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 identifying targets associated with a single cell or cell compartment comprising:
(a) providing a plurality of cells or cell compartments, wherein the cells or cell compartments comprise nucleic acids and are free of rolling circle amplification products,
(b) assembling a cell origination barcode in and/or on individual cells or cell compartments by a method that comprises adding one or more assayable polymer subunit oligonucleotides to the nucleic acids in an ordered manner using a split-pool process; and
(c) sequencing the cell origination barcodes and the nucleic acids, or an amplification product thereof, to produce sequence reads.
2. The method of claim 1 , further comprising analyzing the sequence reads to determine which nucleic acids are associated with which cells or cell compartments.
3. The method of claim 1 , wherein the assembling of (b) is done by ligation, gap-fill/ligation, by extension by a polymerase, hybridization or affinity binding.
4. The method of claim 3 , wherein the ligation is splinted using a bridging oligonucleotide.
5. The method of claim 1 , wherein the cells or cell compartments are mammalian cells or compartments isolated therefrom.
6. The method of claim 1 , wherein the method is performed using a cell suspension.
7. The method of claim 1 , wherein the method is performed using cultured cells, blood cells, cells from a tissue, or a cell compartment isolated therefrom.
8. The method of claim 1 , wherein the method is performed using cells that have been exposed to an agent or genetically modified, or a cell compartment isolated therefrom.
9. The method of claim 1 , wherein the nucleic acids comprise cellular nucleic acids.
10. The method of claim 1 , wherein the nucleic acids comprise genomic DNA or RNA.
11. The method of claim 1 , wherein the nucleic acids comprise cDNA.
12. The method of claim 1 , wherein the nucleic acids comprise methylated nucleic acids.
13. The method of claim 12 , further comprising determining the methylation state of the nucleic acids.
14. The method of claim 1 , wherein the nucleic acids are oligonucleotides.
15. The method of claim 14 , wherein the oligonucleotides are oligonucleotide probes that are directly or indirectly hybridized to nucleic acids in the cells or a compartment thereof.
16. The method of claim 14 , wherein the oligonucleotides are bound to proteins.
17. The method of claim 16 , wherein the oligonucleotides are conjugated to antibodies that are bound to antigens that are in and/or on the cells or cell compartments thereof.
18. The method of claim 1 , wherein the split-pool process comprises multiple rounds of: (i) splitting the cells or cell compartments and adding the assayable polymer subunit oligonucleotides to the nucleic acids while the cells or cell compartments are split; and then (ii) pooling the cells or cell compartments.
19. The method of claim 1 , wherein the nucleic acids are in the cells or cell compartments.
20. The method of claim 19 , wherein the cells or cell compartments are fixed and/or permeabilized.
21. The method of claim 1 , wherein one or more of the assayable polymer subunit oligonucleotides comprises a random sequence.
22. The method of claim 1 , further comprising directly or indirectly adding a biotin to the nucleic acids.
23. The method of claim 22 , wherein the method further comprises enriching for nucleic acids that are biotinylated.
24. The method of claim 1 , wherein the method is performed using cell compartments.
25. The method of claim 1 , wherein the cell or cell compartments of step (a) correspond to a single biomolecular sample.
26. The method of claim 1 , wherein the cell or cell compartments of step (a) correspond to multiple individual biomolecular samples.