Nanopore sequencing using n-mers

• US 9,546,400 B2
• Filed: 09/13/2013
• Issued: 01/17/2017
• Est. Priority Date: 04/10/2009
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Flusberg, Benjamin
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US14/026,906
• Publication Number:
  US 20140061048A1
• Time in Patent Office: 1,222 Days
• US Class Current: 1/1
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

The invention relates to devices and methods for nanopore sequencing. The invention provides for using the signals from n-mers to provide sequence information, for example where the system has less than single base resolution. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS. In some cases, the arrays of nanopores comprise resistive openings for isolating the electronic signals for improved sequencing. Methods for controlling translocation of through the nanopore are disclosed.

Claims

1. A method for sequencing a nucleic acid template comprising:

a) providing a substrate comprising a nanopore in contact with a solution, the solution comprising a template nucleic acid above the nanopore;

b) providing a voltage across the nanopore;

c) measuring a property which has a value that varies for N monomeric units of the template nucleic acid in the pore, wherein the measuring is performed as a function of time, while the template nucleic acid is translocating through the nanopore, wherein N is three or greater; and

d) determining the sequence of the template nucleic acid using the measured property from step (c) by performing a process including comparing the measured property from step (c) to calibration information produced by measuring such property for 4 to the N sequence combinations.

2. The method of claim 1 wherein a property in step (c) comprises current.

3. The method of claim 1 wherein the translocation through the pore is driven by the applied voltage.

4. The method of claim 1 wherein the translocation rate through the pore is enzymatically controlled.

5. The method of claim 3 wherein the translocation through the pore is controlled by a polymerase, a helicase, a translocase, a viral genome packaging motor, or a chromatin remodeling complex.

6. The method of claim 1 wherein N corresponds to n-mers comprising 3-mers, 4-mers or 5-mers.

7. The method of claim 6 wherein N corresponds to n-mers comprising 3-mers.

8. The method of claim 1 wherein the method is carried out on an array of nanopores in the substrate.

9. The method of claim 1 wherein the sequencing comprises peak finding by heuristic decision-tree algorithms, Bayesian networks, hidden Markov models, or conditional random fields.

10. The method of claim 1 wherein the comparing process comprises examining a lookup table for each of the 4 to the N combinations, and keeping only those meeting a threshold value.

11. The method of claim 10 wherein threshold value is within 2 sigma of the expected value.

12. The method of claim 1 wherein some of the values for the 4 to the N sequence combinations are degenerate within the error of the measurement.

13. The method of claim 1 wherein after each single-nucleotide translocation through the nanopore, the possible n-mers for that measurement are looked up, and all the possibilities from the previous measurement that are not consistent with the most recent measurement are thrown away.

14. The method of claim 1 wherein N corresponds to n-mers comprising 4-mers.

15. The method of claim 1 wherein N corresponds to n-mers comprising 5-mers.