The long-term goal of this project is the commercial development of a low-cost Single-Molecule Sequencing (SMS) technology, called Exo-Seq, which employs a sequencing-by-subtraction strategy for high fidelity disassembly of low amounts of unamplified DNAs. In spite of the SMS approach, Exo-Seq possesses superior single-pass base-call accuracy (>95%) using a multi- parameter identification strategy that targets molecular-dependent electrophoretic mobilities and electrical (current transients) signatures to identify individual mononucleotides (dNMPs). In addition, Exo-Seq can generate long reads (>40 kbp) with potentially sequence-independent calling errors as well as reading modified bases without requiring bisulfite conversion. Exo-Seq is comprised of simple hardware with a small instrument footprint, and a novel mixed-scale plastic chip consumable containing nanosensors to accommodate DNA sequencing in parallel for high throughput applications. Exo-Seq requires only minimally pre-processed DNA inputs minimizing extensive hands-on sample preparation by the end-user, while also significantly reducing downstream reagent/consumable costs associated with most commercial sequencing workflows. Exo-Seq consists of a plastic chip with nanofluidic and microfluidic networks, with 3 functional elements: [1] An input/output fluidic network with each channel containing an in-plane pore (~20 nm) to transduce single DNA molecules. [2] Solid-phase bioreactor with an immobilized exonuclease to allow for high fidelity cleavage of single DNA molecules to generate dNMPs. [3] A nanochannel (50 50 nm; length <10 m) with at least 2 in-plane pores (<10 nm effective diameter) positioned a fixed distance apart used to identify them. The use of mixed-scale replication allows for single-step generation of the chip that can allow for low production costs with high manufacturing compliance. Exo-Seq sequencing uses the following steps: (1) Single- stranded DNA molecules are manipulated by electrokinetics in a nanofluidic network and loaded into the nanosensor. (2) The loaded single DNA molecule is captured by an immobilized exonucleases (Exonuclease I and RecJ) that are covalently attached to a support pillar. Using both exonucleases allows for bi-directional sequencing of ssDNA. (3) The DNA that contacts the exonuclease is continuously cleaved one nucleotide at a time in an ordered direction. (3) The released nucleotides electrokinetically enters a nanochannel containing at least 2 in-plane pores positioned at the entrance/exit ends. (4) Each nucleotide base call is deduced from a molecular- dependent mobility and a current amplitude signal obtained from each nanopore providing 2 independent measurements for unprecedented single-molecule identification efficiency.
Advanced single-molecule sequencing (SMS) technologies are needed to simplify the sequencing workflow, but at the same time, do not sacrifice call accuracy associated with short-read NGS. A novel, low-cost SMS technology, called Exo-Seq, is envisioned as the outcome from this application. Exo-Seq employs a sequencing-by-subtraction strategy for direct disassembly of single-stranded DNA molecules using a combination of immobilized exonucleases. Exo-Seq utilizes a simple workflow by requiring low amounts of input DNA without amplification while offering unprecedented single-pass read accuracy (>95%) via a multi-parameter nucleotide identification strategy.
