Spatially addressable molecular barcoding

Patent metadata

Field	Value
Publication number	US-10002316-B2
Application number	US-201715667125-A
Country	US
Kind code	B2
Filing date	Aug 2, 2017
Priority date	Feb 27, 2015
Publication date	Jun 19, 2018
Grant date	Jun 19, 2018

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.

First claim

What is claimed is:

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, comp

Assignees

• Cellular Res Inc

Inventors

• Fodor Stephen P A
• Fan Christina
• Fu Glenn
• Facer Geoffrey

Classifications

• G06K19/06103 Primary

  the marking being embedded in a human recognizable image, e.g. a company logo with an embedded two-dimensional code · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6813 Primary

  Hybridisation assays · CPC title

• C12Q2543/10

  the purpose being "in situ" analysis · CPC title

• C12Q2525/179

  incorporating arbitrary or random nucleotide sequences · CPC title