System and method for automated repeat sequencing

Patent metadata

Field	Value
Publication number	US-11959074-B2
Application number	US-202117525737-A
Country	US
Kind code	B2
Filing date	Nov 12, 2021
Priority date	Nov 14, 2020
Publication date	Apr 16, 2024
Grant date	Apr 16, 2024

Abstract

A method for sequencing a target polynucleotide includes detecting a first series of nucleotide incorporations complementary to at least a portion of the target polynucleotide. The first series of nucleotide incorporations forms a first complementary polynucleotide. The target nucleotide is secured to a substrate disposed in a sequencing zone of an assembly. The method further includes moving the substrate to which the target nucleotide is secured to a templating zone of the assembly; removing the first complementary polynucleotide when the substrate is disposed at the templating zone of the assembly, the target polynucleotide remaining secured to the substrate; following the removing, moving the substrate to which the target polynucleotide is secured to the sequencing zone; and detecting a second series of nucleotide incorporations complementary to at least a portion of the target polynucleotide, the second series of nucleotide incorporations forming a second complementary polynucleotide.

First claim

What is claimed is:

1. A method for sequencing a target polynucleotide, the method comprising:
detecting a first series of nucleotide incorporations complementary to at least a portion of the target polynucleotide, the first series of nucleotide incorporations forming a first complementary polynucleotide, the target nucleotide secured to a substrate disposed in a sequencing zone of an assembly;
moving the substrate to which the target nucleotide is secured to a templating zone of the assembly;
removing the first complementary polynucleotide when the substrate is disposed at the templating zone of the assembly, the target polynucleotide remaining secured to the substrate;
following the removing, moving the substrate to which the target polynucleotide is secured to the sequencing zone; and
detecting a second series of nucleotide incorporations complementary to at least a portion of the target polynucleotide, the second series of nucleotide incorporations forming a second complementary polynucleotide.

2. The method of claim 1 , wherein the second series of nucleotide incorporations is at least 97% the same as the first series of nucleotide incorporations.

3. The method of claim 1 , wherein the second series of nucleotide incorporations is at least 99% the same as the first series of nucleotide incorporations.

4. The method of claim 1 , wherein removing includes denaturing using a change in ionic strength to disassociate the first complementary polynucleotide and the target polynucleotide.

5. The method of claim 1 , wherein removing includes melting by increasing a temperature to disassociate the first complementary polynucleotide and the target polynucleotide.

6. The method of claim 1 , further comprising adding a primer at least partially complementary to the target polynucleotide following the removing.

7. The method of claim 6 , wherein adding the primer includes adding the primer while the substrate is disposed in the templating zone.

8. The method of claim 1 , wherein detecting the first series of nucleotide incorporations is performed using a first flow order of nucleotides and the second series of nucleotide incorporations is performed using a second flow order of nucleotides.

9. The method of claim 1 , wherein the first flow order is different from the second flow order.

10. The method of claim 1 , wherein the detecting the first series of nucleotide incorporations and the detecting the second series of nucleotide incorporations produce first and second sets of signals, the method further comprising using the first and second sets of signals to determine a ordered set of base calls.

11. The method of claim 10 , wherein using the first and second sets of signals includes averaging corresponding signals of the first and second sets of signals.

12. The method of claim 11 , wherein averaging includes weighted averaging.

13. The method of claim 1 , wherein the substrate includes an array of sensors, the target polynucleotide secured in proximity to a sensor of the array of sensors.

14. The method of claim 1 , further comprising engaging an adapter to the substrate when the substrate is disposed in the templating zone.

15. The method of claim 1 , further comprising engaging a fluidic circuit with the substrate in response to moving the substrate to the sequencing zone.

16. The method of claim 1 , further comprising:
securing the target polynucleotide to the substrate when the substrate is in the templating zone; and
moving the substrate to the sequencing zone prior to detecting the first series of nucleotide incorporations.

17. The method of claim 16 , wherein securing the target polynucleotide to the substrate includes:
forming the target polynucleotide on a polymer particle;
depositing a polymer particle into a well of the substrate; and
copying the target polynucleotide onto the polymer particle.

18. The method of claim 1 , wherein detecting includes pH-based detection.

19. The method of claim 18 , wherein pH-based detection includes detecting using an ion-sensitive field effect transistor formed in the substrate.

20. The method of claim 1 , wherein detecting includes fluorescence-based detection.

Assignees

• Life Technologies Corp

Inventors

• Wong Chiu Tai Andrew
• Szeto Kylan
• Shankar Shanti
• Beauchemin Mark

Classifications

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6825

  Nucleic acid detection involving sensors · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• G01N35/1081

  characterised by the means for relatively moving the transfer device and the containers in an horizontal plane (G01N35/1011 takes precedence) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title