Independently removable nucleic acid sequencing system and method

Patent metadata

Field	Value
Publication number	US-11499191-B2
Application number	US-201816105262-A
Country	US
Kind code	B2
Filing date	Aug 20, 2018
Priority date	Jan 26, 2007
Publication date	Nov 15, 2022
Grant date	Nov 15, 2022

Abstract

A technique for sequencing nucleic acids in an automated or semi-automated manner is disclosed. Sample arrays of a multitude of nucleic acid sites are processed in multiple cycles to add nucleotides to the material to be sequenced, detect the nucleotides added to sites, and to de-block the added nucleotides of blocking agents and tags used to identify the last added nucleotide. Multiple parameters of the system are monitored to enable diagnosis and correction of problems as they occur during sequencing of the samples. Quality control routines are run during sequencing to determine quality of samples, and quality of the data collected.

First claim

What is claimed is:

1. A method, comprising
hybridizing a primer to a first region of a first polynucleotide, extending the primer with a polymerase to incorporate a plurality of sequencing nucleotides wherein each of the plurality of sequencing nucleotides comprises a fluorescent tag and a blocking molecule and extending the primer comprises detecting a fluorescent tag, removing the detected fluorescent tag, and removing a blocking molecule, wherein
each of the plurality of sequencing nucleotides is complementary to one of a plurality of sequenced nucleotides of the first polynucleotide adjacent to the first region, and
the first polynucleotide comprises a capture molecule covalently bound to a bead and a second region, wherein the bead is attached to a substrate and the second region is complementary to a region of a second polynucleotide, wherein the second polynucleotide comprises a fragment isolated from a biological source.

2. The method of claim 1 , wherein a plurality of capture molecules are covalently bound to the bead.

3. The method of claim 2 , wherein the plurality of capture molecules comprise a plurality of first regions with identical sequences to each other.

4. The method of claim 1 , wherein the substrate comprises a plurality of beads to each of which a plurality of capture molecules are covalently bound, wherein the plurality of capture molecules bound to each of the plurality of beads comprise a plurality of first regions with identical sequences to each other.

5. The method of claim 1 , further comprising progressively scanning the substrate to produce image data and processing the image data to identify individual sites and a most recently attached nucleotide at each site.

6. The method of claim 4 , further comprising progressively scanning the substrate to produce image data and processing the image data to identify individual sites and a most recently attached nucleotide at each site.

7. The method of claim 4 wherein the plurality of beads are randomly distributed on the substrate.

8. The method of claim 6 wherein the plurality of beads are randomly distributed on the substrate.

9. The method of claim 4 wherein the plurality of beads are regularly spaced on the substrate.

10. The method of claim 6 wherein the plurality of beads are regularly spaced on the substrate.

11. A method, comprising
hybridizing a primer to a first region of a first polynucleotide, extending the primer with a polymerase to incorporate a plurality of sequencing nucleotides wherein each of the plurality of sequencing nucleotides comprises a fluorescent tag and a blocking molecule and extending the primer comprises detecting a fluorescent tag, removing the detected fluorescent tag, and removing a blocking molecule, wherein
each of the plurality of sequencing nucleotides is complementary to one of a plurality of sequenced nucleotides of the first polynucleotide adjacent to the first region, and
the first polynucleotide comprises a capture molecule covalently bound to a bead and a second region, wherein the bead is attached to a substrate and the second region is complementary to a region of a second polynucleotide, wherein the second polynucleotide comprises a fragment isolated from a biological source, and
the substrate comprises a plurality of beads to each of which a plurality of capture molecules are covalently bound, wherein the plurality of capture molecules bound to each of the plurality of beads comprise a plurality of first regions with identical sequences to each other.

12. The method of claim 11 , further comprising progressively scanning the substrate to produce image data and processing the image data to identify individual sites and a most recently attached nucleotide at each site.

13. The method of claim 11 wherein the plurality of beads are randomly distributed on the substrate.

14. The method of claim 12 wherein the plurality of beads are randomly distributed on the substrate.

15. The method of claim 11 wherein the plurality of beads are regularly spaced on the substrate.

16. The method of claim 12 wherein the plurality of beads are regularly spaced on the substrate.

17. A method, comprising
hybridizing a primer to a first region of a first polynucleotide, extending the primer with a polymerase to incorporate a plurality of sequencing nucleotides wherein each of the plurality of sequencing nucleotides comprises a fluorescent tag and a blocking molecule and extending the primer comprises detecting a fluorescent tag, removing the detected fluorescent tag, and removing a blocking molecule, wherein
each of the plurality of sequencing nucleotides is complementary to one of a plurality of sequenced nucleotides of the first polynucleotide adjacent to the first region, and
the first polynucleotide comprises a capture molecule covalently bound to a bead and a second region, wherein the bead is attached to a substrate and the second region is complementary to a region of a second polynucleotide, wherein the second polynucleotide comprises a fragment isolated from a biological source,
the substrate comprises a plurality of beads to each of which a plurality of capture molecules are covalently bound, wherein the plurality of capture molecules bound to each of the plurality of beads comprise a plurality of first regions with identical sequences to each other, and
progressively scanning the substrate to produce image data and processing the image data to identify individual sites and a most recently attached nucleotide at each site.

18. The method of claim 17 wherein the plurality of beads are randomly distributed on the substrate.

19. The method of claim 17 wherein the plurality of beads are regularly spaced on the substrate.

20. The method of claim 18 wherein the plurality of beads are disposed in nanowells.

21. The method of claim 19 wherein the plurality of beads are disposed in nanowells.

Assignees

• Illumina Inc

Inventors

• Kain Robert C
• Heiner David L
• Zhao Chanfeng
• Gunderson Kevin

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

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

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q2565/501

  being an array of oligonucleotides · CPC title