Overview:
Nabsys is pioneering the development of positional sequencing, a novel technology with broad applicability for DNA analysis. The Nabsys positional sequencing platform simultaneously generates information about both DNA sequence as well as its location - in the genome, or with respect to other segments of DNA sequence. Classical sequencing generates dozens or 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 in size. 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.
How it works:
Sample DNA is first isolated by established protocols, then hybridized with probes, incubated with a reaction mixture that prepares the DNA for analysis, and finally loaded into the instrument. Single molecules of DNA with bound probes pass through solid-state nanodetectors that can distinguish DNA from background, and identify the locations of bound probes, by direct electrical detection. Long distance maps are thus generated from each single molecule read, revealing the locations of probe binding. 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 maps are then assembled 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. In addition to genome sequencing, probe libraries of varying complexity can be used to address the entire size scale of DNA variation. The same technology platform can thus be used to query genome structural variation, to create genome maps, and to target specific genes for sequence analysis. The concepts underlying positional sequencing, as well as the general workflow, are described further both in schematic drawings and videos that are displayed on this page.
A non-technical summary of the NABsys approach to DNA sequencing which highlights the benefits of our approach and potential applications.
A more detailed summary of the actual physical processes which we are employing to sequence DNA.
Positional sequencing features and benefits:
• Speed: DNA passes through solid-state detectors at a rate of approximately 1 million bases per second. Whether on a single chip or on multiple chips, as few as 100 detectors per instrument would generate data at rates surpassing existing nextgen sequencing systems
• Scalability and throughput: Thousands of individual detectors can be created 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
• Accuracy: Independent measurements from multiple overlapping probes in multiple runs dramatically increase data accuracy. In order to make an error at any individual base, all of the different probes that hybridize in the area of that base would have to bind incorrectly. The same logic cannot be applied to short-read data even at high coverage
• 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 specific questions, such as analysis of particular SNP variants or detection of large scale translocations. In fact, the system must be effectively untargeted through use of shorter probes which bind in many locations in order to generate the data needed to assemble genome-wide maps and reconstruct whole genome sequence
• Single molecule analysis: Individual DNA molecules are analyzed rather than ensembles. This avoids the need for PCR or amplification and all the biases they can introduce due to base composition and other dependencies. 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, sequencing data burden is reduced by many orders of magnitude. In practical terms, this means that genome 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 hybridization 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 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
Figure 1. Sample preparation for direct electronic detection of hybridized probe locations. Standard DNA extraction procedures are employed, and no library preparation is required. Following isolation, DNA is denatured, tagged probes are hybridized, the remaining second strand is filled in, and the entire complex is coated with RecA protein.
Figure 2. Schematic describing the steps involved in generation and assembly of probe maps, and reconstruction of DNA sequence, by positional sequencing. For targeted DNA analysis, less complex pools of probes are utilized, and depending on the application, map assembly and/or sequence reconstruction may not be necessary.
Figure 3. Example of signal trace resulting from direct electrical detection of a fragment of DNA hybridized with two probes using a solid-state nanodetector. The X axis show time in seconds. The DNA fragment runs from approximately the 0.0035 second mark until the 0.0055 second mark, with peaks representing probes clearly visible at the 0.0047 second mark and the 0.0053 second mark.
