molecule analysis provides quantitative data that can be used to reveal sample heterogeneity. With positional sequencing, experiments can be designed to query the entire size scale of DNA variation.
• Inherently targeted: Long probes can be chosen to target specific sequences in order to answer the question of interest, such as analysis of particular SNP variants or detection of large scale translocations. Assays are readily designed to generate small or large amounts of information, depending on the researcher’s needs.
• Single molecule analysis: Individual DNA molecules are analyzed rather than ensembles of amplified pieces. This avoids the need for PCR or amplification and all the biases they introduce due to base composition and other factors that cause differential copying efficiency. Individual molecules preserve haplotype and long-distance information for more efficient assembly and mapping.
• Reduced data burden: By eliminating the need to analyze optical image files, leveraging knowledge of sequence location to facilitate assembly, and utilizing advanced algorithms for data analysis, the data burden is reduced by many orders of magnitude. In practical terms, this means that genome mapping and sequencing can be performed on a simple laptop computer rather than a complex cluster, and data storage requirements are dramatically reduced.
• Simple workflow: Following DNA extraction, only tagging and incubation reactions are required prior to loading samples for analysis. There is no need for complicated and time-consuming library preparation.
• Low cost: By relying on electrical detection, the need for complex optical systems such as lasers and CCD cameras is eliminated and the cost of instrumentation dramatically reduced. The use of few reagents, all in very small quantities, minimizes the cost of each run.