Analysis of a polynucleotide via a nanopore system

Inventors

Reid, Stuart William • Harper, Gavin

Assignees

Oxford Nanopore Technologies PLC

Abstract

A target polynucleotide is expanded. In respect of each nucleotide in the target polynucleotide, the target polynucleotide comprises clock nucleotides and at least one signal nucleotide in a predetermined order. The clock nucleotides have a predetermined sequence common to each nucleotide in the target polynucleotide. The at least one signal nucleotide is characteristic of the identity of the respective nucleotide in the target polynucleotide. During translocation of the expanded polynucleotide through a nanopore, electrical measurements dependent on the polynucleotide within the pore are made, to derive an analysis signal. Clock signals derived from the clock nucleotides are identified. Relative to the positions of the identified clock signals, nucleotide signals derived from the least one signal nucleotide are derived to analyse the target polynucleotide. The predetermined sequence of the clock nucleotides comprises a restriction site for a restriction enzyme and at least one further nucleotide that extends the predetermined sequence.

Core Innovation

The described invention provides a method of analyzing a target polynucleotide by expanding each nucleotide position into an expanded unit. Each expanded unit ordinally corresponds to a particular nucleotide position of the target polynucleotide and includes a clock nucleotide sequence common among expanded units and at least one signal nucleotide indicative of the member of a set of different nucleotides at the particular nucleotide position. The expanded polynucleotide is prepared as an expanded single-stranded polynucleotide sequence of expanded units so that nucleotide-position information is encoded into clock and signal nucleotides.

To enable nanopore-based reading, two or more single-stranded polynucleotides are hybridised to the expanded single-stranded polynucleotide to form an expanded polynucleotide comprising two or more sections of double-stranded polynucleotide separated by single-stranded polynucleotide. The expanded polynucleotide is then translocated through a nanopore until a first section of double-stranded polynucleotide reaches the nanopore and halts translocation, and the first section is un-hybridised such that translocation continues. Electrical measurements dependent on the expanded polynucleotide within the pore are made throughout the nanopore translocation.

The sequence of one or more regions of the target polynucleotide is determined based on the electrical measurements. The measured electrical signals are converted into an analysis signal in which clock signals are identified to parse the analysis signal and derive nucleotide signals at positions relative to the clock, and nucleotide identity or sequence is determined by comparison to stored reference data or by probabilistic or state-space modeling. The clock nucleotide sequence can include a restriction-site motif plus additional extending nucleotides, and the document further describes feature-vector similarity and Hidden Markov Model analysis with Viterbi to align or infer sequence.

Claims Coverage

The independent claim covers a method with four inventive features: expanded units with a common clock sequence and signal nucleotide(s), hybridisation to create double-stranded sections that halt nanopore translocation, nanopore electrical measurements dependent on the expanded polynucleotide, and determination of one or more target polynucleotide regions from those measurements. The dependent claims further refine the signal/clock nucleotide design and aspects of electrical measurement and translocation registration.

The coverage is directed to an expanded-polynucleotide encoding scheme using a common clock nucleotide sequence and position-indicative signal nucleotide(s), combined with hybridisation-driven halting and continuation of nanopore translocation, and sequence determination of one or more target polynucleotide regions from nanopore-dependent electrical measurements. Additional claim refinements specify how signal/clock nucleotides are constructed, how features are derived and compared, and how the electrical observable and translocation mode are constrained.

Stated Advantages

Documented Applications

No documented applications found