Spatially addressable molecular barcoding

• US 10,002,316 B2
• Filed: 08/02/2017
• Issued: 06/19/2018
• Est. Priority Date: 02/27/2015
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fodor, Stephen P. A., Fan, Christina, Fu, Glenn, Facer, Geoffrey
• Primary Examiner(s): Haupt, Kristy A
• Application Number:
  US15/667,125
• Publication Number:
  US 20170337459A1
• Time in Patent Office: 321 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2543/10 the purpose being "in situ" analysis
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G06K 19/06103 the marking being embedded in a human recognizable image, e.g. a company logo with an embedded two-dimensional code

Abstract

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

Claims

1. A method for determining the numbers and spatial locations of a plurality of targets in a plurality of spatially ordered cells of a sample, comprising:

associating cells of the plurality of spatially ordered cells at two or more spatial locations with a plurality of stochastic barcodes,

wherein each stochastic barcode of the plurality of stochastic barcodes comprises a spatial label and a molecular label, and

wherein the plurality of stochastic barcodes comprises two or more different spatial labels for identifying the two or more spatial locations of the cells;

stochastically barcoding the plurality of targets in the cells of the plurality of spatially ordered cells using the plurality of stochastic barcodes comprising the two or more different spatial labels for identifying the two or more spatial locations of the cells;

estimating the numbers of the plurality of targets in the cells of the plurality of spatially ordered cells at the two or more spatial locations using the molecular labels of the plurality of stochastic barcodes; and

identifying the spatial locations of the plurality of targets in the cells of the plurality of spatially ordered cells at the two or more spatial locations using the two or more different spatial labels of the plurality of stochastic barcodes.

2. The method of claim 1, wherein stochastically barcoding the plurality of targets comprises hybridizing the plurality of stochastic barcodes with the plurality of targets to generate stochastically barcoded targets, and at least one of the plurality of targets is hybridized to one of the plurality of stochastic barcodes.

3. The method of claim 2, wherein the molecular labels of different stochastic barcodes are different from one another.

4. The method of claim 1, wherein the sample is physically divided or is intact during stochastically barcoding the plurality of targets.

5. The method of claim 4, wherein the spatial locations of the plurality of targets are on a surface of the sample, inside the sample, subcellularly in the sample, or any combination thereof.

6. The method of claim 1, wherein estimating the numbers of the plurality of targets using the molecular labels comprises determining sequences of the molecular labels of the plurality of the stochastic labels and counting the number of the molecular labels with distinct sequences associated with each of the plurality of targets at the two or more spatial locations of the cells.

7. The method of claim 6, wherein determining the sequences of the molecular labels of the plurality of the stochastic barcodes comprises sequencing some or all of the plurality of stochastic barcodes.

8. The method of claim 1, wherein identifying the spatial locations of the plurality of targets comprises correlating the spatial labels of the plurality of the stochastic barcodes with the spatial locations of the plurality of targets in the sample.

9. The method of claim 1, further comprising visualizing the plurality of targets in the sample.

10. The method of claim 9, wherein visualizing the plurality of targets in the sample comprises mapping the plurality of targets onto a map of the sample.

11. The method of claim 1, wherein the plurality of targets comprise ribonucleic acids (RNAs), messenger RNAs (mRNAs), microRNAs, small interfering RNAs (siRNAs), RNA degradation products, RNAs each comprising a poly(A) tail, and any combination thereof.

12. The method of claim 1, wherein stochastically barcoding the plurality of targets is performed with a solid support comprising the plurality of stochastic barcodes.

13. The method of claim 12, wherein the solid support comprises a plurality of synthetic particles associated with the plurality of stochastic barcodes.

14. The method of claim 13, wherein two stochastic barcodes associated with a synthetic particle of the plurality of synthetic particles comprise an identical spatial label, and wherein two stochastic barcodes associated with two synthetic particles of the plurality of synthetic particles comprise the two or more different spatial labels.

15. The method of claim 13, wherein each of the plurality of stochastic barcodes further comprises one or more of a universal label and a cellular label, wherein universal labels are the same for the plurality of stochastic barcodes on the solid support and cellular labels are the same for the plurality of stochastic barcodes on the solid support.

16. The method of claim 13, wherein the synthetic particles are beads.

17. A method for determining spatial locations of a plurality of targets in a plurality of spatially ordered cells of a sample, comprising:

associating cells of the plurality of spatially ordered cells at two or more spatial locations with a plurality of stochastic barcodes,

wherein each stochastic barcode of the plurality of stochastic barcodes comprises a spatial label, and

wherein the plurality of stochastic barcodes comprises two or more different spatial labels for identifying the two or more spatial locations of the cells;

stochastically barcoding the plurality of targets in the cells of the plurality of spatially ordered cells using the plurality of stochastic barcodes comprising the two or more different spatial labels for identifying the two or more spatial locations of the cells; and

identifying the spatial locations of the plurality of targets using the two or more different spatial labels.

18. The method of claim 17,

wherein each of the plurality of stochastic barcodes comprises a dimension label,

wherein the dimension labels of the plurality of stochastic barcodes used for stochastic barcoding the plurality of targets at the different time points are different, and

wherein the dimension labels correlate with the different time points.

19. The method of claim 18, wherein stochastically barcoding the plurality of targets comprises contacting the sample with a device at a specific rate, wherein the specific rate correlates the two or more spatial locations of the plurality of targets with the different time points.

20. A method for determining spatial locations of a plurality of targets in a plurality of spatially ordered cells of a sample, comprising:

associating cells of the plurality of spatially ordered cells at two or more spatial locations with a plurality of stochastic barcodes,

wherein each stochastic barcode of the plurality of stochastic barcodes comprises a spatial label and a molecular label, and

wherein the plurality of stochastic barcodes comprises two or more different spatial labels for identifying the two or more spatial locations of the cells;

stochastically barcoding the plurality of targets in the cells of the plurality of spatially ordered cells using the plurality of stochastic barcodes comprising the two or more different spatial labels for identifying the two or more spatial locations of the cells, wherein each of the plurality of stochastic barcodes comprises a pre-spatial label;

concatenating one or more spatial label blocks onto the pre-spatial label to generate a spatial label of the two or more different spatial labels; and

identifying the spatial locations of the plurality of targets in the cells of the plurality of spatially ordered cells at the two or more spatial locations using the two or more different spatial labels of the plurality of stochastic barcodes.

21. A method for determining spatial locations of a plurality of targets in a plurality of spatially ordered cells of a sample, comprising:

associating cells of the plurality of spatially ordered cells at two or more spatial locations with a plurality of stochastic barcodes,

wherein each stochastic barcode of the plurality of stochastic barcodes comprises a spatial label and a molecular label, and

wherein the plurality of stochastic barcodes comprises two or more different spatial labels for identifying the two or more spatial locations of the cells;

imaging the sample to generate a sample image;

stochastically barcoding the plurality of targets in the cells of the plurality of spatially ordered cells using the plurality of stochastic barcodes comprising the two or more different spatial labels for identifying the two or more spatial locations of the cells; and

identifying the spatial locations of the plurality of targets on the sample image using the two or more spatial labels.

22. The method of claim 21, wherein identifying the spatial locations of the plurality of targets using the two or more spatial labels comprises correlating the sample image with the spatial labels of the plurality of targets.

23. A method for distinguishing a plurality of spatially ordered cells of two or more samples, comprising:

associating cells of the plurality of spatially ordered cells at two or more spatial locations with a plurality of stochastic barcodes,

wherein each stochastic barcode of the plurality of stochastic barcodes comprises a spatial label and a molecular label, and

wherein the plurality of stochastic barcodes comprises two or more different spatial labels for identifying the two or more spatial locations of the cells;

stochastically barcoding a plurality of targets in the cells of plurality of spatially ordered cells using the plurality of stochastic barcodes comprising the two or more different spatial labels for identifying the two or more spatial locations of the cells;

estimating the number of the plurality of targets in the cells of the plurality of spatially ordered cells at the two or more spatial locations using the molecular labels of the stochastic barcodes; and

distinguishing the cells of the plurality of spatially ordered cells of the two or more samples from each other using the two or more different spatial labels.

24. The method of claim 23, wherein stochastically barcoding the plurality of targets comprises hybridizing the plurality of stochastic barcodes with the plurality of targets to generate stochastically barcoded targets, and at least one of the plurality of targets is hybridized to one of the plurality of stochastic barcodes.

25. A kit comprising:

a plurality of stochastic barcodes,

wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label,

wherein the plurality of stochastic barcodes comprises two or more different spatial labels for identifying unique spatial locations of cells of a plurality of spatially ordered cells,

and

instructions for using the plurality of stochastic barcodes.

26. The kit of claim 25,

wherein the plurality of stochastic barcodes is associated with a plurality of synthetic particles,

wherein two stochastic barcodes associated with a synthetic particle of the plurality of synthetic particles comprise an identical spatial label, and

wherein two stochastic barcodes associated with two synthetic particles of the plurality of synthetic particles comprise two different spatial labels.