Method and apparatus for volumetric imaging of a three-dimensional nucleic acid containing matrix

• US 10,266,888 B2
• Filed: 05/02/2018
• Issued: 04/23/2019
• Est. Priority Date: 11/03/2015
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Daugharthy, Evan R., Terry, Richard C., Lee, Je-Hyuk, Church, George M., Pruitt, Benjamin W.
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/969,118
• Publication Number:
  US 20180251833A1
• Time in Patent Office: 356 Days
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]

Abstract

Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.

Claims

1. A method for nucleic acid analysis, comprising:

(a) providing a biological sample comprising a plurality of nucleic acid molecules in a three dimensional (3D) matrix, which plurality of nucleic acid molecules have a relative 3D spatial relationship;

(b) subjecting said plurality of nucleic acid molecules to nucleic acid amplification under conditions sufficient to form a plurality of amplicons within said 3D matrix, wherein subsequent to formation, said plurality of amplicons are coupled to said 3D matrix and have said relative 3D spatial relationship;

(c) subjecting said plurality of amplicons to nucleic acid sequencing while said plurality of amplicons are coupled to said 3D matrix, which nucleic acid sequencing comprises (i) sequentially contacting said plurality of amplicons with detectable labels and (ii) using a detector to obtain signals corresponding to said detectable labels from said 3D matrix, which signals are obtained by said detector from a plurality of planes during said nucleic acid sequencing; and

(d) using said signals obtained by said detector from said plurality of planes in (c) to generate a 3D volumetric image of said plurality of amplicons, which 3D volumetric image identifies said relative 3D spatial relationship of said plurality of amplicons.

2. The method of claim 1, wherein (b) further comprises coupling said plurality of amplicons to said 3D matrix.

3. The method of claim 1, wherein subsequent to formation, each of said plurality of amplicons is covalently coupled to said 3D matrix.

4. The method of claim 1, further comprising, prior to (a), (i) introducing a matrix-forming material into said biological sample, and (ii) using said matrix-forming material to generate said 3D matrix.

5. The method of claim 1, wherein said biological sample is a cell.

6. The method of claim 5, wherein said 3D matrix is in a cell, and wherein in (c) said signals are obtained from within said cell.

7. The method of claim 6, wherein said signals are fluorescence signals.

8. The method of claim 1, wherein said biological sample is a tissue.

9. The method of claim 1, wherein said detector obtains said signals from said plurality of planes (i) as a hologram, (ii) simultaneously from said plurality of planes, or (ii) sequentially across individual planes of said plurality of planes.

10. The method of claim 1, wherein said 3D matrix comprises a polymeric material.

11. The method of claim 1, wherein each of said plurality of amplicons is functionalized to form covalent attachments to said 3D matrix.

12. The method of claim 1, wherein said signals are obtained at a plurality of planes within said 3D matrix simultaneously.

13. The method of claim 1, wherein said signals are obtained while said plurality of amplicons are contacted with said detectable labels.

14. The method of claim 1, wherein (d) further comprises using said signals obtained in (c) to identify sequences of said plurality of amplicons, and wherein said 3D volumetric image of said plurality of amplicons identifies said sequences.

15. The method of claim 1, wherein (c) further comprises (i) contacting said plurality of amplicons with a plurality of detection probes each having subsequences, (ii) sequentially coupling said detectable labels to at least a subset of said subsequences, and (iii) determining sequences of said subsequences, thereby determining sequences of at least a subset of said plurality of amplicons.

16. The method of claim 1, wherein said biological sample is disposed adjacent to a stage, and wherein during said sequencing, a position of said detector relative to said stage is adjusted to obtain said signals from said plurality of planes.

17. The method of claim 1, wherein said plurality of planes is a plurality of focal planes of said detector.

18. A system for nucleic acid analysis, comprising:

a support configured to retain a biological sample comprising a plurality of nucleic acid molecules in a three dimensional (3D) matrix, which plurality of nucleic acid molecules have a relative 3D spatial relationship;

a detector configured to detect one or more signals from said biological sample; and

a computer operatively coupled to said detector, wherein said computer is configured to;

(a) subject said plurality of nucleic acid molecules to nucleic acid amplification under conditions sufficient to form a plurality of amplicons within said 3D matrix, wherein subsequent to formation, said plurality of amplicons are coupled to said 3D matrix and have said relative 3D spatial relationship;

(b) subject said plurality of amplicons to nucleic acid sequencing while said plurality of amplicons are coupled to said 3D matrix, which nucleic acid sequencing comprises (i) sequentially contacting said plurality of amplicons with detectable labels and (ii) using said detector to obtain signals corresponding to said detectable labels from said 3D matrix, which signals are obtained by said detector from a plurality of planes during said nucleic acid sequencing; and

(c) use said signals obtained by said detector from said plurality of planes in (b) to generate a 3D volumetric image of said plurality of amplicons, which 3D volumetric image identifies said relative 3D spatial relationship of said plurality of amplicons.

19. The system of claim 18, further comprising an optical assembly comprising said detector, which optical assembly is configured to adjust a position of said detector relative to said stage to obtain said signals from said plurality of planes during said nucleic acid sequencing.

20. The system of claim 19, wherein said optical assembly comprises one or more objective lenses that are configured to be in optical communication with said support.

21. The system of claim 18, further comprising a track and a motor having an encoder feedback for adjusting a position of a focal plane of said detector during said nucleic acid sequencing.

22. The system of claim 18, further comprising a mapping unit configured to map a surface of said support with respect to one or more motion axes such that said biological sample is reproducibly positioned relative to said support.

23. The system of claim 22, wherein said mapping unit comprises an autofocus unit that is configured to acquire one or more distance measurements between said detector and said sample.

24. The system of claim 22, wherein said mapping unit is configured to compute one or more offsets relative to a positional encoder along said one or more motion axes.

25. The system of claim 18, further comprising a heating or cooling unit operatively coupled to said support and configured to subject said plurality of nucleic acid molecules to thermo-cycling to facilitate said nucleic acid amplification.

26. The system of claim 18, further comprising a fluidics dispenser configured to deliver programmed volumes of reagents to said biological sample.

27. The system of claim 18, wherein said plurality of planes is a plurality of focal planes of said detector.

28. The system of claim 18, wherein a position of said detector relative to said stage is adjustable for obtaining said signals from said plurality of planes.

29. The system of claim 18, wherein said computer is configured to use said signals obtained in (b) to identify sequences of said plurality of amplicons, and wherein said 3D volumetric image of said plurality of amplicons identifies said sequences.

30. The system of claim 18, wherein said detector is configured to obtain said signals from said plurality of planes (i) as a hologram, (ii) simultaneously from said plurality of planes, or (ii) sequentially across individual planes of said plurality of planes.