Spatially addressable molecular barcoding

Patent metadata

Field	Value
Publication number	US-2016253584-A1
Application number	US-201615055445-A
Country	US
Kind code	A1
Filing date	Feb 26, 2016
Priority date	Feb 27, 2015
Publication date	Sep 1, 2016
Grant date	—

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 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.

2 . 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.

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 in the sample.

5 . 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.

6 . 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.

7 . 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.

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 in the sample 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 the spatial labels of the plurality of stochastic barcodes on different solid supports differ by at least one nucleotide.

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 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.

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

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.

20 . 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.

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.

22 . 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.

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.

24 . 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.

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.

26 . The method of claim 25 , whe

Assignees

• Cellular Res Inc

Inventors

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

Classifications

• C12Q1/6816

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

• C12Q1/6813 Primary

  Hybridisation assays · CPC title

• 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

• C12Q1/6853 Primary

  using modified primers or templates · CPC title