Systems and methods for high-fidelity capture, threading, and infinite-depth sequencing of single nucleic acid molecules

Inventors

AKSIMENTIEV, Aleksei

Assignees

University of Illinois System

Abstract

Aspects of the subject disclosure may include, for example, an apparatus comprising: a membrane, wherein the membrane has a first side and a second side, wherein the membrane has a first pore disposed therein, wherein the first pore extends through the membrane from the first side of the membrane to the second side of the membrane, wherein the membrane has a second pore disposed therein, and wherein the second pore extends through the membrane from the first side of the membrane to the second side of the membrane; a first channel disposed on the first side of the membrane, wherein the first channel is along a first longitudinal axis; a second channel disposed on the first side of the membrane, wherein the second channel is along a second longitudinal axis, and wherein the first channel and the second channel are disposed side by side adjacent to each other; a third channel disposed on the second side of the membrane, wherein the third channel is along a third longitudinal axis, wherein the third channel is in first fluid communication with the first channel via the first pore, and wherein the third channel is in second fluid communication with the second channel via the second pore; and one or more sensors disposed at one or more locations to facilitate sequencing of a molecule that extends from the first channel, through the first pore across at least a portion of the third channel, and through the second pore into the second channel. Additional embodiments are disclosed.

Core Innovation

The disclosure describes double-nanopore sequencing using a membrane with a first and a second pore. A molecule is moved through both pores such that a first portion of the molecule is detected as a sequence of a plurality of different types of nucleotides at the first pore, while a second portion is detected for a presence of a single particular type of nucleotide at the second pore. The detection on the second portion includes determining a count of adjacent occurrences of the single particular type of nucleotide based at least in part on the sequence detected on the first portion.

The described approach links multi-portion nanopore detection by correlating signals between the first pore and the second pore. Sensing at the first pore from current changes associated with a first voltage differential provides information used to interpret detection at the second pore. The second-pore signal includes counting based on the first-pore sequence to determine a presence and adjacent-occurrence count of a single particular nucleotide type on the second portion.

The disclosure also describes determining homopolymer length across the two pores. It describes using a heterogeneous fragment passing through one pore at a particular time, counting current steps, and using that count to determine a length of a homopolymer fragment associated with the other pore. The disclosure further describes repeated exposure and correlated stepwise displacement to enable increased sequence accuracy and homopolymer recognition in the sequencing context.

Claims Coverage

The independent claim set comprises three independent claims. Across these claims, the main inventive features are two-portion, two-pore nanopore detection where second-portion adjacent-occurrence counting of a single nucleotide type is determined based at least in part on the first-portion detected nucleotide sequence.

The claim coverage focuses on two-pore nanopore detection in which first-pore detection of a sequence of plurality different nucleotide types is used to determine a count of adjacent occurrences of a single particular nucleotide type detected at the second pore. Dependent claims refine signal interpretation, including current-flow/voltage-differential changes and counting current steps, and relate counted signal behavior to homopolymer fragment length.

Stated Advantages

Documented Applications