Spatially addressable molecular barcoding

Patent metadata

Field	Value
Publication number	US-9727810-B2
Application number	US-201615055445-A
Country	US
Kind code	B2
Filing date	Feb 26, 2016
Priority date	Feb 27, 2015
Publication date	Aug 8, 2017
Grant date	Aug 8, 2017

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 spatial locations of a plurality of single 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.

2. The method of claim 1 , wherein the first optical moiety and the second optical moiety are selected from a group comprising two or more spectrally-distinct optical moieties.

3. The method of claim 1 , wherein stochastically barcoding the plurality of single cells using the plurality of synthetic particles comprises contacting the plurality of single cells with the plurality of synthetic particles.

4. The method of claim 3 , wherein a synthetic particle of the plurality of synthetic particles is in close proximity to a single cell or a small number of cells.

5. The method of claim 3 ,
wherein each of the plurality of single cells comprises a plurality of targets,
wherein stochastically barcoding the plurality of single cells further comprises hybridizing the plurality of stochastic barcodes with the plurality of targets to generate stochastically barcoded targets, and
wherein at least one of the plurality of targets is hybridized to one of the plurality of stochastic barcodes.

6. The method of claim 1 , wherein cellular labels of at least two stochastic barcodes of the plurality of stochastic barcodes on one synthetic particle have the same sequence, and wherein cellular labels of at least two stochastic barcodes of the plurality of stochastic barcodes on different synthetic particles have different sequences.

7. The method of claim 1 , wherein molecular labels of at least two stochastic barcodes of the plurality of stochastic barcodes on one synthetic particle have different sequences.

8. The method of claim 1 , wherein the molecular labels are selected from a group comprising at least 100 molecular labels with unique sequences.

9. The method of claim 1 , wherein the molecular labels are selected from a group comprising at least 1000 molecular labels with unique sequences.

10. The method of claim 1 , wherein detecting the optical barcode of each of the plurality of synthetic particles to determine the location of each of the plurality of synthetic particles comprises generating an optical image showing the optical barcodes and the locations of the plurality of synthetic particles.

11. The method of claim 1 , wherein the plurality of single cells comprises cells distributed across a microwell array comprising microwells.

12. The method of claim 11 , comprising:
lysing the plurality of single cells; and
generating an indexed library of stochastically barcoded targets,
wherein each of the stochastically barcoded targets comprises a cellular label sequence, a molecular label sequence, and at least a portion of the complementary sequence of one of the plurality of targets.

13. The method of claim 12 , comprising:
amplifying the stochastically barcoded targets of the indexed library to generate amplified stochastically barcoded targets; and
sequencing the amplified stochastically barcoded targets to determine the number of amplified stochastically barcoded targets with unique molecular label sequences and identical complementary sequence,
wherein the number of amplified stochastically barcoded targets with unique molecular label sequences and identical complementary sequence is substantially the same as the occurrences of targets with sequences complementary of the identical complementary sequence in the single cell or the small number of cells.

14. The method of claim 13 , wherein the labeled target molecules are amplified using bridge amplification, amplification with a gene specific primer, amplification with a universal primer, amplification with an oligo(dT) primer, or any combination thereof.

15. The method of claim 1 , wherein the plurality of single cells comprises a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof.

16. The method of claim 1 , wherein a synthetic particle of the plurality of synthetic particle is a bead.

17. The method of claim 16 , wherein the bead is selected from the group comprising streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and any combination thereof.

18. The method of claim 1 , wherein a synthetic particle of the plurality of synthetic particles comprises a material selected from the group comprising polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.

19. 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; and
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.

20. The synthetic particle of claim 19 , 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.

21. The synthetic particle of claim 19 , wherein cellular labels of at least two stochastic barcodes of the plurality of stochastic barcodes have the same sequence.

22. The synthetic particle of claim 19 , wherein molecular labels of at least two stochastic barcodes of the plurality of stochastic barcodes have different sequences.

23. The synthetic particle of claim 19 , wherein molecular labels of the plurality of stochastic barcodes are selected from a group comprising at least 100 molecular labels with unique sequences.

24. The synthetic particle of claim 19 , wherein molecular labels of the plurality of stochastic barcodes are selected from a group comprising at least 1000 molecular labels with unique sequences.

25. The synthetic particle of claim 19 , wherein the first optical moiety and the second opt

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

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12Q2525/179

  incorporating arbitrary or random nucleotide sequences · CPC title