Spatially addressable molecular barcoding

• US 9,727,810 B2
• Filed: 02/26/2016
• Issued: 08/08/2017
• Est. Priority Date: 02/27/2015
• Status: Expired

• 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:
  US15/055,445
• Publication Number:
  US 20160253584A1
• Time in Patent Office: 529 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 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 optical moiety are selected from a group comprising two or more spectrally-distinct optical moieties.

26. The synthetic particle of claim 19,

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

wherein spatial labels of at least two stochastic barcodes of the plurality of stochastic barcodes differ from each other by at least one nucleotide.

27. The synthetic particle of claim 19,

wherein each of the plurality of stochastic barcodes further comprises a universal label, and

wherein universal labels of at least two stochastic barcodes of the plurality of stochastic barcodes have the same sequence.

28. The synthetic particle of claim 19, wherein the synthetic particle is a bead.

29. The synthetic particle of claim 28, 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.

30. The synthetic particle of claim 19, wherein the synthetic particle 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.