# US 2016/0253584 A1 · Spatially addressable molecular barcoding

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- Number: US 2016/0253584 A1
- Document ID: US20160253584A1
- Status: Published application
- Kind: A1
- Application number: 15/055,445
- Filed: 2016-02-26
- Published: 2016-09-01
- Earliest claimed: 2015-02-27
- Assignees: Cellular Research, Inc.
- Applicants: Cellular Research, Inc.
- Inventors: Stephen P.A. Fodor; Christina Fan; Glenn Fu; Geoffrey Facer
- Web page: https://patentgrep.com/patent/US20160253584A1

## CPC

- C12Q 1/6853: using modified primers or templates
- G06K 19/06103: {the marking being embedded in a human recognizable image, e.g. a company logo with an embedded two-dimensional code}
- C12Q 1/6813: Hybridisation assays
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G06K: GRAPHICAL DATA READING (image or video recognition or understanding G06V); PRESENTATION OF DATA; RECORD CARRIERS; HANDLING RECORD CARRIERS

## 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

### Claim 1

A method for determining the number and spatial locations of a plurality of targets in a sample, comprising:
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.

### Claim 2 (depends on 1)

The method of claim 1, wherein stochastically barcoding the plurality of targets in the sample 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.

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 1)

The method of claim 1, wherein estimating the number of the plurality of targets using the molecular label comprises determining sequences of the spatial labels and molecular labels of the plurality of the stochastic labels and counting the number of the molecular labels with distinct sequences.

### Claim 7 (depends on 6)

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

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 9)

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.

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 12)

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

### Claim 14 (depends on 13)

The method of claim 13, wherein the spatial labels of the plurality of stochastic barcodes on different solid supports differ by at least one nucleotide.

### Claim 15 (depends on 13)

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.

### Claim 16 (depends on 13)

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

### Claim 17

A method for determining spatial locations of a plurality of targets in a sample, comprising:
stochastically barcoding the plurality of targets in the sample at one or more time points using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label; and
identifying the spatial location of each of the plurality of targets using the spatial label.

### Claim 18 (depends on 17)

The method of claim 17, wherein the dimension labels correlate with the different time points.

### Claim 19

A method for determining spatial locations of a plurality of targets in a sample, comprising:
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 pre-spatial label;
concatenating one or more spatial label blocks onto the pre-spatial label to generate a spatial label; and
identifying the spatial location of each of the plurality of targets using the spatial label.

### Claim 20 (depends on 19)

The method of claim 19, wherein the sample comprises a plurality of cells and the plurality of targets are associated with the plurality of cells.

### Claim 21

A method for determining spatial locations of a plurality of targets in a sample, comprising:
imaging the sample to generate a sample image;
stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes to generate stochastically barcoded targets, wherein each of the plurality of stochastic barcodes comprises a spatial label; and
identifying the spatial location of each of the plurality of targets using the spatial label.

### Claim 22 (depends on 21)

The method of claim 21, wherein identifying the spatial location of each of the plurality of targets using the spatial label comprises correlating the sample image with the spatial labels of the plurality of targets in the sample.

### Claim 23

A method for determining spatial locations of a plurality of singles cells, comprising:
stochastically barcoding the plurality of singe cells using a plurality of synthetic particles,
wherein each of the plurality of synthetic particles comprises a plurality of stochastic barcodes, a first group of optical labels, and a second group of optical labels,
wherein each of the plurality of stochastic barcodes comprises a cellular label and a molecular label,
wherein each optical label in the first group of optical labels comprises a first optical moiety and each optical label in the second group of optical labels comprises a second optical moiety, and
wherein each of the plurality of synthetic particles is associated with an optical barcode comprising the first optical moiety and the second optical moiety;
detecting the optical barcode of each of the plurality of synthetic particles to determine the location of each of the plurality of synthetic particles; and
determining the spatial locations of the plurality of single cells based on the locations of the plurality of synthetic particles.

### Claim 24 (depends on 23)

The method of claim 23, wherein the first optical moiety and the second optical moiety are selected from a group comprising two or more spectrally-distinct optical moieties.

### Claim 25

A method for identifying distinct cells in two or more samples, comprising:
stochastically barcoding a plurality of targets in the two or more samples 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 the plurality of targets in the two or more samples using the molecular label; and
distinguishing the two or more samples from each other using the spatial label, wherein the plurality of targets associated with stochastic barcodes with different spatial labels are from different samples.

### Claim 26 (depends on 25)

The method of claim 25, wherein stochastically barcoding the plurality of targets in the two or more samples 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.

### Claim 27

A kit comprising:
a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label, wherein the spatial labels of the plurality of stochastic barcodes differ from one another by at least one nucleotide; and
instructions for using the plurality of stochastic barcodes.

### Claim 28 (depends on 27)

The kit of claim 27, wherein the solid support comprises a plurality of synthetic particles associated with the plurality of synthetic particles.

### Claim 29

A synthetic particle, comprising:
a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a cellular label and a molecular label;
a first group of optical labels;
a second group of optical labels,
wherein each optical label in the first group of optical labels comprises a first optical moiety and each optical label in the second group of optical labels comprises a second optical moiety, and
wherein each of the plurality of synthetic particles is associated with an optical barcode comprising the first optical moiety and the second optical moiety.

### Claim 30 (depends on 29)

The synthetic particle of claim 29, wherein the molecular labels of the plurality of stochastic barcodes are different from one another, and the molecular labels are selected from a group comprising at least 100 molecular labels with unique sequences.