Exo-Seq for the single molecule of DNA molecules

ABSTRACT The long term goal of this project is the commercial development of a low cost Single Molecule SequencingSMStechnologycalled Exo Seqwhich employs a sequencing by subtraction strategy for high fidelity disassembly of low amounts of unamplified DNAsIn spite of the SMS approachExo Seq possesses superior single pass base call accuracyandgtusing a multiparameter identification strategy that targets molecular dependent electrophoretic mobilities and electricalcurrent transientssignatures to identify individual mononucleotidesdNMPsIn additionExo Seq can generate long readsandgtkbpwith potentially sequence independent calling errors as well as reading modified bases without requiring bisulfite conversionExo Seq is comprised of simple hardware with a small instrument footprintand a novel mixed scale plastic chip consumable containing nanosensors to accommodate DNA sequencing in parallel for high throughput applicationsExo Seq requires only minimally pre processed DNA inputs minimizing extensive hands on sample preparation by the end userwhile also significantly reducing downstream reagent consumable costs associated with most commercial sequencing workflowsExo Seq consists of a plastic chip with nanofluidic and microfluidic networkswithfunctional elementsAn input output fluidic network with each channel containing an in plane porenmto transduce single DNA moleculesSolid phase bioreactor with an immobilized exonuclease to allow for high fidelity cleavage of single DNA molecules to generate dNMPsA nanochannelnmlength andltmwith at leastin plane poresandltnm effective diameterpositioned a fixed distance apart used to identify themThe use of mixed scale replication allows for single step generation of the chip that can allow for low production costs with high manufacturing complianceExo Seq sequencing uses the following stepsSinglestranded DNA molecules are manipulated by electrokinetics in a nanofluidic network and loaded into the nanosensorThe loaded single DNA molecule is captured by an immobilized exonucleasesExonuclease I and RecJthat are covalently attached to a support pillarUsing both exonucleases allows for bi directional sequencing of ssDNAThe DNA that contacts the exonuclease is continuously cleaved one nucleotide at a time in an ordered directionThe released nucleotides electrokinetically enters a nanochannel containing at leastin plane pores positioned at the entrance exit endsEach nucleotide base call is deduced from a moleculardependent mobility and a current amplitude signal obtained from each nanopore providingindependent measurements for unprecedented single molecule identification efficiency NARRATIVE Advanced single molecule sequencingSMStechnologies are needed to simplify the sequencing workflowbut at the same timedo not sacrifice call accuracy associated with short read NGSA novellow cost SMS technologycalled Exo Seqis envisioned as the outcome from this applicationExo Seq employs a sequencing by subtraction strategy for direct disassembly of single stranded DNA molecules using a combination of immobilized exonucleasesExo Seq utilizes a simple workflow by requiring low amounts of input DNA without amplification while offering unprecedented single pass read accuracyandgtvia a multi parameter nucleotide identification strategy