A significant obstacle for scientific progress in biotechnology and medicine is the current cost of sequencing a mammalian sized genome, estimated to be in the range of 10-50 MUSD, and the time required for genome sequencing. Many efforts have therefore been recently undertaken in order to develop cheaper and faster alternatives to the current paradigm of DNA sequencing, and it has been identified that techniques based on single molecule sequencing (SMS) may provide the ultimate cost reduction and speed. It has however, proven difficult to scale down DNA analysis to the single molecule level, mainly due to the relatively small differences between the four nucleotides constituting DNA, and due to the inherent noise in single molecule probing. To this end we have undertaken a radical new approach, designed to circumvent these problems and to provide the means for low-cost, high-throughput DNA sequencing performed at the single molecule level. Our approach consists of two steps: (1) As part of the initial preparation step DNA molecules are converted into longer and periodically structured DNA molecule, which we name """"""""Design Polymers"""""""". (2) The Design Polymers are read in a highly parallel way using optical detection of Nanopore arrays. Based on our preliminary experiments and computer simulations, we project that our method will yield an exceptionally high readout rate of >5 M bases per second, using a 100x100 nanopore array.
The specific aims of the current application are threefold: First, we will fabricate a two-color TIRF system for the high-throughput detection of design polymers in 5x5, 10x10, 25x25 and eventually 100x100 nanopore arrays. In parallel, we will develop computer algorithms for the quasi real-time analysis and error correction of the nanopore data. Second, we will improve and extend the biochemical conversion method used to produce design polymers. Our focus will be on cost reduction, extending read length to 80-mers and massive parallelism (converting thousands of different DNA targets in a single tube). Third, we will harness our methodology to real-life studies, during the lifetime of this award: (a) we will re-sequence a viral genome (such as Lambda phage) as a benchmark test for speed, cost, and accuracy, (b) Detect Polymorphism in the human mitochondria genome, (c) Map regulatory regions in the human genome enriched by Chromatin Immunoprecipitation.
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