NUCLEIC ACID SEQUENCING BY SELECTIVE EXCITATION OF MICROPARTICLES

• US 20090061526A1
• Filed: 08/28/2007
• Published: 03/05/2009
• Est. Priority Date: 08/28/2007
• Status: Active Grant

• Current Assignee: Rebus Biosystems, Inc.
• Original Assignee: LightSpeed Genomics, Inc.
• Inventors: Park, Jong Buhm, Ryu, Jekwan, Hong, Stanley S.
• Granted Patent: US 8,222,040 B2
• Time in Patent Office: Days
• US Class Current: 436/94
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G06F 18/00 Pattern recognition
• CPC Class Codes: G06V 2201/04 Recognition of patterns in DNA microarrays
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Nucleic acid microparticles are sequenced by performing a sequencing reaction on the microparticles using one or more reagents, selectively exciting the microparticles in an excitation pattern, optically imaging the microparticles at a resolution insufficient to resolve individual microparticles, and processing the optical images of the microparticles using information on the excitation pattern to determine the presence or absence of the optical signature, which indicates the sequence information of the nucleic acid. An apparatus for optical excitation of the microparticles comprises an optical fiber delivering a first laser beam, and an interference pattern generation module coupled to the optical fiber. The interference pattern generation module splits the first laser beam into second and third laser beams and generates the excitation pattern for selectively exciting the microparticles by interference between the second and third laser beams.

Claims

1. A method for sequencing nucleic acid microparticles, the method comprising the steps of:

performing at least a sequencing reaction on the nucleic acid microparticles using one or more sequencing reagents;

selectively exciting the nucleic acid microparticles in an excitation pattern;

optically imaging the nucleic acid microparticles at a resolution insufficient to resolve individual microparticles; and

processing the optical images of the nucleic acid microparticles using information on the excitation pattern to determine presence or absence of at least an optical signature, the presence or absence of said at least optical signature indicating sequence information of the nucleic acid.

2. The method of claim 1, wherein the nucleic acid microparticles are selectively excited at a resolution sufficient to resolve the individual microparticles.

3. The method of claim 1, wherein the excitation pattern is generated by interference of a plurality of illumination beams.

4. The method of claim 3, wherein the plurality of illumination beams undergo total internal reflection off a substrate on which the microparticles are placed.

5. The method of claim 3, wherein the excitation pattern generated by interference of the plurality of illumination beams produces sampling of a frequency domain in which an extent of the distribution of frequency samples is matched to a feature size of the microparticles.

6. The method of claim 3, wherein the excitation pattern generated by interference of the plurality of illumination beams produces a rectilinear sampling of a frequency domain.

7. The method of claim 3, wherein the excitation pattern generated by interference of the plurality of illumination beams produces a non-rectilinear sampling of a frequency domain.

8. The method of claim 1, further comprising:

performing another sequencing reaction on the nucleic acid microparticles;

selectively exciting the nucleic acid microparticles;

optically imaging the nucleic acid microparticles at a resolution insufficient to resolve the individual microparticles; and

processing the optical images of the nucleic acid microparticles using information on the excitation pattern to determine presence or absence of another optical signature, the presence or absence of said another optical signature indicating different sequence information of the nucleic acid.

9. The method of claim 1, wherein selectively exciting the nucleic acid microparticles comprises:

selectively exciting the nucleic acid microparticles in a first excitation pattern generated by interference of the illumination beams; and

selectively exciting the nucleic acid microparticles in a second excitation pattern different from the first pattern generated by a different interference of the illumination beams.

10. The method of claim 9, wherein a phase of at least one of the illumination beams is modulated to generate the different interference of the illumination beams.

11. The method of claim 1, wherein determining the presence or absence of at least an optical signature comprises determining that at least one of the microparticles exhibits an optical signature brighter or with a different spectral characteristic than other microparticles.

12. The method of claim 1, wherein processing the optical images of the nucleic acid microparticles comprises:

generating a synthetic image of the microparticles with a higher resolution than the optical image based on the information on at least the excitation pattern;

applying a microparticle signature identification algorithm to the synthetic image to generate microparticle optical signature data; and

applying a base calling algorithm to determine the presence or absence of said at least optical signature corresponding to the sequence information of the nucleic acid.

13. The method of claim 1, further comprising repeating the steps of selective excitation, optical imaging, and image processing for a different field of view.

14. The method of claim 1, further comprising:

selectively exciting a calibration target by interference of a plurality of illumination beams;

optically imaging the excited calibration target; and

processing the optical images of the excited calibration target to generate a first optical image of the excited calibration target, a difference between the first optical image of the excited calibration target and a second, predicted optical image of the calibration target predicted based on first calibration parameters for generating the interference of the illumination beams being used to generate second calibration parameters for generating the interference of the illumination beams.

15. A system for sequencing nucleic acid microparticles, the system comprising:

a sequence reaction module configured to apply one or more sequencing reagents to the nucleic acid microparticles;

a selective microparticle excitation module configured to selectively excite the nucleic acid microparticles in an excitation pattern;

an optical imaging module configured to generate one or more optical images of the nucleic acid microparticles at a resolution insufficient to resolve individual microparticles; and

an image processing module configured to process the optical images of the nucleic acid microparticles using information on the excitation pattern and to determine presence or absence of at least an optical signature, the presence or absence of said at least optical signature indicating sequence information of the nucleic acid.

16. The system of claim 15, wherein the selective microparticle excitation module is configured to selectively excite the nucleic acid microparticles at a resolution sufficient to resolve the individual microparticles.

17. The system of claim 15, wherein the selective microparticle excitation module is configured to generate the excitation pattern by interference of a plurality of illumination beams.

18. The system of claim 17, wherein the plurality of illumination beams undergo total internal reflection off a substrate on which the microparticles are placed.

19. The system of claim 17, wherein the selective microparticle excitation module is configured to generate the excitation pattern by interference of a plurality of illumination beams that produces a rectilinear sampling of a frequency domain.

20. The system of claim 17, wherein the selective microparticle excitation module is configured to generate the excitation pattern by interference of a plurality of illumination beams that produces a non-rectilinear sampling of a frequency domain.

21. The system of claim 15, wherein the selective microparticle excitation module is configured to:

selectively excite the nucleic acid microparticles in a first excitation pattern generated by interference of the illumination beams; and

selectively excite the nucleic acid microparticles in a second excitation pattern different from the first excitation pattern generated by a different interference of the illumination beams.

22. The system of claim 21, wherein the selective microparticle excitation module is configured to modulate a phase of at least one of the illumination beams to generate the different interference of the illumination beams.

23. The system of claim 15, wherein the image processing module is configured to determine the presence or absence of an optical signature by determining that at least one of the microparticles exhibits an optical signature brighter or with a different spectral characteristic than other microparticles.

24. The system of claim 15, wherein the image processing module is configured to:

generate a synthetic image of the microparticles with a higher resolution than the optical image based on the information on at least the excitation pattern;

apply a microparticle signature identification algorithm to the synthetic image to generate microparticle optical signature data; and

apply a base calling algorithm to determine the presence or absence of said at least optical signature corresponding to the sequence information of the nucleic acid.