HK40104030A - Methods and compositions for analyzing cellular components - Google Patents

Description

Methods and compositions for analyzing cellular components

This application is a divisional application of Chinese patent application No. 201680017121.4, filed on February 10, 2016, entitled "Method and Composition for Analyzing Cell Components".

Technical Field

Embodiments of this application relate to methods and compositions for analyzing cellular components. In some embodiments, this application relates to methods and compositions for analyzing components of a single cell. In some embodiments, this application relates to methods and compositions for identifying single cell types. In some embodiments, the methods and compositions involve sequencing nucleic acids. Some embodiments of the provided methods and compositions are useful in determining the complex state of such single cells.

Background Technology

Detection of specific nucleic acid sequences present in biological samples has been used, for example, as a method for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to diseases, and measuring responses to various types of treatments. A common technique for detecting specific nucleic acid sequences in biological samples is nucleic acid sequencing.

Nucleic acid sequencing methods have evolved significantly from chemical degradation methods used by Maxam and Gilbert and chain extension methods used by Sanger. Today, several sequencing methods are in use that allow for parallel processing of nucleic acids, all within a single sequencing run. Therefore, the information generated from a single sequencing run can be enormous.

Summary of the Invention

This invention includes the following:

1. A method for analyzing at least two or more analytes in a single cell, the method comprising:

(a) Provide multiple contiguity preserving elements (CEs), each CE containing a single cell;

(b) Lysing the single cell within the CE, wherein the analyte within the single cell is released within the CE;

(c) Provide a first reporter portion of the first analyte to the single cell of each CE;

(d) Provide a second reporter portion of the second analyte to each CE within the single cell;

(e) Modify the analytes such that at least some of the first and second analytes of the CE respectively include the first and second reporter portion;

(f) A combination of a CE containing the analyte, wherein the analyte contains the reporter portion;

(g) The CE containing the first and second analytes is compartmentalized into multiple compartments, wherein the first and second analytes each contain the first and second reporter portion;

(h) Provide a third report portion to the first analyte containing the first report portion of each CE;

(i) Provide a fourth report portion to the second analyte, which contains the second report portion of each CE;

(j) The analytes are further modified such that at least some of the first analytes include portions of the first and third reporting substances and at least some of the second analytes include portions of the second and fourth reporting substances;

(j) Analyze the analyte in each compartment containing the report portion, wherein such analysis detects the analyte in a single cell.

2. The method of item 1, wherein the first and second report portions identify the source of the analyte.

3. The method of item 1, wherein the combination of the report portion identifies the source of the analyte.

4. The method of any one of items 1-3, wherein the analyte is detected simultaneously.

5. The method of any one of items 1-4, wherein the first analyte is genomic DNA and the second analyte is cDNA or RNA.

6. The method of claim 5, wherein modifying at least some of the genomic DNA, cDNA, or RNA to include first and second reporter portions includes contacting the genomic DNA, cDNA, or RNA with a plurality of transposons under conditions, each transposome comprising a transposase and a transposon sequence comprising a first reporter portion or a second reporter portion, such that at least some of the transposon sequences are inserted into the genomic DNA, cDNA, or RNA.

7. The method of item 6, wherein step (g) further includes removing the transposase from the genomic DNA, cDNA or RNA.

8. The method of item 7, wherein the transposase is removed after the analyte is modified in step (e).

9. The method of item 8, wherein removing transposase includes methods selected from the group consisting of: adding detergent, changing temperature, changing pH, adding protease, adding chaperone, changing salt concentration, and adding chain displacement polymerase.

10. The method of claim 6, wherein step (e) includes contacting a target nucleic acid with a plurality of transposons, each transposon containing a first transposon sequence comprising a first reporter portion, a second transposon sequence that is noncontiguous to the first transposon sequence, and a transposon enzyme associated with the first transposon sequence and the second transposon sequence.

11. The method of any one of items 6-10, wherein the first transposon sequence comprises a first primer site and the second transposon sequence comprises a second primer site.

12. The method of item 13, wherein the first primer site further comprises a first barcode and the second primer site further comprises a second barcode.

13. The method of any one of items 1-12, wherein the first, second, third, or fourth report portion comprises a barcode.

14. The method of claim 1, wherein one of the analytes is a protein, and optionally wherein the protein is labeled with a nucleic acid reporter portion, and further optionally wherein the nucleic acid reporter portion comprises a combination-derived set of barcodes.

15. The method of item 14, wherein the protein is derived from a single cell.

16. The method of any one of items 1-15, wherein steps (c)-(j) are repeated at least once more.

17. The method of item 16, wherein the additional report portion in each additional step is different from the first, second, third, and fourth report portions.

18. A method for analyzing an analyte, the method comprising:

(a) Provide adjacent retention elements (CEs), wherein each CE contains at least one analyte;

(b) Dividing the CE containing the at least one analyte into a plurality of first compartments;

(c) In the first compartment, a first set of report portions is provided to the analyte of each CE, wherein the first set of report portions identifies the CE;

(d) Modify the analytes such that at least some of the analytes of the CE include portions of the first reportant;

(e) Combining the CE containing the analyte, the analyte comprising the first reporter portion;

(f) Dividing the CE containing the analyte into a plurality of second compartments, the analyte containing the first report portion;

(g) Provide a second set of report portions to the analyte, the analyte comprising the first report portion of each CE;

(h) Further modify the analytes such that at least some of the analytes contain portions of the second reporter;

(i) Analyze the analyte, which comprises the first and second report portions of each second compartment.

19. The method of item 18, wherein the analyte comprises a nucleic acid.

20. The method of item 19, wherein the nucleic acid comprises DNA.

21. The method of item 20, wherein the DNA comprises genomic DNA.

22. The method of item 19, wherein the nucleic acid comprises RNA or cDNA.

23. The method of item 18, wherein the analyte is selected from the group consisting of tissue sections, organelles, lipids, carbohydrates, and cellular metabolites.

24. The method of item 19, wherein step (d) includes contacting a nucleic acid with a plurality of transposons under conditions, each transposon containing a transposase and a transposon sequence containing a first reporter portion, such that at least some of the transposon sequences are inserted into the target nucleic acid.

25. The method of item 19, wherein step (d) includes contacting a target nucleic acid with a plurality of transposons, each transposon containing a first transposon sequence comprising a first reporter portion, a second transposon sequence discontinuous from the first transposon sequence, and a transposon enzyme associated with the first and second transposon sequences.

26. The method of item 19, wherein step (f) includes removing transposase from the separated first indexed template nucleic acid.

27. The method of item 26, wherein the transposase is removed after step (d).

28. The method of item 26, wherein the transposase is removed prior to step (i).

29. The method of item 26, wherein removing transposases includes methods selected from the group consisting of: adding detergent, changing temperature, changing pH, adding protease, adding protein chaperone, changing salt concentration, and adding chain displacement polymerase.

30. The method of any one of items 24-29, wherein the first transposon sequence comprises a first primer site and the second transposon sequence comprises a second primer site.

31. The method of item 30, wherein the first primer site further comprises a first barcode and the second primer site further comprises a second barcode.

32. The method of item 18, wherein the first reporter portion comprises primers, and wherein step (d) comprises amplifying the nucleic acid with at least one primer.

33. The method of item 18, wherein the first reporter portion comprises primers, and wherein step (d) comprises ligating the nucleic acid with at least one primer.

34. The method of any one of items 18-33, wherein the first report portion of the analyte provided to each first compartment is different.

35. The method of any one of claims 18-34, wherein the analyte is a component of a single cell, wherein each of the first and/or second reporter portions is unique to the first cell, and wherein the first and/or second reporter portions identify a single cell.

36. The method of any one of claims 18-35, further comprising providing a plasmid to the CE in step (c), wherein the plasmid is modified in steps (d)-(h) to include the first and/or second reporter portion, wherein the plasmid including the first or the reporter portion identifies the CE.

37. The method of item 18, wherein the analyte is a protein.

38. The method of item 37, wherein the protein is derived from a single cell.

39. The method of any one of items 18-37, wherein steps (c)-(h) are repeated at least once more.

40. The method of item 39, wherein the additional group of the report portion in each additional step is different from the first and second groups of the report portion.

41. The method of item 18, wherein the analyte is mRNA or cDNA.

42. The method of item 41, wherein the mRNA or cDNA is derived from a single cell.

43. The method of any one of claims 41-42, wherein the CE comprises a solid support capable of immobilizing the mRNA or cDNA.

44. The method of item 43, wherein the solid support comprises an oligomer (dT) probe fixed on the solid support.

45. The method of any one of items 41-44, wherein the first set of report portions comprises a first barcode sequence.

46. The method of any one of items 41-45, wherein the second set of report portion comprises a second barcode sequence.

47. The method of any one of claims 41-44, wherein step (d) comprises contacting a nucleic acid with a plurality of transposons under conditions, each transposon containing a transposase and a transposon sequence containing a first reporter portion, such that at least some of the transposon sequences are inserted into the target nucleic acid.

48. The method of any one of claims 41-44, wherein step (d) includes contacting a target nucleic acid with a plurality of transposons, each transposon containing a first transposon sequence including a first reporter portion, a second transposon sequence discontinuous from the first transposon sequence, and a transposon enzyme associated with the first transposon sequence and the second transposon sequence.

49. The method of any one of items 47-48, wherein step (f) comprises removing the transposase from the separated first indexed template nucleic acid.

50. The method of item 49, wherein the transposase is removed after step (d).

51. The method of item 49, wherein the transposase is removed prior to step (i).

52. The method of item 49, wherein removing transposase includes methods selected from the group consisting of: adding detergent, changing temperature, changing pH, adding protease, adding protein chaperone, changing salt concentration, and adding chain displacement polymerase.

53. The method of any one of items 41-52, wherein the first and second report portions identify the source of the analyte.

54. The method of any one of items 41-52, wherein the combination of the report portion identifies the source of the analyte.

55. The method of items 41-54, wherein step (d)-(i) is repeated at least once more.

56. The method of item 55, wherein the additional group of the report portion in each additional step is different from the first and second groups of the report portion.

57. The method of any one of items 1-56, wherein the single cell is associated with the disease.

58. The method of item 57, wherein the single cell is associated with cancer.

59. The method of item 57, wherein the single cell is associated with a genetic disease.

60. A method for obtaining sequence information from a target nucleic acid in a single cell, the method comprising:

a) Introduce the cell suspension into the first droplet actuator;

b) Use electrode-mediated droplet manipulation to disperse an array of droplets containing a cell suspension, each droplet containing a single cell;

c) Introduce cell lysis buffer into the second droplet actuator;

c) Use electrode-mediated droplet manipulation to dispense an array of droplets containing cell lysis buffer;

d) Use electrode-mediated droplet manipulation to combine arrays of cell lysis buffer droplets and arrays of cell suspension droplets to generate an array of cell lysate droplets;

e) Introducing a reagent solution containing a first reporter portion and an enzyme into a third droplet actuator, wherein at least one enzyme is capable of introducing the first reporter portion into the target nucleic acid, and wherein the first reporter portion identifies the single cell;

f) Use electrode-mediated droplet manipulation to dispense droplet arrays containing reagent solutions;

g) Using electrode-mediated droplet manipulation to combine an array of cell lysate droplets with reagent droplets to generate an array of first reporter fraction droplets, wherein the reporter fraction is introduced into the nucleic acid of the cell lysate droplets;

h) Use electrode-mediated droplet manipulation to dispense an array of droplets containing a washing solution;

i) Using electrode-mediated droplet manipulation to combine an array of first reporter portion droplets with washing solution droplets, wherein the enzyme is removed from the nucleic acid after the reaction to generate an array of droplets containing a target nucleic acid, wherein the target nucleic acid contains a first reporter portion;

(j) Repeat steps (e)-(i) at least once, wherein the subsequent reagent solution contains a reporter portion different from the first reporter portion to generate a droplet array containing a target nucleic acid comprising a plurality of reporter portions;

(k) Obtain sequence information from the target nucleic acid containing one or more reporter portions and associate the sequence information with the single cell.

61. The method of item 60, which also includes:

(l) Introduce a protein labeling reagent solution containing a protein-specific marker into the droplet actuator;

(m) Using electrode-mediated droplet manipulation to dispense an array of droplets containing a protein-labeled reagent solution;

(n) Using electrode-mediated droplet manipulation to combine an array of protein labeling reagent solutions into a droplet array containing a target nucleic acid comprising multiple reporter portions, wherein at least a portion of the protein from the single cell contains a marker unique to the single cell, and identifying at least a portion of the protein containing the marker.

62. The method of claim 61, wherein the method of obtaining sequence information from the target nucleic acid is performed simultaneously with the identification of at least a portion of a protein containing the marker.

63. The method of any one of items 60-62, wherein the target nucleic acid is RNA.

64. The method of any one of items 60-62, wherein the target nucleic acid is DNA.

65. The method of item 64, wherein the target nucleic acid is genomic DNA.

66. The method of any one of items 60-62, wherein the target nucleic acid is both RNA and DNA.

67. The method of any one of claims 60-66, wherein the first reporter portion is introduced into the target nucleic acid via a linker.

68. The method of any one of items 60-67, which further includes:

(i) Introduce a reagent solution containing a reporter portion specific to cell metabolites into the droplet actuator;

(ii) Using electrode-mediated droplet manipulation to dispense an array of reagent droplets containing a reporter portion specific to cell metabolites;

(iii) Using electrode-mediated droplet manipulation to combine the reagent droplet array into a droplet array containing a target nucleic acid comprising multiple reporter portions, wherein at least a portion of the cell metabolites are derived from the single cell, the single cell containing a reporter portion unique to the single cell, and identifying at least a portion of the cell metabolites.

69. A method for obtaining sequence information from cellular nucleic acids, the method comprising:

(a) Introducing one or more cells into a solid support, wherein the solid support includes a cell attachment portion, and wherein, after such introduction, the one or more cells are fixed to the solid support;

(b) Lysing the cells immobilized on the solid support to generate cell lysates;

(c) Provide a reporter portion to the nucleic acid of the cell lysate and modify the nucleic acid to generate a nucleic acid containing the first reporter portion;

(d) Obtain sequence information from the nucleic acid containing the first reporter portion.

70. The method of item 69, wherein the nucleic acid is genomic DNA.

71. The method of item 69, wherein the nucleic acid is RNA.

72. The method in item 71 also includes reverse transcription of RNA into cDNA.

73. The method of any one of items 69-72, further comprising providing an additional reporter portion to the nucleic acid comprising the first reporter portion in step (c), modifying said nucleic acid from step (c) to further comprise one or more additional reporter portions.

74. The method of item 73, wherein the additional reporting portion differs from the first reporting portion.

75. The method of any one of items 69-74, wherein the first reporter portion is unique for each cell, making it possible to identify the cell origin of the nucleic acid containing the first index.

76. The method of any one of items 69-75, wherein the nucleic acid is derived from a single cell.

77. The method of any one of items 69-76, wherein the nucleic acid is both RNA and DNA.

78. The method of any one of items 69-77, wherein the solid support is selected from the group consisting of flow cells, beads, and micropores.

79. The method of any one of claims 69-78, wherein the cell attachment portion is an antibody, wherein the antibody binds to cell surface proteins.

80. The method of item 69, wherein the antibody is a monoclonal antibody.

81. The method of any one of items 61-62, wherein the antibody specifically binds to a cell surface protein specific to cancer cells.

82. The method of any one of claims 69-81, further comprising exposing cell lysate proteins with a protein-specific marker, wherein at least a portion of the proteins from said cells includes a marker unique to said cell, and identifying at least a portion of the proteins containing said marker.

83. The method of claim 82, wherein identifying at least a portion of a protein containing the marker and obtaining sequence information from cellular nucleic acids are performed simultaneously.

84. The method of any one of items 69-83, wherein one or more report portions contain at least one barcode.

85. The method of any one of items 69-84, wherein one or more reporter portions contain primer binding sites.

86. The method of any one of items 69-85, wherein the nucleic acid comprising the first reporter portion is modified by a linker.

87. The method of any one of items 69-84, wherein the nucleic acid comprising the one or more additional reporter portions is modified by connection.

88. The method of any one of items 69-87, wherein the modified nucleic acid comprising the first reporter portion is amplified prior to obtaining sequence information.

89. The method of any one of claims 69-88, wherein the modified nucleic acid comprising two or more reporter moieties is amplified prior to obtaining sequence information.

90. The method of any one of items 69-88, wherein at least a portion of said cells is associated with the disease.

91. The method of item 90, wherein at least a portion of said cells is associated with cancer.

92. A method for analyzing cell composition, the method comprising:

(a) Introducing one or more cells into a solid support, wherein the solid support includes a cell attachment portion, and wherein the one or more cells are fixed to the solid support after such introduction;

(b) Lysing the cells immobilized on the solid support to generate cell lysates;

(c) Provide one or more reporter portions to the cell lysate such that one or more analytes within the cell lysate contain the one or more reporter portions, wherein the one or more reporter portions identify the cells;

(d) Analyze the analyte comprising one or more reporter portions, wherein the analyte identifies the cell and detects the composition of the cell.

93. The method of item 92, wherein a single cell is fixed to the solid support.

94. The method of any one of items 92-93, wherein the solid support is selected from the group consisting of flow cells, beads, and micropores.

95. The method of any one of claims 92-94, wherein the cell attachment portion is an antibody, wherein the antibody binds to cell surface proteins.

96. The method of item 95, wherein the antibody is a monoclonal antibody.

97. The method of any one of claims 95-96, wherein the antibody specifically binds to a cell surface protein specific to cancer cells.

98. The method of item 90, wherein the analyte is selected from the group consisting of proteins, nucleic acids, organelles, lipids, carbohydrates, and cellular metabolites.

99. The method of any one of items 1-13, wherein the first, second, third, or fourth reporter portion comprises a primer binding site.

100. The method of any one of items 1-17, wherein the first, second, or both analytes are nucleic acids.

101. The method of 100, wherein the nucleic acid containing the reporter portion is amplified prior to analysis.

102. The method of any one of items 100-101, wherein nucleic acids are analyzed by sequencing.

Attached Figure Description

Figure 1: Four-tier combinatorial indexing, illustrating a schematic diagram of four-tier combinatorial indexing of DNA proximity retention elements (CEs) created by embedding single-cell contents in a polymer matrix or attaching them to beads. Compartment-specific indexes are attached in the pooling and redistribution steps (layers) of each combination. In the example shown, the four layers result in four indices linked together (via repeated rounds of ligation, polymerase extension, tagging, etc.), enabling easy sequencing readout. Alternatively, proximity retention elements containing DNA can be created by compartmentalized DNA partitions (i.e., DNA dilutions that are secondary samples of the original DNA sample) encapsulated in a matrix or immobilized on beads. This type of dilution is useful in phasing and assembly applications.

Figure 2: Two-layer combinatorial indexing for single-cell libraries, depicting a method for preparing single-cell DNA or cDNA libraries using a two-layer combinatorial indexing scheme, wherein a first-level index is attached via tag fragmentation (compartment-specific indexing in transposons), and a second-level index is attached via PCR (compartment-specific indexing on PCR primers). The contents of the single-cell container (i.e., genomic DNA or cDNA) can be processed using optional whole-genome amplification (WGA) or whole-transcriptome amplification steps.

Figure 3: Single-cell gene expression in a CE (e.g., a droplet), illustrating a method for preparing a cDNA library from the contents of a single cell in a CE, such as a droplet. In the example shown, an index is being used to label different samples.

Figure 4: Single-cell contents in a container, depicting representative contents of a single cell that can be analyzed via the proposed combined indexing scheme.

Figure 5A: Formation of a proximity element (CE), and Figure 5B: Customized assay by the creation of a proximity element (CE), depict exemplary schematic embodiments for creating proximity retention elements (CEs) from the contents of a single cell captured within a CE, such as within a polymer bead, through encapsulation and lysis. The cell is embedded, for example, within a polymer bead. All components from the single cell remain close to each other within the bead. Subsequently, one or more components can be amplified, modified (cDNA synthesis), and subsequently indexed or tagged. Figure 5C: Formation of a proximity element (CE), depicting an exemplary schematic embodiment where sample indexing can be accomplished by adding (spiking) a coding DNA sequence (e.g., a plasmid) during encapsulation, amplification/cDNA, or polymerization stages. Each sample is prepared with different sets of coding plasmids or combinations of coding plasmids. The indexed CEs of each combination will produce an indexed sample of the corresponding combination of coding library elements. In this way, each library element can be located back to its originating CE and originating sample.

Figure 6: Single-cell encapsulation and expansion, depicting a schematic diagram of encapsulating single-cell contents in CE, such as polymer matrix beads.

Figure 7: High-throughput analysis of cellular components via direct surface capture. This diagram illustrates an exemplary high-throughput analysis of cellular components via direct surface capture. "A" represents an aggregate of cells. "B" represents a surface-bound transposon. In "C," cells flow onto the surface. In "D," cells are lysed, allowing cellular components to diffuse in a controlled manner around the site of cell capture. In "E," nucleic acids are captured by transposons (tagmented). Different cellular components are captured depending on whether the cell membrane or nucleus is lysed. Various cellular components can be captured by using component-specific capture portions (i.e., antibodies, receptors, ligands). Analysis of the captured molecules can be performed directly on the capture surface. Alternatively, the captured molecules can be harvested and analyzed on different surfaces. In this case, the first surface consists of multiple regions (i.e., pads), and each pad is coated with an oligomer sharing the same barcode, such that molecules captured on the same pad will share the same identifying barcode.

Figure 8: Streamlined CPT-seq: Workflow on beads, depicting an exemplary schematic of nucleic acid analysis using proximity retention elements on beads.

Figures 9A-9D: These represent the methods for assembling repetitive regions in genomic DNA using segmentation and mutagenesis, respectively, illustrating exemplary modeling strategies.

Figure 10 illustrates a method for creating particles that are useful for creating neighboring elements.

Detailed Implementation

Some aspects of the present invention relate to methods and compositions for evaluating components of single cells that are retained, embedded, or contained within a nearby retention element (CE).

On one hand, this document discloses methods for analyzing multiple analyte types from single cells. In some embodiments, multiple proximity retention elements (CEs) are provided, each CE containing a single cell. The cell is lysed within the CE, causing multiple analytes within the single cell to be released within the CE. In some embodiments, multiple types of reporter portions are provided, such that each type of reporter portion is specific to each type of analyte. In some embodiments, the reporter portions identify the single cell. Multiple analytes are modified such that each type of analyte contains a reporter portion specific to the analyte type. In some embodiments, CEs containing analytes and reporter portions are combined. In some embodiments, CEs containing combinations of analytes and reporter portions are separated. In some embodiments, additional reporter portions are provided and combined with the analytes containing the analytes, such that the analytes contain two or more distinct reporter portions. The analytes containing reporter portions are analyzed, such that the identity of the analytes is detected, and the reporter portions identify the source of the analytes from the single cell.

In some embodiments, exemplary analytes include, but are not limited to, DNA, RNA, cDNA, proteins, lipids, carbohydrates, cellular organelles (e.g., nucleus, Golgi apparatus, ribosomes, mitochondria, endoplasmic reticulum, chloroplasts, cell membranes, etc.), cellular metabolites, tissue sections, cells, single cells, contents derived from cells or single cells, nucleic acids isolated from cells or single cells, or nucleic acids isolated from cells or single cells and further modified, or cell-free DNA (e.g., from placental fluid or plasma). In some embodiments, the analytes include genomic DNA and mRNA. In some embodiments, the mRNA has a poly A-tail. In some embodiments, genomic DNA and mRNA are simultaneously immobilized on a solid support within the CE. In some embodiments, the immobilization of genomic DNA and the immobilization of mRNA to the solid support are sequential. In some embodiments, genomic DNA is combined with a transposon complex, the transposon ends are immobilized on the solid support, and the mRNA is immobilized to the solid by hybridization with oligomeric (dT) probes immobilized on the solid support. In some embodiments, genomic DNA is combined with a transposon complex, and optionally, the transposon ends hybridize to a complementary sequence immobilized on a solid support, such that mRNA is immobilized to the solid by hybridization of oligomeric (dT) probes immobilized on the solid support. Other methods may also be used to immobilize mRNA. In some embodiments, the solid support is a bead. In some embodiments, the solid support is a flow cell surface. In some embodiments, the solid surface is the wall of a reaction vessel.

In some embodiments, the method includes sequencing nucleic acids retained, embedded, or contained within a CE. Specifically, embodiments of the methods and compositions provided herein relate to preparing nucleic acid templates and obtaining sequence data therefrom. The methods and compositions provided herein relate to those provided in U.S. Patent Application Publication No. 2002/0208705, U.S. Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. WO 02/061832, each of which is incorporated herein by reference in its entirety. Some embodiments of the invention relate to preparing DNA within a CE to obtain phasing and sequence assembly information from target nucleic acids, and to obtaining phasing and sequence assembly sequence information from such templates. Specific embodiments provided herein involve using integrase, such as transposases, to maintain physical proximity of relevant ends of fragmented nucleic acids; and using combined indexing to create individual libraries from each CE. Obtaining haplotype information from a CE includes distinguishing different alleles (e.g., SNPs, genetic abnormalities, etc.) in the target nucleic acid. Such methods can be used to characterize different alleles in the target nucleic acid and reduce error rates in sequence information.

In one implementation, the template nucleic acid can be diluted to a CE, such as a droplet. Optional whole-genome amplification can be used, and sequence information can be obtained from an amount of template nucleic acid equivalent to approximately the same haplotype as the target nucleic acid.

In a further embodiment, the template nucleic acid can be separated so that multiple copies of the chromosome can exist in the same compartment, and the haplotype can still be determined as a result of the two or more indexings provided herein. In other words, the template nucleic acid can be prepared using virtual compartments. In such embodiments, nucleic acids can be distributed among several first compartments, providing a first index for the nucleic acids in each compartment, and the nucleic acids can be combined. Nucleic acids can also be distributed among several second compartments, providing a second index for the nucleic acids in each compartment. Advantageously, compared to diluting the nucleic acid only to an amount equivalent to the nucleic acid haplotype in a single compartment, this type of indexing allows haplotype information to be obtained at a higher concentration of nucleic acids.

As used herein, the term "compartment" refers to an area or volume that separates or isolates something from other substances. Exemplary compartments include, but are not limited to, vials, tubes, holes, droplets, boluses, beads, containers, surface features, or areas or volumes separated by physical forces (such as fluid flow, magnetism, electric current, etc.).

An exemplary method for fabricating the compartments is shown in Figure 10. A silicon masterplate with pillars can be used to imprint holes into a hydrogel sheet (the holes in the hydrogel are inverted images of the pillars). The resulting holes in the hydrogel can be filled with a material that forms particles (e.g., a gel or polymer) together with the target analyte or other reagents. The hydrogel sheet can then be dissolved using a technique that does not dissolve the particles. The particles can then be collected and manipulated using the methods described herein.

In some embodiments provided herein, transposons are used to prepare template libraries. In some such libraries, the target nucleic acid can be fragmented. Therefore, some embodiments provided herein relate to sequence information for maintaining the physical proximity of adjacent fragments. Such methods include using integrases to maintain the association of adjacent template nucleic acid fragments in the target nucleic acid. Advantageously, this use of integrases to maintain the physical proximity of fragmented nucleic acids increases the likelihood that fragmented nucleic acids from the same original molecule, such as chromosomes, will appear in the same compartment.

Other implementations provided in this paper involve obtaining sequence information from each strand of a nucleic acid, which can be used to reduce the error rate in sequencing information. Methods for preparing libraries of template nucleic acids to obtain sequence information from each strand of the nucleic acid can be used, making it possible to distinguish each strand and also to distinguish the products of each strand.

Some of the methods described in this article include methods for analyzing nucleic acids. These methods include preparing libraries of template nucleic acids for the target nucleic acid, obtaining sequence data from the template nucleic acid libraries, and assembling sequence representations of the target nucleic acid from such sequence data.

Generally, the methods and compositions provided herein relate to those provided in U.S. Patent Application Publication No. 2002/0208705, U.S. Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. WO 02/061832, each of which is incorporated herein by reference in its entirety. The methods provided herein relate to the use of transposons for inserting features into target nucleic acids. Such features include fragmentation sites, primer sites, barcodes, affinity tags, reporter moieties, etc.

In the methods used in the embodiments provided herein, a template nucleic acid library is prepared from a CE containing a target nucleic acid. The library is prepared by inserting or attaching multiple unique barcodes throughout the target nucleic acid. In some embodiments, each barcode includes a first barcode sequence and a second barcode sequence with fragmentation sites placed therebetween. The first and second barcode sequences can be identified or designated as paired with each other. The pairing can be informative, such that the first barcode is associated with the second barcode. Advantageously, the paired barcode sequences can be used to assemble sequencing data from the template nucleic acid library. For example, identifying a first template nucleic acid containing a first barcode sequence and a second template nucleic acid containing a second barcode sequence paired with the first barcode sequence indicates that the first and second template nucleic acids represent sequences adjacent to each other in the sequence representation of the target nucleic acid. Such methods can be used to assemble the sequence representation of the target nucleic acid de novo without a reference genome.

In some implementations, combinatorial barcoding can be used, such that the target nucleic acid from each single cell contains a unique barcode (e.g., a unique combination of barcodes), and the target nucleic acid can be easily identified from different target nucleic acids from different single cells. In some implementations, the CE may contain the target nucleic acid from a single cell. In some implementations, the target nucleic acid within the CE will have a uniquely identifiable barcode that differs from the target nucleic acids within different CEs.

In some embodiments, in addition to nucleic acids, combinatorial labeling schemes can be used for intracellular components, such as proteins, organelles, lipids, or cell membranes, enabling the identification of intracellular components from different single cells. In some embodiments, the CE may contain intracellular components. In some embodiments, the intracellular components within the CE will have identifiable and unique markers that differ from those of intracellular components within different CEs.

In some implementations, multiple combination barcode encoding schemes can be used for target nucleic acids from single cells, and multiple combination labeling schemes can be used together for components within a single cell. In some implementations, such combination barcode encoding and combination labeling can be performed within a single-cell CE containing a single cell. In some implementations, such combination barcode encoding and combination labeling can be performed in parallel on multiple CEs containing single cells.

In some embodiments, proteins that are retained, embedded, immobilized, or contained within a CE can be sequenced. In some embodiments, such proteins are uniquely labeled. In some embodiments, proteins that are retained, embedded, immobilized, or contained within a CE can be identified using methods known in the art. In some embodiments, protein identification and/or sequencing can be performed in conjunction with the collection of nucleic acid sequence information.

As used herein, the terms “nucleic acid” and/or “oligonucleotide” and/or their grammatical equivalents may refer to at least two nucleotide monomers linked together. Nucleic acids typically contain phosphodiester bonds; however, in some embodiments, nucleic acid analogs may have other types of backbones, including, for example, phosphoramides (Beaucage et al., Tetrahedron, 49:1925 (1993); Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl et al., Eur. J. Biochem., 81:579 (1977); Letsinger et al., Nucl. Acids Res., 14:3487 (1986); Sawai et al., Chem. Lett., 805 (1984); Letsinger et al., J. Am. Chem. Soc., 110:4470 (1988); and Pauwels et al., Chemica Scripta, 26:141 (1986)), thiophosphates (Mag et al. References cited above are incorporated herein by reference. (e.g., 19:1437 (1991); and US Patent No. 5,644,048), dithiophosphates (Briu et al., J. Am. Chem. Soc., 111:2321 (1989)), O-methylphosphoramide linkages (see Eckstein; Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc., 114:1895 (1992); Meier et al., Chem. Int. Ed., Engl, 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature, 380:207 (1996)).

Other similar nucleic acids include those with the following: a positively charged backbone (Denpcy et al., Proc. Natl. Acad. Sci. USA, 92: 6097 (1995)); a non-ionic backbone (US Patent Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English, 30: 423 (1991); Letsinger et al., J. Am. Chem. Soc. 110: 4470 (1988); Letsinger et al., Nucleosides & Nucleotides, 13: 1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modified Nucleic acids may also contain one or more carbocyclic sugars (see Jenkins et al., Chem. Soc. Rev., (1995) pp. 169, 176). Nucleic acids may also contain one or more carbocyclic sugars (see Jenkins et al., Chem. Soc. Rev., (1995) pp. 169, 176). The above references are incorporated into this paper by way of citation.

The ribose-phosphate backbone can be modified to facilitate the addition of additional components, such as markers, or to improve the stability of such molecules under certain conditions. Furthermore, mixtures of naturally occurring nucleic acids and analogues can be prepared. Optionally, mixtures of different nucleic acid analogues, as well as mixtures of naturally occurring nucleic acids and analogues, can be prepared. Nucleic acids can be single-stranded or double-stranded, as specified, or contain a portion of both double-stranded or single-stranded sequences. Nucleic acids can be DNA, such as genomic DNA or cDNA, RNA, or hybrids derived from single-celled, multi-celled, or multi-species samples, such as samples with metagenomic structures, such as environmental samples, and also from mixed samples such as mixed tissue samples or mixed samples for different individuals of the same species, disease samples such as cancer-related nucleic acids, etc. Nucleic acids can contain any combination of deoxyribonucleotides and ribonucleotides, as well as any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, and base analogs such as nitropyrrole (including 3-nitropyrrole) and nitroindole (including 5-nitroindole).

In some embodiments, the nucleic acid may include at least one promiscuous base. The promiscuous base may pair with more than one different type of base. In some embodiments, the promiscuous base may pair with at least two different types of bases and no more than three different types of bases. Examples of promiscuous bases include inosine, which can pair with adenine, thymine, or cytosine. Other examples include hypoxanthine, 5-nitroindole, acyclic 5-nitroindole, 4-nitropyrazole, 4-nitroimidazole, and 3-nitropyrrole (Loakes et al., Nucleic Acid Res. 22: 4039 (1994); Van Aerschot et al., Nucleic Acid Res. 23: 4363 (1995); Nichols et al., Nature 369: 492 (1995). 4); Bergstrom et al., Nucleic Acid Res. 25: 1935 (1997); Loakes et al., Nucleic Acid Res. 23: 2361 (1995); Loakes et al., J. Mol. Biol. 270: 426 (1997); and Fotin et al., Nucleic Acid Res. 26: 1515 (1998). Mixed bases that pair with at least three, four, or more types of bases may also be used. The above references are incorporated herein by reference.

As used herein, the term "nucleotide analog" and/or its grammatical equivalents can refer to synthetic analogs having a modified nucleotide base moiety, a modified pentose moiety, and/or a modified phosphate moiety, and, in the case of polynucleotides, a modified internucleotide linkage, as generally described elsewhere (e.g., Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Englisch, Angew. Chem. Int. Ed. Engl. 30: 613-29, 1991; Agarwal, Protocols for Polynucleotides and Analogs, Humana Press, 1994; and S. Verma and F. Eckstein, Ann. Rev. Biochem. 67: 99-134, 1998). Typically, the modified phosphate moiety comprises an analog of a phosphate ester in which the phosphorus atom is in a +5 oxidation state and one or more oxygen atoms are replaced by a non-oxygen moiety, such as sulfur. Exemplary phosphate ester analogues include, but are not limited to, thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phosphoroanilothioate, phosphoranilidate, aminophosphates, and borate phosphates, including associated counterions such as H ⁺ , _NH4 ⁺ , and Na ⁺ (if such counterions are present). Examples of modified nucleotide base moieties include, but are not limited to, 5-methylcytosine (5mC); C-5-propynyl analogues, including, but not limited to, C-5-propynyl-C and C-5-propynyl-U; 2,6-diaminopurine (also known as 2-aminoadenine or 2-amino-dA); hypoxanthine, pseudouridine, 2-thiopyrimidine, isocytosine (isoC), 5-methylisocytosine, and isoguanine (isoG; see, for example, U.S. Patent No. 5,432,272). Exemplary modifications of the pentose moiety include, but are not limited to, locked nucleic acid (LNA) analogs, including but not limited to Bz-A-LNA, 5-Me-Bz-C-LNA, dmf-G-LNA and T-LNA (see, for example, The Glen Report, 16(2):5, 2003; Koshkin et al., Tetrahedron 54:3607-30, 1998), and 2'- or 3'-modifications, wherein the 2'- or 3'-position is hydrogen, hydroxyl, alkoxy (e.g., methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy and phenoxy), azide, amino, alkylamino, fluorine, chlorine or bromine. Modified internucleotide linkages include phosphate ester analogs, analogs with achiral and uncharged subunit linkages (e.g., Sterchak, EP et al., Organic Chem., 52:4202, 1987), and uncharged morpholino polymers with achiral subunit linkages (see, for example, U.S. Patent No. 5,034,506). Some internucleotide linkage analogs include morpholinic acids, acetals, and polyamide-linked heterocycles. In a class of nucleic acid analogs called peptide nucleic acids, including pseudocomplementary peptide nucleic acids (“PNA”), the conventional sugar-nucleotide linkages have been replaced by a 2-aminoethylglycine backbone polymer (see, for example, Nielsen et al., Science, 254: 1497-1500, 1991; Egholm et al., J. Am. Chem. Soc., 114: 1895-1897, 1992; Demidov et al., Proc. Natl. Acad. Sci. 99: 5953-58, 2002; Peptide Nucleic Acids: Protocols and Applications, Nielsen, ed., Horizon Bioscience, 2004). These references are incorporated herein by reference.

As used herein, the term "sequencing read" and/or its syntactic equivalents may refer to a repetitive process involving physical or chemical steps to obtain a signal indicating the sequence of monomers in a polymer. The signal may indicate the sequence of monomers at a single monomer resolution or lower. In specific embodiments, the steps may be initiated on a nucleic acid target and proceed to obtain a signal indicating the base sequence in the nucleic acid target. This process may proceed to its typical completion, which is generally defined by a point at which the signal from the process can no longer distinguish the bases of the target at a definite and reasonable level. Completion may occur earlier if desired, for example, once the desired amount of sequence information has been obtained. Sequencing reads may be performed on a single target nucleic acid molecule or simultaneously on a group of target nucleic acid molecules having the same sequence, or simultaneously on a group of target nucleic acid molecules having different sequences. In some embodiments, the sequencing read is terminated when no signal is obtained from one or more target nucleic acid molecules from the initiation signal. For example, a sequencing read may be initiated on one or more target nucleic acid molecules present on a solid-phase substrate and terminated after one or more target nucleic acid molecules are removed from the substrate. Sequencing can be terminated by stopping the detection of the target nucleic acid present on the substrate when the sequencing run begins, under other circumstances.

As used herein, the term "seqencing representation" and/or its syntactic equivalents can refer to information indicating the sequence and type of monomeric units in a polymer. For example, this information can indicate the sequence and type of nucleotides in a nucleic acid. This information can be in any of a variety of forms, including, for example, drawings, images, electronic media, a series of symbols, a series of numbers, a series of letters, a series of colors, etc. This information can be at single-monomer resolution or at a lower resolution. Exemplary polymers are nucleic acids containing nucleotide units, such as DNA or RNA. A series of letters "A," "T," "G," and "C" is a well-known sequence representation of DNA, which can be correlated with the actual sequence of the DNA molecule at single-nucleotide resolution. Other exemplary polymers are proteins containing amino acid units and polysaccharides containing sugar units.

As used herein, the term “at least a portion” and/or its grammatical equivalents may refer to any fraction of a total quantity. For example, “at least a portion” may refer to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or 100% of the total quantity.

As used herein, the term “detection” and/or its grammatical equivalents may refer to the presence or existence of an analyte, the identification of individual components of the analyte, such as sequence information, and/or the quantification of the amount of such analyte.

Fragmentation site

In some embodiments that include a cyclic transposon, the linker may include a fragmentation site. The fragmentation site can be used to cleave a physical, rather than informational, association between a first and second barcode sequence. Cleavage may be biochemical, chemical, or otherwise. In some embodiments, the fragmentation site may include a nucleotide or nucleotide sequence that can be fragmented in various ways. For example, the fragmentation site may include a restriction endonuclease site; at least one ribonucleotide cleavable with RNase; a nucleotide analog cleavable in the presence of certain chemical reagents; a diol linkable by periodate treatment; a disulfide bond cleavable with a chemical reducing agent; a cleavable portion that can withstand photochemical cleavage; and a peptide cleavable with peptidase or other suitable methods. See, for example, U.S. Patent Application Publication No. 2002/0208705, U.S. Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. WO 02/061832, each of which is incorporated herein by reference in its entirety.

Primer site

In some embodiments, the reporter portion may include primer sites that can hybridize with primers. In some embodiments, the reporter portion may include at least one first primer site for amplification, sequencing, etc.

In some embodiments, the transposon sequence may include a "sequencing adapter" or "sequencing adapter site," that is, a region containing one or more sites that can hybridize to a primer. In some embodiments, the transposon sequence may include at least one first primer site for amplification, sequencing, etc. In some embodiments containing a circular transposon, the adapter may include a sequencing adapter. In more embodiments containing a circular transposon, the adapter includes at least a first primer site and a second primer site. In such embodiments, the orientation of the primer sites may be such that the primer hybridizing to the first primer site and the primer hybridizing to the second primer site are in the same or different orientations.

In some embodiments, the adapter may include a first primer site, a second primer site, and a non-amplifiable site disposed therebetween. The non-amplifiable site is useful for blocking the elongation of the polynucleotide chain between the first and second primer sites, where the polynucleotide chain hybridizes to one of the primer sites. The non-amplifiable site is also useful for preventing concatamer formation. Examples of non-amplifiable sites include nucleotide analogs, non-nucleotide chemical moieties, amino acids, peptides, and polypeptides. In some embodiments, the non-amplifiable site comprises a nucleotide analog that does not significantly pair with A, C, G, or T bases. Some embodiments include an adapter comprising a first primer site, a second primer site, and a fragmentation site disposed therebetween. Other embodiments may use fork-shaped or Y-shaped adaptor designs for directed sequencing, as described in U.S. Patent No. 7,741,463, the disclosure of which is incorporated herein by reference in its entirety.

Exemplary sequences of primer binding sites include, but are not limited to, AATGATACGGCGACCACCGAGATCTACAC (P5 sequence) and CAAGCAGAAGACGGCATACGAGAT (P7 sequence).

Report Section

As used herein, the term “report portion” and its grammatical equivalents may refer to any identifiable label, marker, index, barcode, or group that can determine the composition, identity, and/or origin of the analyte under study.

Those skilled in the art will understand that many different types of reporter portions can be used in the methods and compositions described herein, either alone or in combination with one or more different reporter portions. In some embodiments, more than one different reporter portion can be used to analyze more than one analyte simultaneously. In some embodiments, multiple different reporter portions can be used simultaneously to uniquely identify single cells or components of a single cell.

In some embodiments, the reporter component may emit a signal. Examples of signals include, but are not limited to, fluorescence, chemiluminescence, bioluminescence, phosphorescence, radioactivity, calorimetry, ionic activity, and electro- or electrochemiluminescence signals. Example reporter molecules are listed, for example, in U.S. Patent Application Publication No. 2002/0208705, U.S. Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. WO 02/061832, each of which is incorporated herein by reference in its entirety.

In some embodiments, the reporter portion may be a connector. In some embodiments of the compositions and methods described herein, the transposon sequence may include the reporter portion. In some embodiments comprising a ring-shaped transposon, the connector or connector may include the reporter portion.

In some embodiments, the reporter portion may not emit a signal. In some embodiments, the reporter portion may be a nucleic acid fragment, such as a barcode, a unique molecular index, or a plasmid. In some embodiments, the reporter portion may contain an antibody that specifically binds to a protein. In some embodiments, the antibody may contain a detectable tag. In some embodiments, the reporter may include an antibody or affinity reagent labeled with a nucleic acid tag. The nucleic acid tag can be detected, for example, via proximity linkage assay (PLA) or proximity extension assay (PEA).

In some embodiments, a set of reporter portions may be used. In some embodiments, the set of reporter portions may comprise a mixture of subgroups of reporter portions, wherein each subgroup of reporter portions is specific to a different type of analyte, such as protein, nucleic acid, lipid, or carbohydrate. In some embodiments, the set of reporter portions may comprise a mixture of subgroups of reporter portions, wherein each subgroup of reporter portions is different from each other but is specific to the same type of analyte.

barcode

Typically, a barcode may comprise one or more nucleotide sequences that can be used to identify one or more specific analytes (e.g., nucleic acids, proteins, metabolites, or other analytes) described herein or known in the art. The barcode may be an artificial sequence or a naturally occurring sequence generated during transposition, such as the same flanking genomic DNA sequence (g-code) at the ends of previously juxtaposed DNA fragments. A barcode may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides. In some embodiments, the barcode contains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more consecutive nucleotides. In some embodiments, at least a portion of the barcodes within the nucleic acid group containing the barcode is distinct. In some implementations, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 99% of the barcodes are different. In many more such implementations, all barcodes are different. The diversity of different barcodes in the nucleic acid population containing the barcodes can be generated randomly or non-randomly.

In some embodiments, the transposon sequence includes at least one barcode. In some embodiments, such as a transposon comprising two discontinuous transposon sequences, the first transposon sequence including a first barcode and the second transposon sequence including a second barcode. In some embodiments, such as in a circumferential transposon, the transposon sequence includes a barcode comprising a first barcode sequence and a second barcode sequence. In some of the foregoing embodiments, the first barcode sequence can be identified or designated to pair with the second barcode sequence. For example, it can be known that, using a reference table containing a plurality of first and second barcode sequences known to pair with each other, a known first barcode sequence is paired with a known second barcode sequence.

In another instance, the first barcode sequence may contain the same sequence as the second barcode sequence. In yet another instance, the first barcode sequence may contain the inverse complement of the second barcode sequence. In some implementations, the first and second barcode sequences are different. The first and second barcode sequences may contain dual codes.

In some embodiments of the compositions and methods described herein, barcodes are used for the preparation of template nucleic acids. As will be understood, a wide range of available barcodes allow each template nucleic acid molecule to contain a unique identifier. The unique identifier of each molecule in a mixture of template nucleic acids can be used for a variety of applications. For example, in samples with multiple chromosomes, in genomes, in cells, in cell types, in cellular disease states, and in species, such as in haplotype sequencing, in parental allele identification, in metagenomic sequencing, and in genome sample sequencing, uniquely identified molecules can be used to identify individual nucleic acid molecules. Exemplary barcode sequences include, but are not limited to, TATAGCCT, ATAGAGGC, CCTATCCT, GGCTCTGA, AGGCGAAG, TAATCTTA, CAGGACGT, and GTACTGAC.

connector

Some embodiments including a cyclic transposon include a transposon sequence comprising a first barcode sequence and a second barcode sequence, with a linker disposed therebetween. In other embodiments, the linker may be absent, or may be a sugar-phosphate backbone linking one nucleotide to another. The linker may comprise one or more of, for example, nucleotides, nucleic acids, non-nucleotide chemical moieties, nucleotide analogs, amino acids, peptides, polypeptides, or proteins. In a preferred embodiment, the linker comprises a nucleic acid. The linker may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, the linker may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more nucleotides.

In some implementations, the adapter can be amplifiable, for example, by PCR, rolling circle amplification, strand displacement amplification, etc. In other implementations, the adapter may contain a non-amplifiable portion. Examples of non-amplifiable adapters include organic chemical adapters such as alkyl, propyl, and PEG; non-natural bases such as IsoC and isoG; or any group that is not amplified in DNA-based amplification protocols. For example, transposons containing isoC and isoG pairs can be amplified using a mixture of dNTPs lacking complementary isoG and isoC, ensuring that amplification does not occur across the inserted transposon.

In some implementations, the adapter comprises a single-stranded nucleic acid. In some implementations, the adapter is coupled with a transposon sequence in a 5'-3', 5'-5', or 3'-3' orientation.

Affinity Label

In some embodiments, the transposon sequence may contain an affinity tag. In some embodiments that include a circular transposon, the adapter may contain an affinity tag. Affinity tags can be used for a variety of applications, such as the mass isolation of target nucleic acids from hybridization to hybridization tags. Additional applications include, for example, using affinity tags for the purification of transposase/transposon complexes and transposon insertions into target DNA, target RNA, or target proteins. As used herein, the term “affinity tag” and its grammatical equivalents may refer to components of a multicomponent complex, wherein the components of the multicomponent complex specifically interact with or bind to each other. For example, an example of an affinity tag may contain biotin or poly-His, which binds to streptavidin protein or nickel, respectively. Other examples of multicomponent affinity tag complexes are listed, such as U.S. Patent Application Publication No. 2002/0208705, U.S. Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. WO 02/061832, each of which is incorporated herein by reference in its entirety.

solid support

Solid supports can be two-dimensional or three-dimensional and can include planar surfaces (e.g., glass slides) or can be shaped. Solid supports can include glass (e.g., controlled-pore glass (CPG)), quartz, plastics (e.g., polystyrene (low-crosslinked and high-crosslinked polystyrene), polycarbonate, polypropylene, and poly(methyl methacrylate)), acrylic copolymers, polyamides, silicon, metals (e.g., alkanethiolate-derived gold), cellulose, nylon, latex, dextran, gel matrices (e.g., silica gel), polyacrolein, or composite materials.

Suitable three-dimensional solid supports include, for example, spheres, microparticles, beads, nanoparticles, polymer matrices such as agarose, polyacrylamide, alginate, membranes, glass slides, plates, micromachined chips, tubes (e.g., capillaries), micropores, microfluidic devices, channels, filters, flow cells, and structures suitable for immobilizing nucleic acids, proteins, or cells. Solid supports can comprise planar arrays or matrices capable of having regions containing clusters of template nucleic acids or primers. Examples include nucleoside-derived CPG and polystyrene slides; derived magnetic slides; and polystyrene grafted with polyethylene glycol, etc.

In some embodiments, the solid support comprises microspheres or beads. The terms "microsphere," "bead," "particle," or grammatical equivalents as used herein refer to small, discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thorium oxide sol, carbon graphite, titanium dioxide, latex, or cross-linked dextran such as agarose (Sepharose), cellulose, nylon, cross-linked micelles, and polytetrafluoroethylene, as well as any other materials outlined herein for the solid support. The "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. is a useful guide. In some embodiments, the microspheres are magnetic microspheres or beads. In some embodiments, the beads may be color-coded. For example, microspheres from Luminex, Austin, TX may be used.

The beads do not need to be spherical; irregular particles can be used. Optionally or additionally, the beads can be porous. The size of the beads ranges from nanometers, i.e., 100 nanometers, to millimeters, i.e., 1 mm, with beads having a size from about 0.2 micrometers to about 200 micrometers being preferred, and from about 0.5 to about 5 micrometers being particularly preferred, although smaller or larger beads can be used in some embodiments. In some embodiments, the beads can be about 1, 1.5, 2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 15, or 20 μm in diameter.

In some embodiments, the beads may contain antibodies or other affinity probes (see *mmobilized Biomolecules in Analysis. A Practical Approach. Cass T, Ligler F S, eds. Oxford University Press, New York, 1998. pp 1-14, incorporated herein by reference for typical attachment schemes). In some embodiments, the antibody may be monoclonal; in other embodiments, the antibody may be polyclonal. In some embodiments, the antibody may be specific to cell surface epitopes. In some embodiments, the antibody may be specific to intracellular proteins.

In some implementations, the nucleic acid templates provided herein can be attached to a solid support. Nucleic acids can be attached, anchored, or fixed to the surface of the solid support using various methods well known in the art.

Analytes

An analyte is a biomolecule used to study its function, composition, identity, and/or origin. Exemplary analytes include, but are not limited to, DNA, RNA, cDNA, proteins, lipids, carbohydrates, cellular organelles (e.g., nucleus, Golgi apparatus, ribosomes, mitochondria, endoplasmic reticulum, chloroplasts, cell membranes, etc.), cellular metabolites, tissue sections, cells, single cells, contents derived from cells or single cells, nucleic acids isolated from cells or single cells, or nucleic acids isolated from cells or single cells and further modified, or cell-free DNA (e.g., from placental fluid or plasma).

Target nucleic acid

The target nucleic acid can include any nucleic acid of interest. In one embodiment, the target nucleic acid can include any nucleic acid of interest contained, captured, embedded, or immobilized in a CE, such as a matrix, droplet, emulsion, solid support, or compartment that keeps the nucleic acid in proximity internally but allows access to liquid and enzyme reagents. The target nucleic acid can include DNA, cDNA, products of WGA, RNA, peptide nucleic acid, morpholinonucleotide, locked nucleic acid, diol nucleic acid, threonine nucleic acid, mixed samples of nucleic acids, polyploid DNA (i.e., plant DNA), mixtures thereof, and hybrids thereof. In a preferred embodiment, a fragment of genomic DNA or an amplified copy thereof is used as the target nucleic acid. In another preferred embodiment, cDNA, mitochondrial DNA, or chloroplast DNA is used.

The target nucleic acid can contain any nucleotide sequence. In some embodiments, the target nucleic acid contains a homopolymer sequence. The target nucleic acid may also include repetitive sequences. The repetitive sequences can be of any length, including, for example, 2, 5, 10, 20, 30, 40, 50, 100, 250, 500, or 1000 nucleotides or more. The repetitive sequences can be repeated, continuously or discontinuously, and any number of repetitions includes, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times or more.

Some embodiments described herein may utilize a single target nucleic acid. Other embodiments may utilize multiple target nucleic acids. In such embodiments, multiple target nucleic acids may include multiple identical target nucleic acids, some of which are multiple different target nucleic acids, or all of which are multiple different target nucleic acids. Embodiments utilizing multiple target nucleic acids may be carried out in multiple forms, such that reagents are delivered simultaneously to the target nucleic acids, for example, in one or more chambers or on an array surface. In some embodiments, multiple target nucleic acids may include substantially all of the genome of a particular organism. Multiple target nucleic acids may include at least a portion of the genome of a particular organism, including, for example, at least about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the genome. In specific embodiments, this portion may have an upper limit of up to about 1%, 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, or 99% of the genome.

The target nucleic acid can be obtained from any source. For example, the target nucleic acid can be prepared from nucleic acid molecules obtained from a single organism or from a group of nucleic acid molecules obtained from natural sources comprising one or more organisms. Sources of nucleic acid molecules include, but are not limited to, organelles, cells, tissues, organs, or organisms. Cells that can be used as sources of target nucleic acid molecules can be prokaryotes (bacterial cells, such as Escherichia, Bacillus, Serratia, Salmonella, Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria, Spirochetes, Mycoplasma, Leptospira, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia, Agrobacterium, Rhizobium, and Streptomyces); archaea, such as Crenarchaeota, Nanoarchaeota, or Euryarchaeotia; or eukaryotes such as fungi (e.g., yeast), plants, protozoa, and other parasites, as well as animals (including insects (e.g., Drosophila species), nematodes (e.g., Caenorhabditis elegans)), and mammals (e.g., rats, mice, monkeys, non-human primates, and humans).

For certain sequences of interest, target and template nucleic acids can be enriched using various methods well known in the art. Examples of such methods are provided in International Publication No. WO/2012/108864, which is incorporated herein by reference in its entirety. In some embodiments, nucleic acids can be further enriched during the method for preparing the template library. For example, for certain sequences, nucleic acids can be enriched before transposon insertion, after transposon insertion, and/or after nucleic acid amplification.

Furthermore, in some embodiments, the target nucleic acid and/or template nucleic acid can be highly purified; for example, the nucleic acid can be at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% free of contaminants prior to use in the methods provided herein. In some embodiments, it is advantageous to use methods known in the art for maintaining the quality and size of the target nucleic acid, such as using an agarose plug for the isolation and/or direct transposition of the target DNA.

In some implementations, the target nucleic acid may be obtained from biological samples or patient samples. As used herein, the terms "biological sample" or "patient sample" include, for example, samples of one or more cells, tissues, or body fluids. "Body fluids" may include, but are not limited to, blood, serum, plasma, saliva, cerebrospinal fluid, pleural fluid, tears, mammary duct fluid, lymph, sputum, urine, amniotic fluid, or semen. Samples may include body fluids that are "acellular." "Cellular body fluids" include less than about 1% (w/w) of whole-cell material. Plasma or serum are examples of cellless body fluids. Samples may include specimens of natural or synthetic origin (i.e., prepared as cell-free cellular samples).

As used herein, the term "plasma" refers to the cell-free fluid found in blood. "Plasma" can be obtained from blood by removing whole-cell material from it using methods known in the art (e.g., centrifugation, filtration, etc.).

Some methods for preparing template nucleic acids

Some implementations include methods for preparing template nucleic acids. As used herein, "template nucleic acid" can refer to a substrate used to obtain sequence information. In some implementations, the template nucleic acid may include a target nucleic acid, a fragment thereof, or any copy thereof, containing at least one transposon sequence, a fragment thereof, or any copy thereof. In some implementations, the template nucleic acid may include a target nucleic acid containing a sequencing adaptor, such as a sequencing primer site. In some implementations, the CE may contain the target nucleic acid.

Some methods for preparing template nucleic acids involve inserting a transposon sequence into a target nucleic acid. Some insertion methods involve contacting the transposon sequence provided herein with the target nucleic acid in the presence of an enzyme such as a transposase or integrase, under conditions sufficient for the integration of one or more transposon sequences into the target nucleic acid. In some embodiments, the CE may comprise such a target nucleic acid.

In some embodiments, the insertion of the transposon sequence into the target nucleic acid may be non-random. In some embodiments, the transposon sequence may contact the target nucleic acid containing proteins that inhibit integration at certain sites. For example, the transposon sequence may inhibit integration into genomic DNA containing proteins, genomic DNA containing chromatin, genomic DNA containing nucleosomes, or genomic DNA containing histones. In some embodiments, the transposon sequence may be associated with an affinity tag to integrate the transposon sequence into the target nucleic acid at a specific sequence. For example, the transposon sequence may be associated with proteins that target a specific nucleic acid sequence, such as histones, chromatin-binding proteins, transcription factors, initiation factors, etc., and antibodies or antibody fragments that bind to specific sequence-specific nucleic acid-binding proteins. In exemplary embodiments, the transposon sequence is associated with an affinity tag such as biotin; the affinity tag may be associated with a nucleic acid-binding protein. In some embodiments, the CE may contain such a target nucleic acid.

It should be understood that during the integration of transposon sequences into the target nucleic acid, several consecutive nucleotides of the target nucleic acid at the integration site are duplicated in the integration product. Therefore, the integration product may include a repeating sequence at each end of the integrated sequence in the target nucleic acid. As used herein, the term "host tag" or "g tag" may refer to a target nucleic acid sequence that is repeated at each end of the integrated transposon sequence. Single-stranded portions of nucleic acids resulting from the insertion of transposon sequences can be repaired using a variety of methods well known in the art, such as by using ligases, oligonucleotides, and/or polymerases.

In some implementations, multiple transposon sequences provided herein are inserted into the target nucleic acid. Some implementations include selecting conditions sufficient to integrate multiple transposon sequences into the target nucleic acid such that the average distance between each integrated transposon sequence comprises a certain number of consecutive nucleotides in the target nucleic acid.

Some implementations include selecting conditions sufficient to allow insertion of one or more transposon sequences into a target nucleic acid rather than into another one or more transposon sequences. Various methods can be used to reduce the likelihood of a transposon sequence inserting into another transposon sequence. Examples of such methods used in the implementations provided herein can be found, for example, in U.S. Patent Application Publication No. 2002/0208705, U.S. Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. WO 02/061832, each of which is incorporated herein by reference in its entirety.

In some embodiments, conditions can be selected such that the average distance in the target nucleic acid between the integrated transposon sequences is at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive nucleotides. In some embodiments, the average distance in the target nucleic acid between the integrated transposon sequences is at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more consecutive nucleotides. In some embodiments, the average distance in the target nucleic acid between the integrated transposon sequences is at least about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 90 kb, 100 kb or more consecutive nucleotides. In some implementations, the average distance between the integrated transposon sequences in the target nucleic acid is at least about 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, or more consecutive nucleotides. As will be understood, some optional conditions include contacting the target nucleic acid with a certain number of transposon sequences.

Some embodiments of the methods described herein include selecting conditions sufficient to achieve the integration of at least a portion of a transposon sequence into different target nucleic acids. In preferred embodiments of the methods and compositions described herein, each transposon sequence integrated into the target nucleic acid is different. Some conditions that can be selected to achieve the integration of a portion of a transposon sequence into different target sequences include selecting the degree of diversity of the transposon sequence groups. As will be understood, the diversity of transposon sequences arises in part from the diversity of barcodes of such transposon sequences. Therefore, some embodiments include providing a transposon sequence group in which at least a portion of the barcode is a different transposon sequence group. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the barcodes in the transposon sequence group are different. In some embodiments, at least a portion of the transposon sequence integrated into the target nucleic acid is identical.

Some embodiments for preparing template nucleic acids may include replicating a sequence containing a target nucleic acid. For example, some embodiments include hybridizing primers to primer sites of transposon sequences integrated into the target nucleic acid. In some such embodiments, primers may be hybridized to primer sites and extended. The replicated sequence may include at least one barcode sequence and at least a portion of the target nucleic acid. In some embodiments, the copied sequence may include a first barcode sequence, a second barcode sequence, and at least a portion of the target nucleic acid disposed therebetween. In some embodiments, at least one replicated nucleic acid may include at least a first barcode sequence of a first replicated nucleic acid that can be identified or designated as pairing with a second barcode sequence of a second replicated nucleic acid. In some embodiments, primers may include sequencing primers. In some embodiments, sequencing data is obtained using sequencing primers. In more embodiments, an adaptor containing a primer site may be ligated to each end of the nucleic acid, and the nucleic acid is amplified from such primer sites.

Some embodiments for preparing template nucleic acids may include amplifying a sequence comprising at least a portion of one or more transposon sequences and at least a portion of a target nucleic acid. In some embodiments, at least a portion of the target nucleic acid may be amplified using primers that hybridize to primer sites that integrate transposon sequences into the target nucleic acid. In some such embodiments, the amplified nucleic acid may include a first barcode sequence and a second barcode sequence with at least a portion of the target nucleic acid disposed therebetween. In some embodiments, at least one amplified nucleic acid may include at least a first barcode sequence of a first amplified nucleic acid that can be identified as pairing with a second barcode sequence of a second amplified sequence.

Some methods for preparing template nucleic acids involve inserting transposon sequences containing a single linker. In one example, a transposon sequence (ME-P1-linker-P2-ME; mosaic end-primer site 1-linker-primer site 2-mosaic end) is inserted into the target nucleic acid. The target nucleic acid with the inserted transposon/linker sequence can be extended and amplified.

In one embodiment of the compositions and methods described herein, transposable transposable ends are used to generate end-tagged target nucleic acid fragments (tagged fragments or tag fragments). Therefore, each tagged fragment contains the same ends and lacks directionality. Single-primer PCR using the transposon end sequences can then be applied to amplify template copy numbers from 2n to 2n* ^2x , where x corresponds to the number of PCR cycles. In subsequent steps, primer-based PCR can be used to add additional sequences, such as sequencing adaptor sequences.

In some implementations, it may be advantageous to incorporate at least one universal primer site into each template nucleic acid. For example, the template nucleic acid may include a first terminal sequence containing a first universal primer site and a second terminal sequence containing a second universal primer site. The universal primer site can have various applications, such as in amplification, sequencing, and/or identification of one or more template nucleic acids. The first and second universal primer sites may be identical, substantially similar, similar, or different. The universal primer site can be introduced into the nucleic acid by various methods well known in the art, such as primer site-to-nucleic acid ligation, amplification of the nucleic acid using a tailed primer, and insertion of a transposon sequence containing the universal primer site.

Rotary body

A transposon comprises an integration enzyme, such as an integrase or transposase, and a nucleic acid containing an integration recognition site, such as a transposase recognition site. In the embodiments provided herein, the transposase can form a functional complex with a transposase recognition site capable of catalyzing a transposition reaction. The transposase can bind to the transposase recognition site and insert the transposase recognition site into a target nucleic acid within the CE in a process sometimes referred to as “tag fragmentation.” In some such insertion events, one strand of the transposase recognition site can be transferred into the target nucleic acid. In one example, the transposon contains a dimeric transposase comprising two subunits and two discontinuous transposon sequences. In another example, the transposase contains a dimeric transposase comprising two subunits and a continuous transposon sequence.

Some implementations may include the use of a highly active Tn5 transposase and a Tn5-type transposase recognition site (Goryshin and Reznikoff, J. Biol. Chem., 273:7367 (1998)) or a MuA transposase and a Mu transposase recognition site containing R1 and R2 terminal sequences (Mizuuchi, K., Cell, 35:785, 1983; Savilahti, H et al., EMBO J., 14:4893, 1995). The ME sequence may also be optimized by those skilled in the art. The above references are incorporated herein by reference.

Further examples of transposable systems that can be used in certain embodiments of the compositions and methods provided herein include Staphylococcus aureus Tn552 (Colegio et al., J. Bacteriol, 183: 2384-8, 2001; Kirby C et al., Mol. Microbiol., 43: 173-86, 2002), Tyl (Devine & Boeke, Nucleic Acids Res., 22: 3765-72, 1994 and International Publication WO 9) 5/23875), transposon Tn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, reviewed in: Curr Top Microbiol Immuno. l, 204: 27-48, 1996), Tn/O and IS10 (Kleckner N et al., Curr Top Microbiol Immunol., 204: 49-82, 1996), Mariner transposase (La mpe D J, et al., EMBO J., 15: 5470-9, 1996), Tcl (Plasterk R H, Curr. Topics Microbiol. Immunol., 204: 125-43, 1996), P element (Gloor, G B, Methods Mol. Biol., 260:97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol. Chem. 265:1 8829-32, 1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses (Brown et al., Proc Natl Acad Sci USA, 86: 2525-9, 1989), and yeast retrotransposons (Boeke & Corces, Aram Rev Microbiol. 43: 403-34, 1989). More examples include IS5, Tn10, Tn903, IS911, and engineered versions of transposase family enzymes (Zhang et al., (2009) PLoS Genet. 5: el000689. Epub 2009 Oct 16; Wilson C. et al. (2007) J. Microbiol. Methods 71: 332-5). The above references are incorporated into this paper by way of citation.

Further examples of integrases that can be used in the methods and compositions provided herein include retroviral integrases and integrase recognition sequences for such retroviral integrases, such as integrases from HIV-1, HIV-2, SIV, PFV-1, and RSV.

Transposon sequence

Some embodiments of the compositions and methods provided herein include transposon sequences. In some embodiments, the transposon sequence includes at least one transposase recognition site. In some embodiments, the transposon sequence includes at least one transposase recognition site and at least one barcode. Transposon sequences for use in the methods and compositions provided herein are provided in U.S. Patent Application Publication No. 2002/0208705, U.S. Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. WO 02/061832, each of which is incorporated herein by reference in its entirety. In some embodiments, the transposon sequence includes a first transposase recognition site, a second transposase recognition site, and a barcode disposed therebetween.

Transposons with discontinuous transposon sequences

Some transposons described herein include transposases comprising two transposon sequences. In some such embodiments, the two transposon sequences are not connected to each other; in other words, the transposon sequences are not consecutive. Examples of such transposons are known in the art, see, for example, U.S. Patent Application Publication No. 2010/0120098, the disclosure of which is incorporated herein by reference in its entirety.

Ring structure

In some embodiments, the transposon contains a transposon sequence nucleic acid that binds two transposase subunits to form a "circular complex" or "circular transposon". In one example, the transposon contains a dimeric transposase and a transposon sequence. The circular complex ensures that the transposon is inserted into the target DNA while maintaining the ordering information of the original target DNA and preventing fragmentation of the target DNA. As will be understood, circular structures can insert primers, barcodes, indexes, etc., into the target nucleic acid while maintaining the physical connectivity of the target nucleic acid. In some embodiments, the CE may contain the target nucleic acid. In some embodiments, the transposon sequence of the circular transposon may include a fragmentation site, allowing the transposon sequence to be fragmented to produce a transposon containing two transposon sequences. Such transposons are useful for ensuring that adjacent target DNA fragments in which the transposon is inserted receive a codon combination that can be definitively assembled at a later stage of assay.

Some methods for preparing transposon sequences

The transposon sequences provided herein can be prepared by a variety of methods. Exemplary methods include direct synthesis and hairpin extension methods. In some embodiments, the transposon sequences can be prepared by direct synthesis. For example, transposon sequences containing nucleic acids can be prepared by methods involving chemical synthesis. Such methods are well known in the art, such as solid-phase synthesis using phosphoramide precursors such as phosphoramide precursors derived from protected 2'-deoxynucleosides, ribonucleosides, or nucleoside analogs. Exemplary methods for preparing transposon sequences can be found, for example, U.S. Patent Application Publication No. 2002/0208705, U.S. Patent Application Publication No. 2012/0208724, and International Patent Application Publication No. WO 02/061832, each of which is incorporated herein by reference in its entirety.

In some embodiments involving a cyclic transposon, a transposon sequence comprising a single linker can be prepared. In some embodiments, the linker couples the transposon sequence of the transposon such that the transposon sequence containing a first transposase recognition sequence is coupled to a second transposon sequence containing a second transposase recognition sequence in a 5' to 3' orientation. In some embodiments, the linker couples the transposon sequence containing the first transposase recognition sequence to a second transposon sequence containing the second transposase recognition sequence in a 5' to 5' or 3' to 3' orientation. Coupled transposon sequences of the transposon in a 5' to 5' or 3' to 3' orientation can be advantageous in preventing the transposase recognition elements, particularly the mosaic elements (ME or M), from interacting with each other. The coupled transposon sequence can be prepared by preparing a transposon sequence containing an aldehyde or oxyamine group. Aldehydes and oxyamine groups can interact to form a covalent bond, thereby coupling the transposon sequence.

In some embodiments, transposons containing complementary sequences can be prepared. In one embodiment, the transposase carries a transposon sequence containing a complementary tail. Tail hybridization is performed to form linked transposon sequences. Hybridization can occur under dilution conditions to reduce the possibility of hybridization between transposons.

Targeted insertion

In some embodiments of the methods and compositions provided herein, transposon sequences can be inserted into specific targeted sequences of the target nucleic acid. Transposition to dsDNA can be more efficient than transposition to ssDNA. In some embodiments, dsDNA is denatured into ssDNA and annealed with oligonucleotide probes (20-200 bases). These probes generate sites on the dsDNA that can be efficiently used as integration sites with the transposons provided herein. In some embodiments, dsDNA can be targeted using D-loop formation and subsequent triplet formation with recA-coated oligomeric probes. In some such embodiments, the D-loop is a preferred substrate for transposons containing the Tn4430 transposase. In many more embodiments, regions of interest in the dsDNA can be targeted using sequence-specific DNA-binding proteins such as zinc finger complexes and other affinity ligands for specific DNA regions.

In some embodiments, transposons containing a transposase can be used for targeted insertion into a target nucleic acid, the transposase having a preferred substrate at a mismatch site in the target nucleic acid. For example, some MuA transposases, such as HYPERMU (Epicenter), are biased towards mismatched targets. In some such embodiments, the mismatched oligonucleotide probe is annealed to a single-stranded target nucleic acid. Transposons containing MuA transposases such as HYPERMU can be used to target mismatched sequences in the target nucleic acid.

Proximity retention element (CE)

A proximity retention element (CE) is a physical entity that retains at least two or more analytes in close proximity (or adjacent) through one or more assay steps, provides proximity to the assay reagent, and can be combined and separated multiple times without losing the proximity of the analytes.

In some embodiments, the CE can be a solid support. In one embodiment, the CE can be an emulsion or droplets. In some embodiments, the CE is a gel, hydrogel, or gel bead. In some embodiments, the CE can contain a solid support, such as beads. In some embodiments, the beads can further contain antibodies, oligonucleotides, and/or barcodes. In another embodiment, the CE can constitute DNA nanospheres produced by condensation of WGA, RCA, or any nucleic acid reagent.

In some embodiments, CEs can be prepared by encapsulating nucleic acids from cells or single cells, or their amplified products (from WGA, etc.), in a polymer matrix such as agarose, polyacrylamide, alginate, etc. In some embodiments, the proximity of the contents of cells or single cells within the CE is maintained by encapsulating (e.g., in a polymer matrix), immobilizing on beads, or trapping to maintain the physical proximity of components to each other, and by repeatedly merging and redistributing them to effectively maintain proximity information within the CE. The ability of CE sets to be independently merged and separated, reacted with assay reagents, merged and separated again, etc., while also maintaining the continuity of the analytes constituting a single CE, enables combinatorial indexing through different separation and merging steps.

In some embodiments, the analyte in the adjacent retention element is accessible to the assay reagent, which includes aqueous solutions, enzymes (e.g., fragmentases, polymerases, ligases, transposases, kinases, restriction endonucleases, proteases, phosphatases, lipases), nucleic acid adaptors, nucleic acid barcodes, and markers.

In some embodiments, the CE comprises cells or single cells. In some embodiments, the CE comprises nucleic acids, such as DNA, mRNA, or cDNA, from cells or single cells; macromolecules from cells or single cells, including proteins, polysaccharides, lipids, and nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and natural products from cells or single cells. In some embodiments, the nucleic acids undergo amplification, such as PCR or whole-genome amplification, prior to the formation of the nucleic acid-containing CE. In some embodiments, DNA and mRNA analysis can be performed in parallel.

In some implementations, one or more analytes in the CE are labeled with one or more markers. Exemplary markers include, but are not limited to, DNA barcodes or indexes, fluorescent markers, chemiluminescent markers, RNA barcodes or indexes, radioactive markers, antibodies containing markers, and beads containing markers.

In some implementations, the method may include the following steps: (a) dividing a CE containing a target nucleic acid into multiple first containers; (b) providing a first index to the target nucleic acid in each first container to obtain a first-indexed nucleic acid; (c) combining the first-indexed nucleic acids; (d) dividing the first-indexed template nucleic acid into multiple second containers; and (e) providing a second index to the first-indexed template nucleic acid in each second container to obtain a second-indexed nucleic acid. Steps a-e can be continued with additional loops from one or more steps in the series a-e to derive additional virtual compartments. This method of combining indexes can be used to efficiently create a large number of virtual compartments from a limited number of physical compartments.

In some implementations, the method may include the following steps: (a) providing a CE containing a non-nucleic acid analyte (e.g., a protein) with an attached nucleic acid reporter; (b) partitioning the CE into a plurality of first containers; (c) providing a first index to a target nucleic acid reporter in each first container to obtain a first-indexed target nucleic acid reporter; (d) combining the first-indexed nucleic acid reporters; (e) partitioning the first-indexed CE into a plurality of second containers; and (f) providing a second index to a first-indexed nucleic acid reporter in each second container to obtain a second-indexed nucleic acid reporter. Steps a-e can be continued with additional cycles from one or more steps in the series a-e to derive additional virtual compartments. The partitioning steps may further include nucleic acid amplification or capture steps, such as PLA, PEA, or other techniques for capturing or amplifying nucleic acids.

In some implementations, formalin-fixed, paraffin-embedded tissue can be sectioned, with each section added to a CE (cell electrode assembly). The contents or sequences of each CE can then be analyzed, and 2D or 3D images of the contents of each section can be obtained at a later stage.

In some implementations, one or more nucleic acids may be embedded in a matrix that confines the nucleic acids within a defined space but allows reagents to enter for steps including, but not limited to, amplification (PCR, whole genome amplification, random primer extension, etc.), ligation, transposition, hybridization, restriction digestion, and DNA mutagenesis. Examples of mutagenesis include, but are not limited to, error-prone extension, alkylation, bisulfite conversion, and activation-induced (cytidine) deaminase.

In some implementations, the methods and compositions using CEs can be combined with mutagenesis assembly methods to significantly improve the assembly of DNA sequence information. Genomic DNA can be fragmented and divided into multiple CEs, each containing a fraction of the genome. Different fractions of the genome receive different barcodes, allowing each fraction of the genome to be assembled independently. One of the greater challenges is the assembly of repetitive sequences. One method for assembling repetitive sequences is outlined by Levy, D. and Wigler, M. (2014) Facilitated sequence counting and assembly by template mutagenesis. Proc. of the Natl. Acad. Sci., Ill(43). E4632-E4637. ISSN 0027-8424. Assembly methods are also discussed in US20140024537, entitled: Methods and Systems for Determining Haplotypes and Phasing of Haplotypes, and this application is incorporated herein by reference in its entirety. The above references are incorporated into this paper by way of citation.

For the partitioning and mutagenesis of combined DNA fragments, methods such as CE (cell partitioning), wells, indexing, virtual indexing, physical compartments, and droplets can be used. Mutagenesis can be induced by several methods, including but not limited to error-prone extension, alkylation, bisulfite conversion, and activation-induced (cytidine) deaminase. In cases where conventional methods make assembling repetitive or dissimilar regions challenging, partitioning nucleic acids into CE methods and mutagenesis can be useful.

In some implementations, the methods described herein can be used for variant phasing, (de novo) genome assembly, screening cell populations to determine heterogeneity across populations, and identifying cell-to-cell differences.

In some implementations, cDNA from cells or single cells is isolated in a container and converted into CEs indexed using a virtual compartmentalization method as described above. This enables gene expression and transcript profiling from 1,000, 10,000, 100,000, or even more differently indexed single-cell libraries.

In some implementations, due to Poisson sampling, the number of analyzable single cells is approximately 10% of the total number of virtual compartments. For a four-layer indexing scheme with 96-well compartments in each step, a total of 4 × 96 = 384 physical compartments can be used in a single experiment to analyze a total of 10% × 96 × 96 × 96 × 96 = over 8 million single cells. In the example of Figure 3, a large number of virtual compartments (containing a set of molecules or DNA library elements with unique index combinations) are created using four combined dilution and merging steps. In this example, a continuous DNA container is created by encapsulating the contents of single cells in a polymer matrix (e.g., PAM = polyacrylamide). In a preferred specific embodiment for genomic analysis, the genomic DNA contents of single cells are amplified by MDA (WGA multiple displacement amplification reaction). This single-cell MDA product constitutes a DNA container prepared by the combined indexing scheme. For gene expression, as described by Picelli (Picelli, 2014), single-cell cDNA can be prepared from the single-cell container. In a preferred embodiment, an initial index is attached to genomic DNA or cDNA using standard library preparation techniques, either by fragmentation (enzymatic) and adaptor ligation, or by tagging of a transposase complex. In a preferred embodiment, subsequent indexes are attached to the library via ligation or PCR. Ligation is preferred because adding indexed adapters sequentially is straightforward. The final step may involve only indexing PCR or ligation and PCR.

In some implementations, the target nucleic acid is histone/protein protected (see Buenrostro et al., Nature Methods 10, 1213-1218 (2013) doi: 10.1038/nmeth.2688, incorporated herein by reference). Applications include epigenetic sequence analysis, as well as analysis of open chromatin and the location of DNA-binding proteins and nucleosomes.

In some implementations, the proximity-preserving element can contain a single cell and can amplify nucleic acids from the cell. Each proximity-preserving element can then be uniquely indexed using a combined indexing scheme. Short sequencing reads can be grouped based on unique indexes. Long synthetic reads can be assembled de novo individually based on unique indexes (McCoy et al., Plosone 2014 (DOI: 10.1371/journal.pone.0106689) Illumina TruSeq Synthetic Long-Reads Empower De Novo Assembly and Resolve Complex, Highly-Repetitive Transposable Elements, incorporated herein by reference).

In some implementations, the CE may contain cellular contents such as proteins, organelles, RNA, DNA, ribosomes, antibodies, steroids, specialized structures, glycans, lipids, small molecules, molecules that may affect biological pathways, monosaccharides and polysaccharides, alkaloids, and primary and secondary metabolites.

In some implementations, organelles within the CE can be differentially stained. Examples of organelle staining reagents are organelle-targeting fluorescent proteins (Cellular Lights ^™ ), classic organelle staining agents, or dye conjugates that can selectively or non-selectively label organelles or cellular structures.

In some implementations, the analyte of interest in the CE is a protein. Proteins can be labeled using barcodes or alternative markers. Barcodes or markers can be read using conventional array or sequence-based methods. Proximity-linking methods and antibody indexing sequences can be used in conjunction with the detection of barcode sequences to detect proteins (Fredriksson et al., Nature Biotechnology 20, 473-477 (2002), incorporated herein by reference) to establish protein identity and abundance in each individual cell. Proteins can be labeled by technicians using a variety of known methods (www.piercenet.com/cat/protein-antibody-labeling), including in vivo and in vitro site-specific chemical labeling strategies.

Proximity ligation (Duo-link PLA, www.sigmaaldrich.com/life-science/molecular-biology/molecular-biology-products.html?TablePage=l12232138, Multiplex proximity ligation detection EP 2714925 A1) is an example of a method for detecting proteins, protein-protein interactions, and post-translational modifications that can be adapted for use in proximity retention elements. This method can be used to detect and quantify specific proteins or protein complexes in proximity retention elements. An example workflow is as follows: (1) Prepare one or more proximity retention elements, (2) Wash and add one or more pairs of primary antibodies specific to the protein of interest, (3) Wash and stain with barcode-labeled antibodies. Each group of proximity retention elements in the container receives a different barcode-labeled antibody. Through proximity ligation, one or more pairs of primary antibodies, an amplifiable product, can be formed containing a unique barcode for a specific protein. One barcode can be specific to the protein of interest, while other barcodes are used to assign the protein to specific neighboring retention elements and/or cells. Individual portions can be differentially labeled through one or more separate and merge steps. In this way, the contents of a single neighboring retention element can be analyzed without processing each element separately in many parallel steps. Processing 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000 and more neighboring retention elements in this manner is a particularly significant advantage. Steroids and small molecules can be detected in a similar manner to that described for proteins. Barcode-labeled antibodies can be developed for use with steroids (Hum Reprod. 1988 Jan; 3(l): 63-8. Antibodies against steroids. Bosze P et al.). Optionally, fluorescent dyes and radioconjugates have been described (www.jenabioscience.eom/cms/en/l/catalog/2305_fluorescent_hormones.html). These antibody conjugates for steroids can be processed as described above. Various methods can be used to detect one or more components adjacent to the retained element. One or more components adjacent to the retained element can be labeled with chemiluminescence, fluorescence, radioactive probes, DNA tags, barcodes, and indexes. Amplification strategies can be used to enhance the signal. For example, rolling circle amplification (RCA) can be used to detect the analyte. The RCA product can then be detected by sequencing and a fluorescence decoder (probe). In addition, microarrays, protein arrays, sequencing, nanopore sequencing, next-generation sequencing, capillary electrophoresis, and bead arrays can be used for readout.

Establishing proximity of cell contents

In some implementations, the proximity of cells or contents derived from single cells (e.g., but not limited to DNA, RNA, proteins, organelles, metabolites, small molecules) can be preserved in proximity retention elements (CEs). CEs can be created using several methods, including, but not limited to, encapsulating the contents within droplets, embedding the contents in a polymer matrix (after encapsulation), and attaching the contents to beads. In a preferred embodiment, the CE is permeable to detection reagents such as aqueous buffers, enzymes (polymerases, ligases, transposases, etc.), nucleotides, oligonucleotide adaptors, transposons, and primers. As described above, an indexed library is created from this CE. Repeated rounds of dilution into physical compartments, attachment of compartment-specific indexes, merging, and re-dilution into additional compartments result in the exponential creation of numerous virtual compartments. If properly designed, the contents of each CE will ultimately be virtually indexed with a unique barcode. As an example in Figure 1, a four-layer indexing scheme results in a large number of virtual compartments and indexes (>84 million), with only 4 x 96 = 384 total physical compartments. In a preferred embodiment, a compartment-specific index is added to each compartmentation layer via tag fragmentation, ligation, or PCR. In a preferred embodiment, each physical compartment has a unique index at each step. Subsequent compartmentation can use the same or different indexes. If the same index is used from one compartmentation layer to the next, the position of the index within the final sequence string will identify the compartment and the compartmentation layer.

Analyzing cell components using droplets

In one embodiment, the CE can be an emulsion or droplets. In one embodiment, the CE is a droplet in contact with oil. In one example, a CE containing nucleic acids includes diluting and dispersing a nucleic acid sample into droplets, compartments, or beads. In one embodiment, the droplets include cells or single cells. In one embodiment, a CE containing single cells includes diluting and dispersing the single cells into droplets, compartments, or beads.

In some embodiments, a "droplet" can be a volume of liquid on a droplet actuator that is at least partially defined by a filler fluid. For example, a droplet can be completely surrounded by the filler fluid, or it can be defined by the filler fluid and one or more surfaces of the droplet actuator. Droplets can take on a variety of shapes; non-limiting examples include generally disk-shaped, block-shaped, truncated sphere, ellipsoidal, spherical, partially compressed sphere, hemispherical, oval, cylindrical, and various other shapes that form during droplet operation, such as merging or separating, or due to contact between such shapes and one or more surfaces of the droplet actuator.

Droplet actuators are used to perform a wide variety of droplet manipulations. A droplet actuator typically comprises two substrates spaced apart. The substrates include electrodes for performing droplet manipulations. The space is typically filled with a filler fluid immiscible with the fluid to be manipulated on the droplet actuator. The surfaces exposed to the space are typically hydrophobic. Analysis of genetic material (genomics) and its expression (functional genomics), proteomics, combinatorial library analysis, and other multiplexed bioanalytical applications can be performed on droplets, and the following manipulations can be performed on the analytical droplet actuator. Methods for manipulating droplets using droplet actuators are disclosed in U.S. Patent Application Publications 20100130369 and 20130203606, each of which is incorporated herein by reference.

A droplet actuator is a device used to manipulate droplets. For examples of droplet actuators, see Pamula et al., U.S. Patent No. 6,911,132, entitled "Apparatus for Manipulating Droplets by Electrorowetting-Based Techniques," published June 28, 2005; Pamula et al., U.S. Patent Publication No. 20060194331, entitled "Apparatus and Methods for Manipulating Droplets on a Printed Circuit Board," published August 31, 2006; Pollack et al., International Publication No. WO/2007/120241, entitled "Droplet-Based Biochemistry," published October 25, 2007; Shenderov, U.S. Patent No. 6,773,566, entitled "Electrostatic Actuators for Microfluidics and Methods for Using Same," published August 10, 2004; and Shenderov, U.S. Patent No. 6,565,727, entitled "Actuators for...""Microfluidics Without Moving Parts," published May 20, 2003; Kim et al., U.S. Patent Publication No. 20030205632, entitled "Electrowetting-driven Micropumping," published November 6, 2003; Kim et al., U.S. Patent Publication No. 20060164490, entitled "Method and Apparatus for Promoting the Complete Transfer of Liquid Drops from a Nozzle," published July 27, 2006; Kim et al., U.S. Patent Publication No. 20070023292, entitled "Small Object Moving on Printed Circuit Board," published February 1, 2007; Shah et al., U.S. Patent Publication No. 20090283407, entitled "Method for Using Magnetic Particles in Droplet.""Microfluidics", published November 19, 2009; Kim et al., U.S. Patent Publication No. 20100096266, entitled "Method and Apparatus for Real-time Feedback Control of Electrical Manipulation of Droplets on Chip", published April 22, 2010; Velev, U.S. Patent No. 7,547,380, entitled "Droplet Transportation Devices and Methods Having a Fluid Surface", published June 16, 2009; Sterling et al., U.S. Patent No. 7,163,612, entitled "Method, Apparatus and Article for Microfluidic Control via Electrowetting, for Chemical, Biochemical and Biological Assays and the Like", published January 16, 2007; Becker et al., U.S. Patent No. 7,641,779, entitled "Method and Apparatus for Programmable Fluidic U.S. Patent Publication No. 6,977,033, entitled "Method and Apparatus for Programmable Fluidic Processing," was published on January 5, 2010; Becker et al., U.S. Patent No. 6,977,033, entitled "Method and Apparatus for Programmable Fluidic Processing," was published on December 20, 2005; Decree et al., U.S. Patent No. 7,328,979, entitled "System for Manipulation of a Body of Fluid," was published on February 12, 2008; Yamakaawa et al., U.S. Patent Publication No. 20060039823, entitled "Chemical Analysis Apparatus," was published on February 23, 2006; Wu, U.S. Patent Publication No. 20110048951, entitled "Digital Microfluidics Based Apparatus for Heat-exchanging Chemical Processes," was published on March 3, 2011; Fouillet et al., U.S. Patent Publication No. 20090192044, entitled "Electrode Addressing," was published on March 3, 2011. Method, published July 30, 2009; Fouillet et al., U.S. Patent No. 7,052,244, entitled "Device for Displacement of Small Liquid Volumes Along a Micro-catenary Lineby Electrostatic Forces", published May 30, 2006; Marchand et al., U.S. Patent Publication No. 20080124252, entitled "Droplet Microreactor", published May 29, 2008; Adachi et al., U.S. Patent Publication No. 20090321262, entitled "Liquid Transfer Device", published December 31, 2009; Roux et al., U.S. Patent Publication No. 20050179746, entitled "Device for Controlling the Displacement of a Drop Between Two or Several Solid Substrates", published August 18, 2005; and Dhindsa et al., "Virtual Electrowetting Channels: Electronic Liquid Transport with Continuous Channel". Functionality, Lab Chip, 10:832–836 (2010), the entire disclosure of which is incorporated herein by reference. Some droplet actuators will include one or more substrates disposed therebetween with droplet operating gaps and electrodes associated with (e.g., layered on, attached to, and/or embedded in) the one or more substrates and arranged to perform one or more droplet operations. For example, some droplet actuators will include a base (or bottom) substrate, droplet operating electrodes associated with the substrates, one or more dielectric layers on top of the substrates and/or electrodes, and optionally one or more hydrophobic layers forming droplet operating surfaces on top of the substrates, dielectric layers, and/or electrodes. A top substrate may also be provided, which is separated from the droplet operating surface by gaps (commonly referred to as droplet operating gaps). Various electrode arrangements on the top and/or bottom substrates are discussed in the foregoing referenced patents and applications, and certain novel electrode arrangements are discussed in the description of this disclosure. During droplet operation, the droplet preferably maintains continuous or frequent contact with a ground or reference electrode. Grounding or reference electrodes can be associated with the top substrate facing the gap and the bottom substrate facing the gap. When electrodes are disposed on two substrates, electrical contacts for coupling the electrodes to the droplet actuator device for controlling or monitoring the electrodes can be associated with one or both substrates. In some cases, electrodes on one substrate are electrically coupled to the other substrate, such that only one substrate contacts the droplet actuator. In one embodiment, a conductive material (e.g., epoxy resin, such as MASTER BOND ^™ PolymerSystem EP79 available from Master Bond, Inc., Hackensack, NJ) provides electrical connections between electrodes on one substrate and electrical paths on other substrates; for example, a grounding electrode on the top substrate can be coupled to an electrical path on the bottom substrate via such a conductive material. When using multiple substrates, spacers can be provided between the substrates to determine the height of the gap and define allocated storage on the actuator. The spacer height can be, for example, at least about 5 μm, 100 μm, 200 μm, 250 μm, 275 μm, or greater. Optionally or additionally, the height of the spacer can be up to about 600 μm, 400 μm, 350 μm, 300 μm, or less. The spacer can be formed, for example, from a protruding layer of the top or bottom substrate and/or from a material inserted between the top and bottom substrates. One or more openings can be provided in one or more substrates to form fluid paths through which liquid can be delivered into the droplet-operating gap. In some cases, one or more openings can be aligned for interaction with one or more electrodes, e.g., aligned such that liquid flowing through the openings will become sufficiently close to one or more droplet-operating electrodes to allow droplet operation using the liquid by the droplet-operating electrodes. In some cases, the substrate (or bottom) and top substrate can be formed as an integral component. One or more reference electrodes can be provided on the substrate (or bottom) and/or top substrate and/or in the gap. Examples of reference electrode arrangements are provided in the foregoing reference patents and patent applications. In various embodiments, the manipulation of droplets via the droplet actuator can be electrode-mediated, such as electrowetting-mediated, dielectrophoresis-mediated, or Coulomb force-mediated. Examples of other techniques for controlling droplet manipulation that can be used in the droplet actuators of this disclosure include the use of devices that induce hydrodynamic fluid pressure, such as those based on mechanical principles (e.g., external injection pumps, pneumatic membrane pumps, vibrating membrane pumps, vacuum devices, centrifugal force, piezoelectric/ultrasonic pumps, and acoustic force); electro- or magnetic principles (e.g., electroosmosis, electric pumps, ferrofluid plugs, electrodynamic pumps, magnetic attraction or repulsion, and magnetohydrodynamic pumps); thermodynamic principles (e.g., volume expansion induced by bubble generation/phase change); other types of surface wetting principles (e.g., electrowetting, photoelectric wetting, and chemically, thermally, structurally, and radioactively induced surface tension gradients); gravity; surface tension (e.g., capillary action); electrostatic forces (e.g., electroosmosis); centrifugal flow (a substrate placed on a disc and rotating); magnetic forces (e.g., flow induced by oscillating ions); magnetohydrodynamics; and vacuum or pressure difference. In some embodiments, a combination of two or more of the foregoing techniques may be used to perform droplet operation in the droplet actuators of this disclosure. Similarly, one or more of the foregoing techniques may be used to deliver liquid into the droplet operation gap, for example from a reservoir in another device or an external reservoir of the droplet actuator (e.g., a reservoir associated with the droplet actuator substrate and a flow path from the reservoir to the droplet operation gap). The droplet operation surfaces of some droplet actuators of this disclosure may be made of hydrophobic materials, or may be coated or treated to make them hydrophobic. For example, in some cases, some or all of the droplet operation surfaces may be derivatized with low surface energy materials or chemicals, for example by deposition or in-situ synthesis, using, for example, poly or perfluorinated compounds in solution, or polymerizable monomers. Examples include (available from DuPont, Wilmington, DE), members of the cytop series of materials, coatings in the series of hydrophobic and superhydrophobic coatings (available from Cytonix Corporation, Beltsville, MD), silane coatings, fluorosilane coatings, hydrophobic phosphonate derivatives (e.g., those sold by Aculon, Inc.), and NOVEC ^™ electronic coatings (available from 3M Company, St. Paul, MN), other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxanes for PECVD (e.g., SiOC). In some cases, the droplet operating surface may include a hydrophobic coating having a thickness ranging from about 10 nm to about 1,000 nm. Furthermore, in some embodiments, the top substrate of the droplet actuator comprises a conductive organic polymer, which is then coated with a hydrophobic coating or otherwise treated to make the droplet operating surface hydrophobic. For example, the conductive organic polymer deposited on a plastic substrate may be poly(3,4-ethylenedioxythiophene)poly(styrene sulfonic acid) (PEDOT: PSS). Other examples of conductive organic polymers and alternative conductive layers are described in Pollack et al., International Patent Publication No. WO/2011/002957, entitled "Droplet Actuator Devices and Methods," published January 6, 2011, the entire disclosure of which is incorporated herein by reference. One or two substrates can be fabricated using printed circuit boards (PCBs), glass, indium tin oxide (ITO) coated glass, and/or semiconductor materials as substrates. When the substrate is ITO-coated glass, the ITO coating is preferably at least about 20 nm, 50 nm, 75 nm, 100 nm, or greater. Optionally or additionally, the thickness may be at most about 200 nm, 150 nm, 125 nm, or less. In some cases, the top and/or bottom substrates comprise PCB substrates coated with a dielectric such as a polyimide dielectric, which may in some cases also be coated or otherwise treated to make the droplet-operating surface hydrophobic. When the substrate includes a PCB, the following materials are examples of suitable materials: MITSUI ^™ BN-300 (available from MITSUI Chemicals America, Inc., San Jose, CA); ARLON ^™ 11N (available from Arlon, Inc., Santa Ana, CA); N4000-6 and N5000-30/32 (available from Park Electrochemical Corp., Melville, NY); ISOLA ^™ FR406 (available from Isola Group, Chandler, AZ), especially IS620; fluoropolymer series (suitable for fluorescence detection due to its low background fluorescence); polyimide series; polyester; polyethylene naphthalate (polyethylene naphthalate). Naphthalate; polycarbonate; polyether ether ketone; liquid crystal polymer; cyclic olefin copolymer (COC); cyclic olefin polymer (COP); aromatic polyamide; nonwoven aromatic polyamide reinforced material (available from DuPont, Wilmington, DE); branded fiber (available from DuPont, Wilmington, DE); and paper. Various materials are also suitable for use as dielectric components of substrates. Examples include: vapor-deposited dielectrics, such as PARYLENE ^™ C (particularly on glass), PARYLENE ^™ N, and PARYLENE ^™ HT (for high temperatures, around 300°C) (available from Parylene Coating Services, Inc., Katy, TX); AF coatings; CYTOP; solder masks, such as liquid photoimageable solder masks (e.g., on PCBs) like the TAIYO ^™ PSR4000 series, TAIYO ^™ PSR, and AUS series (available from TaiyoAmerica, Inc., Carson City, NV) (for good thermal properties in applications involving thermal control); and PROBIMER ^™ 8165 (for good thermal properties in applications involving thermal control) (available from Huntsman Advanced Materials Americas Inc., Los Angeles). Los Angeles, CA); dry film soldering masks, such as those in the dry film soldering mask series (available from DuPont, Wilmington, DE); film dielectrics, such as polyimide films (e.g., polyimide films available from DuPont, Wilmington, DE), polyethylene and fluoropolymers (e.g., FEP), polytetrafluoroethylene; polyesters; polyethylene naphthalate; cyclic olefin copolymers (COC); cyclic olefin polymers (COP); any other PCB substrate materials listed above; black matrix resin; polypropylene; and black flexible circuit materials, such as DuPont ^™ Pyralux HXC and DuPont ^™ Kapton. MBC (available from DuPont, Wilmington, DE). The droplet delivery voltage and frequency can be selected to suit the performance of the reagents used in a particular assay. Design parameters can be varied, for example, the number and arrangement of reservoirs on the actuator, the number of independent electrode connections, the size (volume) of different reservoirs, the arrangement of the magnet/bead washing zones, electrode dimensions, inter-electrode spacing, and gap height (between the top and bottom substrates) can be varied for specific reagents, assays, droplet volumes, etc. In some cases, the substrates of this disclosure can be derivatized with low surface energy materials or chemicals, for example, using deposition or in-situ synthesis, which utilizes poly or perfluorinated compounds in solution or polymerizable monomers. Examples include coatings and coatings for dip coating or spray coating, other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxanes (e.g., SiOC) for PECVD. Furthermore, in some cases, some or all of the droplet operating surfaces can be coated with a substance to reduce background noise, such as background fluorescence from the PCB substrate. For example, the noise-reducing coating may include a black matrix resin, for example, available from Toray. Black matrix resins from industries, Inc., Japan. The electrodes of droplet actuators are typically controlled by a controller or processor, which is provided as part of the system and may include processing functions, data and software storage, and input and output capabilities. Reagents may be provided on the droplet actuator in the droplet operating gap or in a reservoir fluidly coupled to the droplet operating gap. Reagents may be in liquid form, such as droplets, or in the droplet operating gap or in a reservoir fluidly coupled to the droplet operating gap, and they may be provided in a reconfigurable form. Reconfigurable reagents can typically be combined with a liquid for reconfiguration. Examples of reconfigurable reagents suitable for use with the methods and apparatus described herein include those described in U.S. Patent No. 7,727,466, entitled “Disintegratable Films for Diagnostic Devices,” published June 1, 2010, by Meathrel et al., the entire disclosure of which is incorporated herein by reference.

"Droplet operation" refers to any operation of a droplet on a droplet actuator. Droplet operations may include, for example: loading a droplet into a droplet actuator; dispensing one or more droplets from a source droplet; splitting, separating, or dividing a droplet into two or more droplets; transporting a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; holding a droplet in place; incubating a droplet; heating a droplet; evaporating a droplet; cooling a droplet; handling a droplet; transporting a droplet away from a droplet actuator; other droplet operations described herein; and/or any combination of the foregoing. The terms "merge," "merging," "combine," "combing," etc., are used to describe the formation of a single droplet from two or more droplets. It should be understood that when such terms are used with respect to two or more droplets, any combination of droplet operations sufficient to result in the combination of two or more droplets into a single droplet may be used. For example, “merging droplet A with droplet B” can be achieved by transporting droplet A to contact a stationary droplet B, transporting droplet B to contact a stationary droplet A, or transporting droplets A and B to contact each other. The terms “split,” “separate,” and “separate” do not imply any particular result relative to the volume of the resulting droplets (i.e., the volumes of the resulting droplets can be the same or different) or the number of resulting droplets (the number of resulting droplets can be 2, 3, 4, 5, or more). The term “mixing” refers to a droplet manipulation that results in a more uniform distribution of one or more components within the droplet. Examples of “loading” droplet manipulation include microdialysis loading, pressure-assisted loading, robotic loading, passive loading, and pipette loading. Droplet manipulation can be electrode-mediated. In some cases, droplet manipulation is further facilitated by the use of hydrophilic and/or hydrophobic regions on a surface and/or by physical barriers. For examples of droplet manipulation, see the patents and patent applications cited above under the definition of “droplet actuator.” Impedance or capacitance sensing or imaging techniques can sometimes be used to determine or confirm the results of droplet manipulation. An example of such a technique is described in U.S. Patent Publication No. 20100194408 to Sturmer et al., entitled “Capacitance Detection in a Droplet Actuator,” published August 5, 2010, the entirety of which is incorporated herein by reference. Generally, sensing or imaging techniques can be used to confirm the presence or absence of a droplet at a specific electrode. For example, the presence of a dispensed droplet at a target electrode after a droplet dispensing operation confirms that the droplet dispensing operation was effective. Similarly, the presence of a droplet at a detection point at an appropriate step in a assay confirms that a previous set of droplet operations has successfully generated a droplet for detection. Droplet transport time can be quite fast. For example, in various embodiments, transport from one electrode to the next droplet can exceed about 1 second, or about 0.1 seconds, or about 0.01 seconds, or about 0.001 seconds. In one embodiment, the electrode operates in AC mode but is switched to DC mode for imaging. Having a droplet footprint area similar to the electrowetting area is helpful for droplet manipulation; in other words, 1, 2, and 3 electrodes are typically used to effectively control 1x-, 2x-, and 3x- droplets, respectively. If the droplet footprint is larger than the number of electrodes available for droplet manipulation at a given time, the difference between the droplet size and the number of electrodes should generally not exceed 1; that is, 1 electrode effectively controls 2 droplets, and 2 electrodes effectively control 3 droplets. When the droplet includes beads, it is useful for the droplet size to equal the number of electrodes controlling the droplet, for example, in droplet transport.

In some aspects, CE (cell-mediated droplet manipulation) can be used to prepare nucleic acid libraries from cells or single cells. In some embodiments, cells can be suspended in a buffer. In some embodiments, the cell suspension can be introduced into a droplet actuator. Electrode-mediated droplet manipulation can dispense an array of droplets containing cell suspension, such that each droplet contains a single cell. Electrode-mediated droplet manipulation can dispense an array of reagent droplets containing cell lysis buffer (lysis buffer droplets). Electrode-mediated manipulation can be used to combine an array of lysis buffer droplets and an array of cell suspension droplets containing single cells to form cell lysate droplets, such that the cell lysate droplets contain components of single cells. Reaction reagents containing unique nucleic acid barcodes, transposons, and suitable enzymes (e.g., fragmentases, polymerases, ligases, transposases, reverse transcriptases, etc.) can be introduced into the droplet actuator. In some embodiments, transposons and/or barcodes can contain primer binding sites. Electrode-mediated droplet manipulation can dispense an array of reagent droplets containing reaction reagents, such that each reagent droplet contains a unique nucleic acid barcode and a suitable enzyme. Electrode-mediated manipulation can be used to combine cell lysate droplets and reagent droplets to form an array of first-barcode droplets, wherein nucleic acids from a single cell are acted upon by an enzyme from the reagent droplet, causing the nucleic acid to contain a barcode. In some embodiments, when cell lysate droplets and reagent droplets are combined and cDNA can contain a barcode, mRNA in the cell lysate droplets can be reverse transcribed. In some embodiments, the barcode can contain a primer binding site and a unique molecular index. Using electrode-mediated droplet manipulation, the first-barcode-encoded droplets can be further combined with reagent droplets multiple times to produce arrays of second-barcode droplets, third-barcode droplets, and so on. In some embodiments, the barcode is different for each round of combination. Therefore, multiple rounds of combination of barcode droplets with reagent droplets will produce combined barcode encodings. Finally, nucleic acids from different droplets can be collected and sequenced. The sequencing information can reveal the sequencing information of nucleic acids from the cell and optionally identify the origin of the nucleic acids (e.g., cells or single cells). Information is valuable if nucleic acids contain mutations that are associated with diseases such as hereditary genetic disorders or cancer.

In some aspects, the methods of this application can be applied to proteomics. Arrays of beads comprising droplets can be prepared by introducing a bead suspension into a droplet actuator to dispense an array of droplets from the bead suspension, such that each droplet in the array contains a single bead (see U.S. Application Publication 20100130369, incorporated herein by reference). The beads may contain antibodies or other affinity probes (see *Immobilized Biomolecules in Analysis*, *A Practical Approach*, Cass T, Ligler FS, eds., Oxford University Press, New York, 1998, pp. 1-14, incorporated herein by reference, for typical attachment schemes). In some embodiments, the antibody may be specific to a cell surface epitope. In some embodiments, the antibody may be a monoclonal antibody, and in other embodiments, the antibody may be polyclonal. Using electrode-mediated droplet manipulation, an array of bead-suspended droplets can be combined with an array of droplets containing single cells to produce an array of cells on the bead droplets, allowing antibodies on the beads to bind to cell surface proteins. In some embodiments, the antibodies may be specific to intracellular proteins. Using electrode-mediated droplet manipulation, an array of bead-suspended droplets can be combined with an array of droplets containing single-cell lysates, allowing antibodies on the beads to bind to intracellular proteins to produce an array of proteins on the bead droplets. Optionally, using electrode-mediated droplet manipulation, the array of proteins on the bead droplets can be combined with an array of reagent droplets containing protein labeling reagents, allowing the proteins to be uniquely labeled. The bound proteins can be detected from the associated label or by other means (SDS-polyacrylamide gel electrophoresis, ELISA, etc.). The identity and origin of the proteins can be determined. In some embodiments, proteomics data may be correlated with sequencing data.

In some embodiments, the antibody may be specific to other biomolecules, not limited to proteins. Such biomolecules may include, but are not limited to, polysaccharides or lipids. In some embodiments, the identity and origin of such biomolecules may be associated with the sequence data generated above.

In situ cell analysis

In some implementations, cells and their components can be analyzed in situ. In some implementations, cells can be passed through a flow cell.

As used herein, the term "flow cell" is intended to refer to a chamber having a surface through which one or more fluid reagents can flow. Typically, a flow cell will have an inlet and an outlet to facilitate fluid flow. Examples of flow cells and related fluid systems and detection platforms readily applicable to the methods of this disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; US 7,057,026; WO 91/06678; WO 07/123744; US7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 and US 2008/0108082, each of which is incorporated herein by reference.

In some implementations, a flow cell can house an array. Arrays used for nucleic acid sequencing typically have a random spatial pattern of nucleic acid features. For example, HiSeq ^™ or MiSeq ^™ sequencing platforms available from Illumina Inc. (San Diego, CA) utilize a flow cell to form nucleic acid arrays by random seeding followed by bridging amplification. However, patterned arrays can also be used for nucleic acid sequencing or other analytical applications. Exemplary patterned arrays, methods of their fabrication, and methods of use are described in U.S. Serial No. 13/787,396; U.S. Serial No. 13/783,043; U.S. Serial No. 13/784,368; U.S. Patent Application Publication No. 2013/0116153A1; and U.S. Patent Application Publication No. 212/0316086A1, each of which is incorporated herein by reference. Features of such patterned arrays can be used to capture individual nucleic acid template molecules for seeding followed by homogeneous colonies, such as by bridging amplification. Such patterned arrays are particularly useful for nucleic acid sequencing applications.

In some embodiments, the flow cell surface may include a capture portion, such as an antibody, to immobilize cells passing through it onto the flow cell surface. In some embodiments, the antibody on the flow cell surface may specifically bind to cell surface proteins. In some embodiments, the antibody may specifically bind to cell surface proteins of cancer cells, thereby enriching cancer cells on the flow cell surface.

In some implementations, cells can be sorted into various types using cell sorting techniques known in the art before being fed into a flow cell. Exemplary cell sorting techniques include, but are not limited to, fluorescence-activated cell sorting or FACS using flow cytometry, magnetic activated cell sorting (MACS) (Miltenyi Biotec Inc., San Diego, CA), or column-free cell separation techniques in which tubes of labeled cells are placed within a magnetic field. Positively selected cells are retained in the tubes, while negatively selected cells are in a liquid suspension (STEMCELL Technologies Inc., Vancouver, BC, Canada).

In some embodiments, cells passing through a flow cell can be lysed within the flow cell, thereby releasing the cell's nucleic acids (DNA and RNA) into the flow cell. In some embodiments, cells are immobilized on the flow cell prior to lysis. Methods of cell lysis are known in the art and include, but are not limited to, sonication, protease treatment, osmotic shock, and high-salt treatment. In some embodiments, whole RNA can be reverse transcribed. In some embodiments, unique barcodes can be introduced into nucleic acids from the cell, such as DNA, RNA, or cDNA. Methods for introducing barcodes into nucleic acids have been discussed above and include, but are not limited to, tagging and fragmentation using Nextera ^™ technology, ligases, and polymerases. In some embodiments, barcodes can be used to identify the cell origin. In some embodiments, barcodes may have primer binding sites. In some embodiments, multiple barcodes can be introduced into the nucleic acids. In some embodiments, the multiple barcodes are different from each other. In some embodiments, nucleic acids with barcodes can be diffused; re-merged, and potentially additional barcodes can be introduced. In some embodiments, nucleic acids can be fragmented after or during the introduction of barcodes. In some implementations, fragmented nucleic acids can be amplified before diffusion into the flow cell. In some implementations, fragmented nucleic acids containing barcodes can diffuse into different portions of the flow cell containing capture probes immobilized on the flow cell. In some implementations, the immobilized fragmented nucleic acids can undergo bridging amplification.

In some embodiments of the above aspects, the cells passing through the flow cell are single cells. In some embodiments, the entire transcriptome can be evaluated. In some embodiments, DNA and RNA from cells or single cells can be evaluated simultaneously for sequence information. In some embodiments, the identity or sequence information of proteins from cells or single cells can be evaluated. In some embodiments, other analytes from cells or single cells, such as lipids, carbohydrates, and cellular organelles, can be evaluated.

Fragmentation of template nucleic acids

Some embodiments for preparing template nucleic acids may include fragmentation of the target nucleic acid. In some embodiments, a barcode-encoded or indexed adaptor is attached to the fragmented target nucleic acid. The adaptor can be attached using any method well known in the art, such as ligation (enzymatic or chemical), tag fragmentation, polymerase extension, etc. In some embodiments, insertion of a transposon containing a discontinuous transposon sequence can lead to fragmentation of the target nucleic acid. In some embodiments containing a circular transposon, the target nucleic acid containing the transposon sequence can be fragmented at a fragmentation site of the transposon sequence. Other examples of methods for fragmenting the target nucleic acid used in the embodiments provided herein can be found, for example, U.S. Patent Application Publication No. 2002/0208705, U.S. Patent Application Publication No. 2012/0208724, and International Patent Application Publication WO 02/061832, each of which is incorporated herein by reference in its entirety.

Tagging of single molecules

This invention provides a method for molecular tagging, enabling the tracking and identification of individual molecules. Batch data can then be deconvolved and transformed back to individual molecules. The ability to distinguish individual molecules and return information to the original molecules is particularly important when the process from the original molecule to the final product alters the (stoichiometric) representation of the original population. For example, amplification can lead to repetitions that can be biased towards the original representation (e.g., PCR duplicates or biased amplification). This can alter methylation state calls, copy numbers, and allele ratios due to uneven amplification and/or amplification bias. By identifying individual molecules, codon-tagged differentiation distinguishes identical molecules after processing. In this way, repetitions and amplification bias can be filtered out, allowing for precise determination of the original representation of molecules or populations of molecules.

The advantage of uniquely tagging single molecules is that identical molecules in the original pool are uniquely identified due to their tagging. In further downstream analyses, these uniquely tagged molecules can now be distinguished. This technique can be developed for use in assay protocols where amplification is employed. For example, amplification is known to distort the original representation of a mixed population of molecules. Without unique tagging, the original representation (such as copy number or allele ratio) would need to account for the bias (known or unknown) of each molecule in the representation. With unique tagging, the representation can be accurately determined by removing duplicates and counting the original representation of molecules (each with a unique tag). Therefore, cDNA can be amplified and sequenced without concern for bias, as the data can be filtered to select only true sequences or sequences of interest for further analysis. Accurate reads can be constructed by agreeing on many reads with the same barcode.

In some embodiments of the compositions and methods described herein, the original population is preferably tagged at an early stage of the assay, although tagging can be performed at a later stage if the earlier steps do not cause bias or are insignificant. In any of these applications, the complexity of the barcode sequence should be greater than the number of individual molecules to be tagged. This ensures that different target molecules receive different and unique tags. Therefore, a random oligonucleotide library of a certain length (e.g., 5, 10, 20, 30, 40, 50, 100, or 200 nucleotides) is desirable. The random pool of tags represents a high complexity of tags with a code space of ⁴ⁿ , where n is the number of nucleotides. Additional codes (whether designed or randomized) can be incorporated at different stages for use as further checks, such as parity checks for error correction.

In one embodiment of the compositions and methods described herein, a single molecule (e.g., target DNA) is attached to a unique marker, such as a unique oligomeric sequence and/or barcode. Attachment of the marker can be performed by ligation, coupling chemistry, adsorption, insertion of transposon sequences, etc. Other methods include amplification (e.g., via PCR, RCA, or LCR), replication (e.g., via the addition of polymerase), and non-covalent interactions.

Specific methods involve incorporating barcodes (e.g., designed or random sequences) into PCR primers, such that each template receives a separate codon within the codon space, thereby producing unique amplicons that can be distinguished from other amplicons. This concept can be applied to any method using polymerase amplification, such as the GoldenGate ^™ assay and the assays disclosed in U.S. Patent Nos. 7,582,420, 7,955,794, and 8,003,354, each of which is incorporated herein by reference in its entirety. The codon-tagged target sequence can be circularized and amplified, for example, using rolling circle amplification, to produce codon-tagged amplicons. Similarly, codons can also be added to RNA.

Methods for analyzing template nucleic acids

Some embodiments of the technology described herein include methods for analyzing template nucleic acids. In such embodiments, sequencing information can be obtained from the template nucleic acid, and this information can be used to generate sequence representations of one or more target nucleic acids.

In some embodiments of the sequencing methods described herein, a linked read strategy may be used. A linked read strategy may include identifying sequencing data that are linked from at least two sequencing reads. For example, a first sequencing read may include a first marker, and a second sequencing read may include a second marker. The first and second markers may identify sequencing data from each adjacent sequencing read in the sequence representation of the target nucleic acid. In some embodiments of the compositions and methods described herein, the marker may include a first barcode sequence and a second barcode sequence, wherein the first barcode sequence may be paired with the second barcode sequence. In other embodiments, the marker may include a first host tag and a second host tag. In further embodiments, the marker may include a first barcode sequence having a first host tag and a second barcode sequence having a second host tag.

An exemplary implementation of a method for sequencing template nucleic acids may include the steps of: (a) sequencing a first barcode sequence using sequencing primers hybridized to a first primer site; and (b) sequencing a second barcode sequence using sequencing primers hybridized to a second primer site. The result is two sequence reads that facilitate the linking of the template nucleic acid to its genomic neighbors. Given sufficiently long reads and sufficiently short library fragments, these two reads can be informationally merged to prepare a single long read covering the entire fragment. Using the barcode sequence reads and 9-nucleotide repeat sequences present in the insert, the reads can now be linked to their genomic neighbors to form a much longer “linked read” in a computer.

As will be understood, a library containing template nucleic acids may include repetitive nucleic acid fragments. Sequencing repetitive nucleic acid fragments is advantageous in methods that include creating shared sequences for the repetitive fragments. Such methods can improve the accuracy of providing shared sequences to template nucleic acids and/or libraries of template nucleic acids.

In some embodiments of the sequencing technology described herein, real-time sequence analysis is performed. For example, real-time sequencing can be performed by simultaneously acquiring and analyzing sequencing data. In some embodiments, the sequencing method for acquiring sequencing data can terminate at various points, including after acquiring at least a portion of the target nucleic acid sequence data or before the entire nucleic acid read is sequenced. Exemplary methods, systems, and further embodiments are provided in International Patent Publication No. WO 2010/062913, the disclosure of which is incorporated herein by reference in its entirety.

In an exemplary embodiment of a method for assembling short sequencing reads using a ligation-based read strategy, transposon sequences containing barcodes are inserted into genomic DNA, a library is prepared, and sequencing data for the library of template nucleic acids is obtained. Template blocks can be assembled by identifying paired barcodes, and then larger contigs can be assembled. In one embodiment, the assembled reads can be further assembled into larger contigs by using codon pairings of overlapping reads.

Some implementations of the sequencing technologies described herein include error detection and correction features. Examples of errors may include errors in base calls during the sequencing process, as well as errors in assembling fragments into larger contigs. As will be understood, error detection may include detecting the presence or probability of errors in the dataset, and thus may not require detecting the location or number of errors. For error correction, information about the location and/or number of errors in the dataset is useful. Methods for error correction are well known in the art. Examples include the use of Hamming distance and the use of checksum algorithms (see, for example, U.S. Patent Application Publication No. 2010/0323348; U.S. Patent No. 7,574,305; and U.S. Patent No. 6,654,696, the disclosures of which are incorporated herein by reference in their entirety).

Nested libraries

Another approach involves combining the junction tagging method described above with the preparation of nested sequencing libraries. Nested sublibraries are created from codon-tagged DNA fragments. This allows for less frequent transposon tagging across the genome. It also creates greater diversity in (nested) sequencing reads. These factors can improve coverage and accuracy.

Secondary sampling and whole-genome amplification can create numerous copies of certain populations of the starting molecule. DNA fragments are then generated through transposon-specific fragmentation, where each fragment receives a codon that allows the fragment to be linked back to its original neighbor with a matching codon (whether identical, complementary, or otherwise informationally linked). The tagged fragments are fragmented at least a second time by random or sequence-specific methods (e.g., enzyme digestion, random scissing, transposon-based scissing, or other methods), resulting in a sub-library of codon-tagged DNA fragments. In a useful variation of the aforementioned methods, codon-tagged fragments can be preferentially isolated by using transposons containing biotin or other affinity functionalities for downstream enrichment purposes. Subsequent library preparation converts the nested DNA fragments into sequencing templates. Paired-end sequencing leads to the determination of the sequences of the codon-tags of the DNA fragments and the target DNA. Because nested libraries for the same codon-tags are created, long DNA fragments can be sequenced using short reads.

sequencing methods

The methods and compositions described herein can be used in conjunction with various sequencing technologies. In some embodiments, the method for determining the nucleotide sequence of the target nucleic acid can be automated.

Some implementations of the sequencing methods described herein include sequencing via synthesis (SBS) techniques, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate ( _PPi ) as specific nucleotides are incorporated into the nascent strand (Ronaghi et al., Analytical Biochemistry 242(1): 84-9 (1996); Ronaghi, M. Genome Res. 11(1): 3-11 (2001); Ronaghi et al., Science 281(5375): 363 (1998); U.S. Patent No. 6,210,891; U.S. Patent No. 6,258,568 and U.S. Patent No. 6,274,320, each of which is incorporated herein by reference in its entirety).

In another exemplary type of SBS, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, cleavable or photobleachable dye labels, as described in U.S. Patent Nos. 7,427,67, 7,414,163, and 7,057,026, each of which is incorporated herein by reference in its entirety. This method, commercialized by Illumina Inc., is also described in International Patent Application Publications WO 91/06678 and WO 07/123744, each of which is incorporated herein by reference in its entirety. The availability of fluorescently labeled terminators (where termination is reversible and the fluorescent label can be cleaved) facilitates efficient cycle reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

Other exemplary SBS systems and methods that can be used with the methods and compositions described herein are described in U.S. Patent Application Publication No. 2007/0166705, U.S. Patent Application Publication No. 2006/0188901, U.S. Patent No. 7057026, U.S. Patent Application Publication No. 2006/0240439, U.S. Patent Application Publication No. 2006/0281109, PCT Publication No. WO 05/065814, U.S. Patent Application Publication No. 2005/0100900, PCT Publication No. WO 06/064199 and PCT Publication No. WO 07/010251, each of which is incorporated herein by reference in its entirety.

Some embodiments of the sequencing technologies described herein can utilize sequencing via ligation techniques. Such techniques utilize DNA ligases to incorporate nucleotides and identify the incorporation of such nucleotides. Exemplary SBS systems and methods that can be used with the methods and compositions described herein are described in U.S. Patent Nos. 6,969,488, 6,172,218, and 6,306,597, each of which is incorporated herein by reference in its entirety.

The sequencing methods described herein can advantageously be performed in a variety of forms, enabling the simultaneous manipulation of multiple different target nucleic acids. In specific embodiments, the different target nucleic acids can be processed in common reaction vessels or on the surface of a specific substrate. This allows for convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of incorporation events in multiple ways. In embodiments using surface-bound target nucleic acids, the target nucleic acids can be in the form of an array. In array form, the target nucleic acids can typically be coupled to the surface in a spatially distinguishable manner. For example, the target nucleic acids can be bound by direct covalent attachment, attachment to beads or other particles, or association with polymerases or other molecules attached to the surface. The array can include a single copy (also called a feature) of the target nucleic acid at each site, or multiple copies with the same sequence can be present at each site or feature. Multiple copies can be generated by amplification methods such as bridging amplification or emulsion PCR, as described further in detail herein.

The method proposed in this paper can use arrays with features at any of various densities, including, for example, at least about 10 features/ ^cm² , 100 features/ ^cm² , 500 features/ ^cm² , 1,000 features/ ^cm² , 5,000 features/ ^cm² , 10,000 features/ ^cm² , 100,000 features/ ^cm² , 100,000 features/ ^cm² , 5,000,000 features/ ^cm² , ^10⁷ features/ ^cm² , 5 × ^10⁷ features/ ^cm² , ^10⁸ features/ ^cm² , 5 × ^10⁸ features/ ^cm² , ^10⁹ features/ ^cm² , 5 × ^10⁹ features/ ^cm² , or higher.

Methods to reduce error rate in sequencing data

Some embodiments of the methods and compositions provided herein include reducing error rates in sequencing data. In some such embodiments, the sense and antisense strands of a double-stranded target nucleic acid are each associated with a different barcode. Each strand is amplified, sequence information is obtained from multiple copies of the amplified strand, and a common sequence representation of the target nucleic acid is generated from the redundant sequence information. Thus, sequence information can be derived from and identified from each strand. Therefore, sequence errors can be identified and reduced when sequence information derived from one strand is inconsistent with sequence information from another strand.

In some embodiments, the sense and antisense strands of the target nucleic acid are associated with different barcodes. Barcodes can be associated with the target nucleic acid by various methods, including the ligation of adaptors and the insertion of transposon sequences. In some such embodiments, a Y-adaptor can be ligated to at least one end of the target nucleic acid. The Y-adaptor can include a double-stranded sequence and a non-complementary strand, each strand containing a different barcode. The target nucleic acid with the ligated Y-adaptor can be amplified and sequenced, such that each barcode can be used to identify the original sense or antisense strand. A similar method is described in Kinde I et al. (2011) PNAS 108:9530-9535, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the sense and antisense strands of the target nucleic acid are associated with different barcodes by inserting transposon sequences provided herein. In some such embodiments, the transposon sequences can contain non-complementary barcodes.

Some implementations of such methods include obtaining sequence information from the strand of a target double-stranded nucleic acid, comprising (a) obtaining sequence data from a template nucleic acid containing a first sequencing adaptor and a second sequencing adaptor (having at least a portion of the double-stranded target nucleic acid disposed therebetween), wherein: (i) the first sequencing adaptor contains a double-stranded first barcode, a single-stranded first primer site, and a single-stranded second primer site, wherein the first and second primer sites are non-complementary; and (ii) the second sequencing adaptor contains a double-stranded second barcode, a single-stranded third primer site, and a single-stranded fourth primer site, wherein the third and fourth primer sites are non-complementary. In some implementations, the first primer site of the sense strand of the template nucleic acid and the third primer site of the antisense strand of the template nucleic acid contain the same sequence. In some implementations, each barcode is different. In some implementations, the first sequencing adaptor contains a single-stranded hairpin conjugating the first and second primer sites.

In another implementation, each end of the target nucleic acid is associated with an adaptor containing a different barcode, allowing extensions of the sense and antisense strands of the nucleic acid to be distinguishable from each other. In some implementations, the primer site sequence and barcode are selected such that extensions of the primer annealed to the sense strand produce products distinguishable from extensions of the primer annealed to the antisense strand. In an example, the 3' sense primer site is the same as the 3' antisense primer site, but different from both the 5' sense and 5' antisense primer sites. Extensions of the primers annealed to the 3' sense primer site and the 3' antisense primer site will produce the following products from each strand:

Meaningful chain: (5') barcode2-[target sequence]-barcode1(3')

Antonym chain: (5') barcode 1 - [target sequence] - barcode 2 (3')

Therefore, the extensions of the sense and antisense strands of a nucleic acid can be distinguished from each other. An exemplary method is illustrated in Schmitt M.W., et al., PNAS (2012) 109:14508-13, the disclosure of which is incorporated herein by reference in its entirety. In some such methods, barcodes and primer sites can be associated with the target nucleic acid through a variety of methods, including the ligation of an adaptor and the insertion of a transposon sequence. In some embodiments, the transposon sequence can be engineered to provide an adaptor with a hairpin. The hairpin provides the ability to maintain the physical proximity of the sense and antisense strands of the target nucleic acid. A template nucleic acid containing a hairpin can be prepared using a transposon sequence containing the adapter described herein. Examples of adapters include single-stranded nucleic acids.

Some embodiments for preparing a library of template nucleic acids for obtaining sequence information from each strand of a double-stranded target nucleic acid include (a) providing a group of transposons comprising a transposon and a first transposon sequence comprising: (i) a first transposon recognition site, a first primer site, and a first barcode; and (ii) a second transposon sequence comprising a second transposon recognition site, a second primer site, and a second barcode, wherein the first transposon sequence and the second transposon sequence are discontinuous; and (b) contacting the transposon with the double-stranded nucleic acid under conditions such that the first and second transposon sequences are inserted into the double-stranded target nucleic acid, thereby preparing a library of template nucleic acids for obtaining sequence information from each strand of the double-stranded target nucleic acid. In some embodiments, the transposon population further comprises a transposon containing a transposon enzyme and a transposon sequence, the transposon sequence containing a third transposon recognition site and a fourth transposon recognition site, having a barcode sequence disposed therebetween, the barcode sequence including a third barcode and a fourth barcode, having a sequencing adaptor disposed therebetween, the sequencing adaptor containing a third primer site and a fourth primer site, having an adapter disposed therebetween. In some embodiments, the first primer site of the sense strand of the template nucleic acid and the third primer site of the antisense strand of the template nucleic acid contain the same sequence. Some embodiments further include step (c) selecting the template nucleic acid containing the transposon sequence, wherein the first transposon sequence is discontinuous with the second transposon sequence containing the adapter. In some embodiments, the adapter contains an affinity tag adapted to bind to a capture probe. In some embodiments, the affinity tag is selected from the group consisting of His, biotin, and streptoavidin proteins. In some embodiments, each barcode is different. In some embodiments, the adapter comprises a single-stranded nucleic acid. In some embodiments, the target nucleic acid comprises genomic DNA.

Methods for obtaining haplotype information

Some embodiments of the methods and compositions provided herein include methods for obtaining haplotype information from a target nucleic acid. Haplotype information may include determining the presence or absence of distinct sequences at specific loci in the target nucleic acid (e.g., the genome). For example, sequence information for maternal and paternal copies of alleles may be obtained. In polyploid organisms, sequence information for at least one haplotype may be obtained. Such methods can also be used to reduce the error rate in obtaining sequence information from a target nucleic acid.

Typically, methods for obtaining haplotype information involve distributing nucleic acids into one or more compartments, such that each compartment contains an amount of nucleic acid equivalent to approximately one haplotype of the target nucleic acid, or an amount of nucleic acid equivalent to less than approximately one haplotype of the target nucleic acid. Sequence information can then be obtained from each compartment, thus obtaining haplotype information. Distributing the template nucleic acid into multiple containers increases the likelihood that a single container contains a single copy of the allele or SNP, or that the shared sequence information obtained from a single container reflects the sequence information of the allele or SNP. As will be understood, in some such embodiments, the template nucleic acid may be diluted before being divided into multiple containers. For example, each container may contain an amount of target nucleic acid equal to approximately one haplotype equivalent of the target nucleic acid. In some embodiments, the container may contain approximately one haplotype equivalent less than the target nucleic acid.

Methods for determining haplotype information, methods for haplotype analysis using virtual compartments, and methods for preparing target nucleic acids for haplotype analysis are described in WIPO Publication WO/2014/142850, which is incorporated herein by reference.

Example

Example 1: Maintaining Template Proximity

This embodiment illustrates a method for maintaining proximity information of template nucleic acids within a CE (Cellular Enzyme-Coefficient). The template nucleic acid is prepared using a transposon containing discontinuous transposon sequences, wherein a Tn5 transposase remains bound to the transposed template DNA. The target nucleic acid is contacted with a transposon containing the Tn5 transposase and the discontinuous transposon sequences. Samples further treated with SDS may appear as smears of various fragments of the template nucleic acid; samples not treated with SDS may show retention of the presumed high molecular weight template nucleic acid. Therefore, even if the nucleic acid can be fragmented, adjacent sequences can still be correlated with each other via the transposase.

In yet another exemplary method, a template nucleic acid library is prepared using a transposon containing discontinuous transposon sequences, with the target nucleic acid comprising the human chromosome. The CE contains the target nucleic acid. For samples in which the transposase has been removed by SDS after dilution, haplotype blocking of the DNA can be observed. Therefore, by implementing the methods described herein, the target nucleic acid can maintain its target integrity during transposition, dilution, and conversion into a sequencing library.

As used herein, the term “comprising” is a synonym for “including,” “containing,” or “characterized by,” and is inclusive or open-ended, and does not exclude additional unlisted elements or methodological steps.

All figures used in the specification to indicate the amount of components, reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters described herein are approximate values that may vary depending on the desired properties sought to be obtained. At least, as is not an attempt to limit the application of the doctrine of equivalence to the scope of any claim in any application asserting priority to this application, each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods.

The foregoing specification discloses several methods and materials of the present invention. The present invention is readily modifiable in its methods and materials, and readily adaptable in its manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art upon consideration of this disclosure or practice of the invention as disclosed herein. Therefore, the present invention is not intended to be limited to the specific embodiments disclosed herein, but rather encompasses all modifications and alternatives that fall within the true scope and spirit of the invention.

All references cited herein, including but not limited to published and unpublished applications, patents, and references, are incorporated herein in their entirety by reference and are thus part of this specification. Where a publication or patent or patent application incorporated by reference contradicts the disclosure contained in this specification, this specification is intended to supersede and/or give precedence to any such contradictory material.

Example 2: Single-cell whole transcriptome sequencing

This embodiment describes a method for uniformly barcoding cDNA along its entire length and using the barcodes to determine cDNA proximity information and identify cell origin, i.e., identifying single cells associated with mRNA.

This embodiment illustrates a method for sequencing single-cell transcriptomes. In this embodiment, droplet microfluidics are used to capture the transcriptomes of multiple single cells on a single capture bead, and then cDNA derived from the transcriptome of each single cell is barcoded using proximity-preserving transposation and combinatorial indexing (CPT-seq). In one embodiment, the method of the present invention uses multiple barcoding methods to index single-cell cDNA, wherein a first barcode is added in the tag fragmentation reaction and a second barcode is added in the PCR amplification reaction.

In one instance, poly-A+ RNA is captured from a single cell, and the captured poly-A+ RNA is processed in batches to generate multiplex sequencing libraries.

This method may include the following steps. In step 1, RNA from a single cell is captured on a capture bead. For example, multiple single cells (e.g., approximately 1000 single cells) are encapsulated in a single droplet containing a lysis buffer and a capture bead (i.e., one cell and one bead per droplet on average). Multiple capture probes, including poly-dT capture sequences and PCR primer sequences, are immobilized on the surface of the capture bead. The lysis buffer composition of the droplet dissociates the single cell cytoplasmic membrane, releasing cytoplasmic RNA. The released poly-A+ RNA is captured by hybridizing a poly-A+ sequence on the RNA to an oligo-dT capture sequence immobilized on the surface of the co-encapsulated capture bead. Each capture bead now contains poly-A+ RNA from the transcriptome of the single cell. All poly-A+ RNA from the single cell remains close to each other on the capture bead.

In step 2, capture beads bearing single-cell poly-A+ RNA are merged from multiple droplets (e.g., approximately 1000 capture beads) and double-stranded cDNA is synthesized. For example, the capture beads are merged, washed, and first-stranded cDNA is synthesized using an RNase H subtracted from a reverse transcriptase capable of strand switching. A strand switching primer is included during first-stranded cDNA synthesis, allowing the placement of a universal primer site at the 3' end of the cDNA. Double-stranded cDNA is then prepared using universal primers and a high-fidelity DNA polymerase in a PCR reaction (e.g., 1 to 2 cycles of PCR). Each capture bead now contains cDNA reverse transcribed from single-cell poly-A+ RNA.

In step 3, capture beads with double-stranded cDNA are distributed in the wells of a 96-well plate, so that there are about 10 capture beads per well.

In step 4, the double-stranded cDNA in each well is tagged and fragmented using 96 uniquely indexed transposons. Tagging and fragmentation is used to modify the cDNA with adaptors and index sequences while maintaining single-cell contiguity. The assembly of the 96 uniquely indexed transposon composition used in the tagging and fragmentation reaction is described in more detail below. The tagging and fragmentation reaction adds the first part of a bi-barcode to each future cDNA fragment. Each capture bead now contains tagged and fragmented cDNA from a single cell.

In step 5, the capture beads from all wells are collected, combined, washed, and redistributed into the wells of another 96-well plate, so that there are approximately 10 capture beads per well. The mRNA/cDNA from the single cell remains on the surface of the individual beads, and the transposase still binds to the fragmented cDNA and keeps the fragment from dissociation.

In step 6, transposase and tagged fragmented cDNA are released from the capture beads. For example, an aliquot of SDS (1% SDS) solution is added to each well to release the bound transposase and tagged fragmented cDNA from the capture beads.

In step 7, the tagged fragmented cDNA in each well is amplified using PCR primers that include a P5 or P7 sequence and a unique barcode sequence. For example, one of 96 unique combinations of barcode-encoded P5 and P7 PCR primers is added to each well, and the tagged fragmented cDNA fragment is amplified. The PCR reaction adds the remaining portion of the bi-barcode to each cDNA fragment. Each cDNA fragment now includes four barcode sequences: two sequences added during the tagging and fragmentation reaction and two sequences added during PCR amplification. Thus, mRNA/cDNA from a single cell is identified by a combination of the tagging and fragmentation index and the PCR index added during the amplification step.

In step 8, the bar-coded cDNA fragments from each well are merged and sequenced.

In this embodiment, 96×96 combined indexing is used to barcode approximately 1000 single cells, with an approximately 5% probability of two cells having the same barcode. Throughput can be easily increased by increasing the number of "compartments." For example, by using 384×384 combined barcode encoding (approximately 147,456 virtual compartments), approximately 10,000 single cells are barcode-encoded in parallel, with an approximately 3% probability of two cells having the same barcode.

This embodiment also describes a process for assembling 96 unique barcode transposable complexes to add the first part of a binary barcode to a combined barcode encoding scheme. This process includes, but is not limited to, the following steps.

In step A, 20 uniquely indexed transposons are formed by annealing the respective indexed oligonucleotides (each containing a Tn5 mosaic terminus (ME) at its 3' end) to universal 5' phosphorylated ME complementary oligonucleotides (pMENTS). For example, indexed oligonucleotide 1110, which includes a P5 sequence, a unique 8-base "i5" index sequence, a universal connector sequence Universalconnector A-C15, and the ME sequence, is annealed to the ME complementary sequence 1115. The ME complementary sequence 1115 is a universal 5' phosphorylated oligonucleotide (pMENTS) that is complementary to the ME sequence in indexed oligonucleotide 1110. Custom index 2 sequencing primers are then annealed using the universal connector sequence A-C15.

A second set of annealing reactions (i.e., 12 separate annealing reactions) is performed to form a second set of 12 transposons, each containing a unique 8-base "i7" index sequence adjacent to the P7 sequence. For example, the indexed oligonucleotide 1120, which includes the P7 sequence, the unique 8-base i7 index sequence, the universal linker sequence B-D15, and the ME sequence, is annealed to the ME complementary sequence 1115. The universal linker sequence B-D15 is then used to anneal the custom index 1 sequencing primers.

In step B, annealed P5_i5 transposons 1125 (i.e., eight P5_i5 transposons 1125, each with a unique 8-base i5 index sequence) and annealed P7_i7 transposons 1130 (i.e., twelve transposons 1130, each with a unique 8-base i7 index sequence) are assembled using Tn5 transposase in separate reactions to form transposome complexes. For example, each annealed P5_i5 transposon 1125 is incubated with Tn5 transposase 1135 at approximately 37°C for approximately 1 hour to form P5_i5 transposome complex 1140. Similarly, each annealed P7_i7 transposon 1130 is incubated with Tn5 transposase 1135 at approximately 37°C for approximately 1 hour to form P7_i7 transposome complex 1145.

In step C, 96 unique transposon complexes are prepared by combining aliquots of P5_i5 transposon complex 1140 with aliquots of P7_7 transposon complex 1145. For example, P5_i5 transposon complex 1140 is aliquoted into rows A through H of a 96-well plate, and P7_i7 transposon complex 1145 is aliquoted into columns 1 through 12 of the same 96-well plate. Combinations of 8 P5_i5 transposon complexes 1140 and 12 P7_i7 transposon complexes 1145 produce 96 different index combinations.

To evaluate the assembled transposon complex, sequencing libraries from 10 single cells were prepared using a single-segment tagging and fragmentation reaction and a single-segment PCR reaction. Ten capture beads containing cDNA from each of the 10 single cells were combined and tagged using a P5_i5_1 + P7_i7_1 transposon mixture. The tagged cDNA was then released from the capture beads and amplified by PCR using barcode-encoded P5 and P7 primers to generate the sequencing library. The fragment size distribution in the sequencing library was then analyzed using a bioanalyzer. In some embodiments, cleanup is performed after PCR. In some embodiments, a second SPRI cleanup is performed after the first SPRI cleanup. In some embodiments, the samples are diluted 10-fold before analysis in the bioanalyzer.

In another example, two different transposon complex mixtures were used to prepare sequencing libraries from 100 single cells. In this example, a separation and merging experimental design was used to evaluate the transposon complexes. 100 capture beads containing cDNA from 100 single cells were distributed to two tag fragmentation reactions: a first tag fragmentation reaction using a P5_i5_2 + P7_i7_2 transposon mixture, and a second tag fragmentation reaction using a P5_i5_3 + P7_i7_3 transposon mixture. Following the tag fragmentation reactions, the capture beads from each reaction were merged and redistributed for PCR amplification using two unique combinations of barcode-encoded P5 and P7 PCR primers (i.e., a first combination of P5 and P7 PCR primers and a second combination of P5 and P7 PCR primers) to produce two sequencing libraries. The fragment size distribution in each sequencing library was then analyzed using a bioanalyzer. The barcode-encoded libraries were analyzed after a single 0.7x SPRI cleanup step.

Claims (29)

1. A method for analyzing one or more analytes of a plurality of adjacent retention elements (CEs), the method comprising: (a) Provide a plurality of CEs, wherein the CEs are physical entities that are in close proximity to the retained analyte; (b) Dividing the CE into a plurality of first compartments, wherein each compartment contains a plurality of CEs; (c) Provide a set of first report portions to the analyte of each CE in each of the plurality of first compartments, wherein each first report portion in the set of first report portions provided to the analyte of each of the first compartments is different from the first report portions provided to the analyte of each of the other first compartments; (d) Combining the CEs that include the first report portion; (e) The CE containing the first report portion is divided into a plurality of second compartments; (f) Providing a set of second report portions to the analyte of each CE in each of the plurality of second compartments, wherein each second report portion in the set of second report portions provided to the analyte of each of the second compartments is different from the second report portions provided to the analyte of each of the other second compartments; (g) The CE combination comprising the first and second report portions; (h) Analyze the analytes comprising the report portion of each compartment. In this type of analysis, the detection is performed on single cells from which each analyte originates. 2. The method of claim 1, wherein the CE is a cell, an embedded cell, or a retained cell. 3. The method of claim 1, wherein each compartment in step (e) comprises a plurality of CEs. 4. The method of claim 1, wherein the analyte comprises nucleic acid, and the first reporter portion is introduced via tag fragmentation, ligation, or PCR. 5. The method of claim 1, wherein the analyte comprises nucleic acid, and the first reporter portion is introduced by transcribing RNA into cDNA. 6. The method of claim 1, wherein the analyte comprises nucleic acid, and the second reporter portion is introduced via tag fragmentation, ligation, or PCR. 7. The method of claim 1, wherein the first reporter portion comprises a nucleic acid barcode. 8. The method of claim 1, wherein the second reporter portion comprises a nucleic acid barcode. 9. The method of any one of claims 1-8, wherein prior to step (h), the method further comprises: splitting the CE containing the first and second report portions into a plurality of third compartments; providing a set of third report portions to the analyte of each CE in each of the plurality of third compartments, wherein each third report portion in the set of third report portions provided to the analyte of each third compartment is different from the third report portions provided to the analyte of each of the other third compartments; and combining the CE containing the first, second, and third report portions. 10. The method of claim 9, wherein the analyte comprises nucleic acid, and the third reporter portion is introduced via tag fragmentation, ligation, or PCR. 11. The method of claim 9, wherein the third reporter portion comprises a nucleic acid barcode. 12. The method of any one of claims 1-8, wherein steps (b)-(g) are repeated with one or more sets of additional report portions. 13. The method of claim 1, wherein the analyte comprises DNA, RNA or cDNA. 14. The method of claim 13, wherein the DNA comprises genomic DNA. 15. The method of any one of claims 1-8, 13 or 14, wherein the first reporter portion is used to identify the sample origin of each single cell. 16. The method of any one of claims 1-8, 13 or 14, wherein the detection of analytes from the same CE is performed simultaneously. 17. A composition comprising a plurality of compartments, each compartment containing a plurality of CEs, Each CE contains an analyte, which includes a first reporter portion and a second reporter portion. Essentially all CE analytes contain the first report portion and the second report portion in a unique combination, unique location, or combination thereof. 18. The composition of claim 17, wherein each CE comprises the first reporter portion and the second reporter portion in a unique combination, a unique location, or a combination thereof. 19. The composition of claim 17, wherein the compartment comprises a three-dimensional solid support. 20. The composition of claim 19, wherein the three-dimensional solid support comprises a porous plate. 21. The composition of claim 17, wherein the CE is a cell, an embedded cell, or a retained cell. 22. The composition of any one of claims 17-21, wherein the analyte comprises nucleic acid. 23. The composition of claim 22, wherein the analyte comprises DNA, RNA or cDNA. 24. The composition of claim 23, wherein the DNA comprises genomic DNA. 25. The composition of any one of claims 17-21, wherein the first reporter portion comprises a nucleic acid barcode. 26. The composition of any one of claims 17-21, wherein the second reporter portion comprises a nucleic acid barcode. 27. The composition of any one of claims 17-21, wherein the plurality of CEs comprises at least 100,000 CEs. 28. The composition of any one of claims 17-21, wherein the plurality of CEs comprises at least 1,000,000 CEs. 29. The composition of any one of claims 17-21, wherein the CE comprises an additional set of reporter portions. Each CE's analyte comprises, in a unique combination, in a unique location, or in a combination thereof, the first report portion, the second report portion, and an additional set of the report portions.