Methods of identifying multiple epitopes in cells
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
1. A method for barcoding target molecules, comprising:
(a) associating target molecules from cells or cell organelles with solid-phase particles, wherein at least some of the particles each become associated with target molecules from a single cell or cell organelle; and
(b) adding cell-origination barcodes onto the target molecules that are associated with the particles by a split-pool barcoding process.
2. The method of claim 1 , wherein step (a) comprises:
(i) associating the particles with the cells or cell organelles, wherein at least some of the particles and cells or cell organelles associate on a one-to-one basis; and
(ii) dissociating target molecules from the cells or cell organelles and allowing the dissociated target molecules to associate with the particles.
3. The method of claim 1 , wherein the split-pool barcoding process comprises at least one round of:
splitting the particles into a plurality of reaction volumes,
adding assayable oligonucleotide subunits onto the target molecules that are in or on the particles in the reaction volumes, and then
pooling the particles,
wherein the added assayable oligonucleotide subunits form all or part of the cell-origination barcode.
4. The method of claim 1 , wherein the particles are beads.
5. The method of claim 1 , wherein the particles are porous or magnetic.
6. The method of claim 5 , wherein in (b) the target molecules are in or on porous particles.
7. The method of claim 1 , wherein the target molecules comprise nucleic acids, polypeptides, proteins, or genomic DNA.
8. The method of claim 1 , further comprising (c) sequencing the target molecules and the added cell-origination barcodes, a copy thereof, or an amplification product thereof.
9. The method of claim 1 , wherein the target molecules of (a) comprise genomic DNA.
10. The method of claim 1 , wherein the target molecules of (a) comprise RNA or cDNA.
11. The method of claim 1 , wherein the target molecules of (a) comprise oligonucleotide probes that are directly or indirectly hybridized to nucleic acid molecules that are in or on the cells or cell organelles.
12. The method of claim 1 , wherein the target molecules of (a) are oligonucleotides that are conjugated to antibodies.
13. The method of claim 1 , wherein the target molecules are nucleic acids.
14. The method of claim 1 , wherein the cells or cell organelles of (a) are fixed and/or permeabilized.
15. The method of claim 1 , wherein the cells or cell organelles are lysed prior to step (b).
16. The method of claim 1 , wherein during the split pool barcoding process the particles are compartmentalized into microbubbles, microdroplets, wells, microwells, or enclosures in a microfluidics device.
17. The method of claim 1 , wherein the method is performed using a cell suspension, cultured cells, blood cells, cells from a tissue, or organelles isolated therefrom.
18. The method of claim 1 , wherein the cells or cell organelles have been exposed to an agent or have been genetically modified.
19. The method of claim 1 , wherein the method comprises directly or indirectly adding a biotin moiety to the target molecules to produce biotinylated target molecules and enriching for the biotinylated target molecules.
20. The method of claim 1 , wherein the adding is done by a method that comprises ligation, gap-fill/ligation, hybridization or extension by a polymerase.
21. The method of claim 1 , wherein the method produces a mixture of nucleic acid molecules, wherein at least some of the nucleic acid molecules comprise a cell-origination barcode and a target molecule or fragment thereof, or a copy of either the target molecule or fragment thereof.
22. The method of claim 1 , wherein the method is performed using cell organelles.
23. The method of claim 1 , wherein the method is performed using cells.
24. The method of claim 1 , wherein the split-pool barcoding process comprises:
(i) splitting the particles and associated target molecules into multiple reaction volumes, wherein at least some of the volumes receive multiple particles;
(ii) appending assayable oligonucleotide subunits onto the target molecules in the multiple reaction volumes, wherein different reaction volumes receive different assayable oligonucleotide subunits; and
(iii) pooling the contents of the multiple reaction volumes to produce a pool,
wherein the assayable oligonucleotide subunits added to the target molecules in (ii) become part or all of the cell origination barcode.
25. The method of claim 24 , wherein in step (i) the particles and associated target molecules are in at least 12 reaction volumes.
26. The method of claim 24 , further comprising:
(iv) splitting the pool of (iii) into multiple reaction volumes, wherein at least some of the volumes receive multiple particles; and
(v) appending additional assayable oligonucleotide subunits onto the target molecules in the multiple reaction volumes of (iv);
wherein the different reaction volumes of (iv) receive different assayable oligonucleotide subunits and the assayable oligonucleotide subunits of (v) are appended to products made in step (ii), and
wherein the assayable oligonucleotide subunits added to the target molecules in steps (ii) and (v) become part or all of the cell origination barcode.
27. The method of claim 1 , wherein the cell or cell organelles of (a) are from a single biomolecular sample.
28. The method of claim 1 , wherein the cell or cell organelles of (a) are from a plurality of biomolecular samples.