Improving read length, accuracy, and availability of single-molecule DNA sequencing

The ability to completely sequence a single molecule of DNA by reading it directly is one of the greatest challenges of biotechnology. It also unlocks the ability to directly read RNA sequences. Two techniques in particular are showing great progress towards this goal. The first one (MinIon, Oxford Nanopore Technologies) is essentially reading the size of short sequential segments of DNA using a protein nanopore. The second technique (SMRT, PacBIO) is using a zero-mode waveguide to record an immobilized DNA polymerase complex as it transcribes a DNA molecule. SMRT sequencing has high startup and consumable costs and has a limited read length which limits the ability to analyze large-scale structural variations. The MinIon system is more cost effective, extremely portable, and has a larger read length, but the error rate without resampling or a priori knowledge is rather large. In addition, since it cannot record the articulation of single nucleotides, it is complicated to measure single-nucleotide variations or homopolymeric sequences accurately. The ability to accurately read an entire strand of DNA is a conditio sine qua non for complete singlemolecule direct read sequencing. Reading nucleotides electronically using a pair of nanoelectrodes promises to yield single-nucleotide resolution and long read length while requiring the smallest possible amount of consumables. We have developed the technology to create small graphene nanogaps that can be used as tunneling electrodes for direct-read DNA sequencing. Here, we propose to develop a single-molecule direct read DNA sequencing platform based on graphene nanogaps. In Aim 1 we will develop a cost-effective process to create graphene nanogap tunneling electrode devices for use in direct read DNA sequencing. We will integrate our graphene devices with high frequency waveguides which enables accurate high speed performance. We aim to increase singlenucleotide accuracy by at least a factor 10 beyond MinIon technology. In Aim 2 we will develop the protocols to accurately measure long segments of single DNA molecules with single-nucleotide accuracy. We will record high frequency electronic traces of molecules passing through the nanogap and develop the signal processing pipeline to generate DNA sequences from it. We will demonstrate this system?s expected unique capability by sequencing long homopolymeric sequences and sequences with long-range structural variations. A guiding principle of our design is that it should be straightforward to implement with modest investment by leveraging cost-effective materials and fabrication techniques that have surfaced during the recent maker revolution.