阅读说明：本技术 用于关联生物颗粒的物理和遗传特性的方法和系统 (Method and system for correlating physical and genetic characteristics of biological particles ) 是由 拉吉夫·巴拉德瓦杰 瑟奇·萨克森诺夫 于 2018-11-16 设计创作，主要内容包括：本文提供了用于处理来自生物颗粒(例如,细胞)的核酸分子的方法和系统。可产生多个分配区(例如,液滴),以使多个分配区中的分配区各自包括包含核酸分子的生物颗粒(例如,细胞)和颗粒(例如,珠粒)。可对分配区进行处理(例如,成像),以获得它们相应的生物颗粒的一种或多种物理和/或光学特性。可对包括在分配区中的核酸分子进行条形码化和测序(例如,使用偶合于分配区的颗粒的核酸条形码分子)以产生核酸分子的核酸序列。核酸序列可与生物颗粒的一种或多种光学特性以电子方式关联。(Provided herein are methods and systems for processing nucleic acid molecules from biological particles (e.g., cells). A plurality of distribution regions (e.g., droplets) can be generated such that distribution regions in the plurality of distribution regions each include a biological particle (e.g., a cell) and a particle (e.g., a bead) comprising a nucleic acid molecule. The distribution areas may be processed (e.g., imaged) to obtain one or more physical and/or optical properties of their respective biological particles. Nucleic acid molecules included in the partition region can be barcoded and sequenced (e.g., using nucleic acid barcode molecules coupled to particles of the partition region) to generate nucleic acid sequences of the nucleic acid molecules. The nucleic acid sequence may be electronically associated with one or more optical properties of the biological particle.)

1. A method for processing nucleic acid molecules of a biological particle, comprising:

(a) providing (1) a plurality of partition regions, wherein a partition region of the plurality of partition regions comprises (i) a particle comprising a plurality of nucleic acid barcode molecules, wherein a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules comprises a barcode sequence, and (ii) a bioparticle comprising the nucleic acid molecule; and (2) a first data set stored in a computer memory comprising data indicative of one or more physical characteristics of the biological particle, the data generated upon sensing the biological particle;

(b) generating barcoded nucleic acid molecules using the nucleic acid barcode molecules of the plurality of nucleic acid barcode molecules and the nucleic acid molecules of the biological particles;

(c) generating a second data set comprising data identifying the nucleic acid sequence of the barcoded nucleic acid molecule or derivative thereof; and

(d) correlating said one or more physical properties of said biological particle with said nucleic acid sequence using said first data set and said second data set.

2. The method of claim 1, wherein the one or more physical characteristics of the biological particle comprise a size of the biological particle, a shape of the biological particle, a surface marker on the biological particle, an inclusion in the biological particle, a structure of an organelle in the biological particle, a number of organelles in the biological particle, secretion or excretion relative to the biological particle, or localization of organelles in the biological particle.

3. The method of claim 1, wherein the biological particle comprises a plurality of nucleic acid molecules.

4. The method of claim 3, wherein the plurality of nucleic acid molecules comprises a plurality of ribonucleic acid molecules.

5. The method of claim 3, wherein the plurality of nucleic acid molecules comprises a plurality of deoxyribonucleic acid molecules.

6. The method of claim 1, wherein the nucleic acid molecule is a ribonucleic acid molecule.

7. The method of claim 1, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule.

8. The method of claim 1, wherein the one or more physical characteristics comprise phenotypic information of the biological particle.

9. The method of claim 1, further comprising optically detecting the particle to generate a third data set comprising data indicative of one or more physical characteristics of the particle or the plurality of nucleic acid barcode molecules.

10. The method of claim 9, wherein the one or more physical characteristics of the particle comprise an optical characteristic of the particle.

11. The method of claim 9, wherein the physical property of the particle comprises a size, shape, roundness, hardness, or symmetry of the particle or a component thereof.

12. The method of claim 10, wherein the optical characteristic of the particle comprises absorbance, birefringence, color, fluorescence characteristic, luminosity, photosensitivity, reflectivity, refractive index, scattering, or transmittance of the particle or a component thereof.

13. The method of claim 1, wherein a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules comprises barcode sequences that are identical.

14. The method of claim 1, wherein the plurality of dispensing regions are a plurality of droplets.

15. The method of claim 1, wherein the plurality of distribution areas are a plurality of wells.

16. The method of claim 1, wherein the particles comprise one or more optical barcodes.

17. The method of claim 16, wherein the plurality of nucleic acid barcode molecules of the particle comprises the one or more optical barcodes.

18. The method of claim 16, wherein the one or more optical barcodes comprise a fluorescent dye, a nanoparticle, a microparticle, or any combination thereof.

19. The method of claim 16, wherein the one or more optical barcodes have a different associated optical intensity or frequency relative to other optical barcodes of the plurality of dispensing regions.

20. The method of claim 16, wherein (a) - (d) are repeated for additional distribution regions of the plurality of distribution regions, wherein the additional distribution regions comprise (i) additional particles comprising an additional plurality of nucleic acid barcode molecules, wherein nucleic acid barcode molecules of the additional plurality of nucleic acid barcode molecules comprise an additional barcode sequence different from the barcode sequence, and (ii) additional biological particles comprising additional nucleic acid molecules, wherein the distribution regions and the additional distribution regions produce optical signals of different intensities or frequencies upon sensing.

21. The method of claim 18, wherein the nanoparticles comprise quantum dots.

22. The method of claim 18, wherein the nanoparticle comprises a Janus particle.

23. The method of claim 1, wherein the plurality of nucleic acid barcode molecules are configured in a geometric configuration.

24. The method of claim 23, wherein the geometric structure is a nucleic acid origami.

25. The method of claim 1, wherein the one or more physical properties of the biological particle are identified by imaging the distribution region using (i) bright field microscopy, (ii) fluorescence microscopy, (iii) phase contrast microscopy, (iv) multispectral microscopy, or (v) polarized light microscopy.

26. The method of claim 1, wherein the particles are beads.

27. The method of claim 26, wherein the bead is a gel bead.

28. The method of claim 27, wherein the plurality of nucleic acid barcode molecules are releasably attached to the gel beads.

29. The method of claim 27, wherein the gel bead comprises a polyacrylamide polymer.

30. The method of claim 1, wherein the one or more physical characteristics are one or more optical characteristics.

31. The method of claim 30, further comprising, after (a), imaging the distribution region to optically detect the biological particles to identify the one or more optical properties of the biological particles.

32. The method of claim 30, further comprising, prior to (a), imaging the biological particle to identify the one or more optical properties of the biological particle.

33. The method of claim 32, wherein the biological particles are imaged prior to being provided in the distribution region.

34. The method of claim 1, wherein the particles are coupled to additional particles comprising one or more optical barcodes.

35. The method of claim 1, wherein (a) - (d) are repeated for additional partitioning regions of the plurality of partitioning regions, wherein the additional partitioning regions each comprise (i) an additional plurality of nucleic acid barcode molecules, wherein nucleic acid barcode molecules of the additional plurality of nucleic acid barcode molecules comprise an additional barcode sequence different from the barcode sequence, and (ii) an additional biological particle comprising an additional nucleic acid molecule, and wherein the partitioning regions and the additional partitioning regions comprise a combination of a particle comprising a molecular barcode and a particle comprising an optical barcode, the combination being different between the partitioning regions and the additional partitioning regions.

36. The method of claim 1, wherein (a) - (d) are repeated for additional partitioning regions of the plurality of partitioning regions, wherein the additional partitioning regions each comprise (i) an additional plurality of nucleic acid barcode molecules, wherein nucleic acid barcode molecules of the additional plurality of nucleic acid barcode molecules comprise an additional barcode sequence different from the barcode sequence, and (ii) an additional biological particle comprising an additional nucleic acid molecule, the additional partitioning regions comprising at least 1,000 partitioning regions.

37. The method of claim 36, wherein the additional allocation zones comprise at least 10,000 allocation zones.

38. The method of claim 37, wherein the additional allocation zones comprise at least 100,000 allocation zones.

39. The method of claim 36, wherein the additional distribution region comprises a plurality of particles comprising nucleic acid barcode molecules comprising at least 1,000 barcode sequences, the at least 1,000 barcode sequences being different in the distribution region and the additional distribution interval.

40. The method of claim 39, wherein the additional distribution region comprises a plurality of particles comprising nucleic acid barcode molecules comprising at least 10,000 barcode sequences, the at least 10,000 barcode sequences being different in the distribution region and the additional distribution interval.

41. The method of claim 40, wherein the additional distribution region comprises a plurality of particles comprising nucleic acid barcode molecules comprising at least 100,000 barcode sequences, the at least 100,000 barcode sequences being different in the distribution region and the additional distribution interval.

42. The method of claim 1, wherein the biological particle is a cell.

43. The method of claim 1, wherein the biological particle comprises cells or one or more components thereof in a matrix.

44. The method of claim 43, wherein the matrix is a polymer matrix.

45. The method of claim 43, wherein the matrix is a gel matrix.

46. The method of claim 1, further comprising associating a protein of the biological particle having the one or more physical characteristics of the biological particle with the nucleic acid sequence.

47. The method of claim 47, wherein the protein is a cell surface protein.

48. The method of claim 1, further comprising correlating one or more ribonucleic acid sequences or one or more deoxyribonucleic acid (DNA) sequences of the biological particle to the one or more physical characteristics.

49. The method of claim 48, wherein the one or more DNA sequences comprise epigenetic information.

50. The method of claim 48, wherein the one or more DNA sequences comprise chromatin information.

51. A kit comprising a plurality of particles comprising (i) a plurality of nucleic acid barcode molecules coupled thereto, wherein the plurality of barcode molecules comprises a plurality of barcode sequences, and (ii) a plurality of optical barcodes, wherein the plurality of optical barcodes comprises a plurality of optical codes,

wherein each particle of the plurality of particles comprises (i) a subset of the plurality of nucleic acid barcode molecules coupled thereto, and (ii) a subset of the plurality of optical barcodes,

wherein the barcode sequences of the subsets of the plurality of nucleic acid barcode molecules are different between particles of the plurality of particles, and wherein the optical codes of the subsets of the plurality of optical barcodes are different between the particles.

52. The kit of claim 51, wherein each of the subsets of the plurality of optical barcodes comprises a single optical barcode.

53. The kit of claim 51, wherein each of the subsets of the plurality of optical barcodes comprises two or more optical barcodes.

54. The kit of claim 51, wherein the plurality of optical barcodes impart optical properties to the plurality of particles.

55. The kit of claim 54, wherein the optical property is selected from the group consisting of: absorbance, birefringence, color, fluorescence characteristics, luminosity, photosensitivity, reflectivity, refractive index, scattering or transmittance of the particle or a component thereof.

56. The kit of claim 51, wherein the plurality of optical barcodes comprise one or more fluorescent dyes, nanoparticles, microparticles, or any combination thereof.

57. The kit of claim 56, wherein the plurality of optical barcodes comprise a plurality of nanoparticles.

58. The kit of claim 57, wherein the plurality of optical barcodes comprise a plurality of quantum dots.

59. The kit of claim 57, wherein the plurality of optical barcodes comprise a plurality of Janus particles.

60. The kit of claim 56, wherein the plurality of optical barcodes comprise a plurality of fluorescent dyes.

61. The kit of claim 60, wherein the plurality of fluorescent dyes comprises between 2-10 fluorescent dyes.

62. The kit of claim 60, wherein the plurality of fluorescent dyes comprises a plurality of fluorescent dyes having the same emission wavelength and different intensities.

63. The kit of claim 62, wherein the plurality of fluorescent dyes comprise at least 20 different intensities.

64. The kit of claim 51, wherein each of the subsets of the plurality of optical barcodes comprises a different optical code.

65. The kit of claim 53, wherein each of the subsets of the plurality of optical barcodes comprises a different combination of optical codes.

66. The kit of claim 51, wherein the plurality of optical barcodes have different associated optical intensities or frequencies relative to each other.

67. The kit of claim 51, wherein the plurality of optical barcodes comprises at least 1,000 different optical codes.

68. The kit of claim 67, wherein the plurality of optical barcodes comprises at least 10,000 different optical codes.

69. The kit of claim 68, wherein the plurality of optical barcodes comprises at least 100,000 different optical codes.

70. The kit of claim 51, wherein nucleic acid barcode molecules within a given subset of the subsets of the plurality of nucleic acid barcode molecules comprise the same barcode sequence.

71. The kit of claim 51, wherein the plurality of barcode sequences comprises at least 1,000 different barcode sequences.

72. The kit of claim 71, wherein the plurality of barcode sequences comprises at least 10,000 different barcode sequences.

73. The kit of claim 72, wherein the plurality of barcode sequences comprises at least 100,000 different barcode sequences.

74. The kit of claim 51, wherein the plurality of particles comprises at least 10,000 particles.

75. The kit of claim 74, wherein the plurality of distribution areas comprise at least 100,000 particles.

76. The kit of claim 51, wherein the plurality of nucleic acid barcode molecules comprises a plurality of functional sequences configured to interact with a plurality of target molecules.

77. The kit of claim 76, wherein the plurality of target molecules comprises a plurality of deoxyribonucleic acid molecules.

78. The kit of claim 76, wherein the plurality of target molecules comprises a plurality of ribonucleic acid molecules.

79. The kit of claim 76, wherein the plurality of functional sequences are configured to capture the plurality of target molecules.

80. The kit of claim 51, wherein the plurality of particles comprises a plurality of beads.

81. The kit of claim 80, wherein the plurality of particles comprises a plurality of gel beads.

82. The kit of claim 81, wherein the plurality of nucleic acid barcode molecules are releasably coupled to the plurality of gel beads.

83. The kit of claim 81, wherein the plurality of gel beads comprises a polyacrylamide polymer.

84. The kit of claim 51, wherein the plurality of optical barcodes are included on the surface of the plurality of particles.

85. The kit of claim 51, wherein the plurality of optical barcodes are included within the plurality of particles.

86. The kit of claim 51, wherein a first particle of the plurality of particles is coupled to a second particle of the plurality of particles, wherein the first particle comprises a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules and the second particle comprises an optical barcode of the plurality of optical barcodes.

Background

The sample may be processed for various purposes, such as identifying the type of sample portion within the sample. The sample may be a biological sample. Biological samples can be processed for various purposes, such as detecting a disease (e.g., cancer) or identifying a particular substance. Various methods exist for processing samples, such as Polymerase Chain Reaction (PCR) and sequencing.

Biological samples can be processed in various reaction environments, such as partitioning zones. The dispensing region may be an aperture or a droplet. The droplets or wells can be used to process the biological sample in a manner that enables the biological sample to be dispensed and processed independently. For example, such droplets may be fluidically isolated from other droplets, thereby enabling precise control of various environments within the droplets.

The biological sample in the distribution area may be subjected to various processes, such as chemical processes or physical processes. The sample in the distribution region may be subjected to heating or cooling, or a chemical reaction, such as to produce a substance that can be processed qualitatively or quantitatively.

Biological samples can be processed using existing methods, where sample information (e.g., phenotypic information) can be lost. Such sample processing is not useful for analyzing inter-cellular variation in a sample. Furthermore, existing methods may suffer from inefficient sample preparation methods, such as time consuming procedures that may include multiple steps.

Disclosure of Invention

There is recognized herein a need for improved methods of sample preparation for analyzing intercellular variations (e.g., phenotypic and sequence variations) in a biological sample.

In one aspect, the present disclosure provides a method for processing nucleic acid molecules of a biological particle, comprising: (a) providing (1) a plurality of distribution regions, wherein a distribution region of the plurality of distribution regions comprises (i) a particle comprising a plurality of nucleic acid barcode molecules, wherein a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules comprises a barcode sequence, and (ii) a biological particle comprising the nucleic acid molecule, and (2) a first data set stored in a computer memory comprising data indicative of one or more physical characteristics of the biological particle, the data generated upon sensing the biological particle; (b) generating barcoded nucleic acid molecules using the nucleic acid barcode molecules of the plurality of nucleic acid barcode molecules and the nucleic acid molecules of the biological particles; (c) generating a second data set comprising data identifying the nucleic acid sequence of the barcoded nucleic acid molecule or derivative thereof; and (d) correlating the one or more physical characteristics of the biological particle with the nucleic acid sequence using the first data set and the second data set.

In some embodiments, the one or more physical characteristics of the biological particle comprise a size of the biological particle, a shape of the biological particle, a surface marker on the biological particle, an inclusion in the biological particle, a structure of an organelle in the biological particle, a number of organelles in the biological particle, secretion or excretion relative to the biological particle, or localization of an organelle in the biological particle.

In some embodiments, the biological particle comprises a plurality of nucleic acid molecules. In some embodiments, the plurality of nucleic acid molecules comprises a plurality of ribonucleic acid molecules. In some embodiments, the plurality of nucleic acid molecules comprises a plurality of deoxyribonucleic acid molecules.

In some embodiments, the nucleic acid molecule is a ribonucleic acid molecule. In some embodiments, the nucleic acid molecule is a deoxyribonucleic acid molecule.

In some embodiments, the one or more physical characteristics comprise phenotypic information of the biological particle.

In some embodiments, the method further comprises optically detecting the particle to generate a third data set comprising data indicative of one or more physical characteristics of the particle or the plurality of nucleic acid barcode molecules. In some embodiments, the one or more physical properties of the particle comprise an optical property of the particle. In some embodiments, the physical property of the particle comprises the size, shape, roundness, hardness, or symmetry of the particle or a component thereof. In some embodiments, the optical characteristic of the particle comprises absorbance, birefringence, color, fluorescence characteristic, luminosity, photosensitivity, reflectivity, refractive index, scattering, or transmittance of the particle or a component thereof.

In some embodiments, the nucleic acid barcode molecules of the plurality of nucleic acid barcode molecules comprise barcode sequences, the barcode sequences being identical.

In some embodiments, the plurality of dispensing regions is a plurality of droplets. In some embodiments, the plurality of distribution areas is a plurality of wells.

In some embodiments, the particles comprise one or more optical barcodes. In some embodiments, the plurality of nucleic acid barcode molecules of the particle comprises the one or more optical barcodes. In some embodiments, the one or more optical barcodes comprise a fluorescent dye, a nanoparticle, a microparticle, or any combination thereof. In some embodiments, the one or more optical barcodes have a different associated optical intensity or frequency relative to other optical barcodes of the plurality of distribution areas. In some embodiments, repeating (a) - (d) for additional distribution regions of the plurality of distribution regions, wherein the additional distribution regions comprise (i) additional particles comprising an additional plurality of nucleic acid barcode molecules, wherein nucleic acid barcode molecules of the additional plurality of nucleic acid barcode molecules comprise an additional barcode sequence different from the barcode sequence, and (ii) additional biological particles comprising additional nucleic acid molecules, wherein the distribution regions and the additional distribution regions produce optical signals of different intensities or frequencies upon sensing. In some embodiments, the nanoparticle comprises a quantum dot. In some embodiments, the nanoparticle comprises a Janus particle.

In some embodiments, the plurality of nucleic acid barcode molecules are configured in a geometric configuration. In some embodiments, the geometric structure is a nucleic acid origami.

In some embodiments, the one or more physical properties of the biological particle are identified by imaging the distribution region using (i) bright field microscopy, (ii) fluorescence microscopy, (iii) phase contrast microscopy, (iv) multispectral microscopy, or (v) polarized light microscopy.

In some embodiments, the particle is a bead. In some embodiments, the bead is a gel bead. In some embodiments, the plurality of nucleic acid barcode molecules are releasably attached to the gel beads. In some embodiments, the gel beads comprise a polyacrylamide polymer.

In some embodiments, the one or more physical properties are one or more optical properties. In some embodiments, the method further comprises, after (a), imaging the distribution region to optically detect the biological particle, thereby identifying the one or more optical properties of the biological particle. In some embodiments, the method further comprises, prior to (a), imaging the biological particle to identify the one or more optical properties of the biological particle. In some embodiments, the biological particles are imaged prior to providing the biological particles in the distribution region.

In some embodiments, the particle is coupled to another particle comprising one or more optical barcodes.

In some embodiments, repeating (a) - (d) for additional partitioning regions of the plurality, wherein the additional partitioning regions each comprise (i) an additional plurality of nucleic acid barcode molecules, wherein nucleic acid barcode molecules of the additional plurality of nucleic acid barcode molecules comprise an additional barcode sequence different from the barcode sequence, and (ii) an additional biological particle comprising an additional nucleic acid molecule, and wherein the partitioning regions and the additional partitioning regions comprise a combination of a particle comprising a molecular barcode and a particle comprising an optical barcode, the combination being different between the partitioning regions and the additional partitioning regions.

In some embodiments, repeating (a) - (d) for additional partitioning regions of the plurality, wherein the additional partitioning regions each comprise (i) an additional plurality of nucleic acid barcode molecules, wherein nucleic acid barcode molecules of the additional plurality of nucleic acid barcode molecules comprise an additional barcode sequence different from the barcode sequence, and (ii) an additional biological particle comprising an additional nucleic acid molecule, the additional partitioning regions comprising at least 1,000 partitioning regions. In some embodiments, the additional distribution areas comprise at least 10,000 distribution areas. In some embodiments, the additional distribution area comprises at least 100,000 distribution areas. In some embodiments, the additional partitioning region comprises a plurality of particles comprising nucleic acid barcode molecules comprising at least 1,000 barcode sequences, the at least 1,000 barcode sequences being different between the partitioning region and the additional partitioning interval. In some embodiments, the additional partitioning region comprises a plurality of particles comprising nucleic acid barcode molecules comprising at least 10,000 barcode sequences, the at least 10,000 barcode sequences being different between the partitioning region and the additional partitioning interval. In some embodiments, the additional partitioning region comprises a plurality of particles comprising nucleic acid barcode molecules comprising at least 100,000 barcode sequences, the at least 100,000 barcode sequences being different between the partitioning region and the additional partitioning interval.

In some embodiments, the biological particle is a cell.

In some embodiments, the biological particle comprises cells or one or more components thereof in a matrix. In some embodiments, the matrix is a polymer matrix. In some embodiments, the matrix is a gel matrix.

In some embodiments, the method further comprises associating a protein of the biological particle having the one or more physical properties of the biological particle with the nucleic acid sequence. In some embodiments, the protein is a cell surface protein.

In some embodiments, the method further comprises correlating one or more ribonucleic acid sequences or one or more deoxyribonucleic acid (DNA) sequences of the biological particle to the one or more physical characteristics. In some embodiments, the one or more DNA sequences comprise epigenetic information. In some embodiments, the one or more DNA sequences comprise chromatin information.

In another aspect, the present disclosure provides a kit comprising a plurality of particles comprising (i) a plurality of nucleic acid barcode molecules coupled thereto, wherein the plurality of barcode molecules comprises a plurality of barcode sequences, and (ii) a plurality of optical barcodes, wherein the plurality of optical barcodes comprise a plurality of optical codes, wherein each particle of the plurality of particles comprises (i) a subset of the plurality of nucleic acid barcode molecules coupled thereto and (ii) a subset of the plurality of optical barcodes, wherein the barcode sequences of the subsets of the plurality of nucleic acid barcode molecules are different between particles of the plurality of particles, and wherein the optical codes of the subsets of the plurality of optical barcodes are different between the particles.

In some implementations, each of the subsets of the plurality of optical barcodes comprises a single optical barcode.

In some embodiments, each of the subsets of the plurality of optical barcodes comprises two or more optical barcodes.

In some embodiments, the plurality of optical barcodes impart optical properties to the plurality of particles. In some embodiments, the optical characteristic is selected from the group consisting of: absorbance, birefringence, color, fluorescence characteristics, luminosity, photosensitivity, reflectivity, refractive index, scattering or transmittance of the particle or a component thereof.

In some embodiments, the plurality of optical barcodes comprise one or more fluorescent dyes, nanoparticles, microparticles, or any combination thereof. In some embodiments, the plurality of optical barcodes comprises a plurality of nanoparticles. In some embodiments, the plurality of optical barcodes comprises a plurality of quantum dots. In some embodiments, the plurality of optical barcodes comprises a plurality of Janus particles. In some embodiments, the plurality of optical barcodes comprises a plurality of fluorescent dyes. In some embodiments, the plurality of fluorescent dyes comprises between 2-10 fluorescent dyes. In some embodiments, the plurality of fluorescent dyes comprises a plurality of fluorescent dyes having the same emission wavelength and different intensities. In some embodiments, the plurality of fluorescent dyes comprises at least 20 different intensities.

In some implementations, each of the subsets of the plurality of optical barcodes comprises a different optical code.

In some implementations, each of the subsets of the plurality of optical barcodes comprises a different combination of optical codes.

In some implementations, the plurality of optical barcodes have different associated optical intensities or frequencies relative to each other.

In some embodiments, the plurality of optical barcodes comprises at least 1,000 different optical codes. In some embodiments, the plurality of optical barcodes comprises at least 10,000 different optical codes. In some implementations, the plurality of optical barcodes comprises at least 100,000 different optical codes.

In some embodiments, the nucleic acid barcode molecules within a given subset of the plurality of nucleic acid barcode molecules comprise the same barcode sequence.

In some embodiments, the plurality of barcode sequences comprises at least 1,000 different barcode sequences. In some embodiments, the plurality of barcode sequences comprises at least 10,000 different barcode sequences. In some embodiments, the plurality of barcode sequences comprises at least 100,000 different barcode sequences.

In some embodiments, the plurality of particles comprises at least 10,000 particles. In some embodiments, the plurality of distribution areas comprise at least 100,000 particles.

In some embodiments, the plurality of nucleic acid barcode molecules comprises a plurality of functional sequences configured to interact with a plurality of target molecules. In some embodiments, the plurality of target molecules comprises a plurality of deoxyribonucleic acid molecules. In some embodiments, the plurality of target molecules comprises a plurality of ribonucleic acid molecules. In some embodiments, the plurality of functional sequences are configured to capture the plurality of target molecules.

In some embodiments, the plurality of particles comprises a plurality of beads. In some embodiments, the plurality of particles comprises a plurality of gel beads. In some embodiments, the plurality of nucleic acid barcode molecules is releasably coupled to the plurality of gel beads. In some embodiments, the plurality of gel beads comprises a polyacrylamide polymer.

In some embodiments, the plurality of optical barcodes are included on the surface of the plurality of particles.

In some embodiments, the plurality of optical barcodes are included within the plurality of particles.

In some embodiments, a first particle of the plurality of particles is coupled to a second particle of the plurality of particles, wherein the first particle comprises a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules and the second particle comprises an optical barcode of the plurality of optical barcodes.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only exemplary embodiments of the present disclosure are shown and described. It is to be understood that the present disclosure is capable of other and different embodiments and its several details are capable of modification in various, different respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also "figures" herein), of which:

fig. 1 shows an example of a microfluidic channel structure for dispensing individual biological particles.

Fig. 2 shows an example of a microfluidic channel structure for delivering barcode-bearing beads to droplets.

Fig. 3 shows an example of a microfluidic channel structure for co-dispensing biological particles and reagents.

Fig. 4 shows an example of a microfluidic channel structure for controlled dispensing of beads into discrete droplets.

Fig. 5 shows an example of a microfluidic channel structure for increasing droplet generation throughput.

Fig. 6 shows another example of a microfluidic channel structure for increasing droplet generation throughput.

Figure 7A shows a cross-sectional view of another example of a microfluidic channel structure having geometry for controlled dispensing. Fig. 7B shows a perspective view of the channel structure of fig. 7A.

Fig. 8 shows an example of a microfluidic channel structure for optical detection of distribution areas.

Fig. 9 shows an example of a procedure for electronically correlating genetic and phenotypic information of a biological particle.

FIG. 10 illustrates a computer system that has been programmed or otherwise configured to implement the methods provided herein.