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 comprising:
a) binding to mRNA targets in a plurality of cells a plurality of unique binding agents (UBAs) that each comprise a probe oligonucleotide that is complementary to an mRNA target; and
b) assembling a cell originating barcode (COB) onto the probe oligonucleotides or ligation products containing the same by adding one or more assayable polymer subunit (APS) oligonucleotides to the bound UBAs in an ordered manner during successive rounds of split-pool synthesis to create unique codes that represent the identities of at least some of the individual cells in which the UBAs are bound, wherein the method does not include a step of isolating each cell in the plurality of cells.
2. The method of claim 1 , wherein the probe oligonucleotides further comprise a common linker sequence and wherein the first APS oligonucleotide added in step (b) anneals to the common linker sequence.
3. The method of claim 1 , wherein the APS oligonucleotides added in successive rounds of split-pool synthesis hybridize to a splint oligonucleotide.
4. The method of claim 2 , wherein the splint oligonucleotide used in the initial round of split-pool synthesis comprises: (i) a first region that is complementary to a region in the common linker oligonucleotide and (ii) a second region that is complementary to a region in the APS oligonucleotides added in the initial round of split-pool synthesis.
5. The method of claim 1 , wherein the APS oligonucleotides added in successive rounds of split-pool synthesis hybridize to one another.
6. The method of claim 1 , wherein at least some of APS oligonucleotides in a given round of split-pool synthesis comprises a unique subcode sequence that is different from the rest of the APS in that round.
7. The method of claim 3 , wherein at least some of the APS oligonucleotides comprise, in order, a region that is complementary to a first splint oligonucleotide, a subcode region, and a region that is complementary to a second splint oligonucleotide.
8. The method of claim 1 , wherein up to 20 APS oligonucleotides are used in each round of split-pool synthesis, each of the APS oligonucleotides characterized by a different subcode region.
9. The method of claim 1 , further comprising amplifying nucleic acids comprising a cell originating barcode (COB) and at least a part of a unique binding agent (UBA).
10. The method of claim 9 , wherein one amplification primer is capable of hybridizing to a common sequence in the last-added APS oligonucleotide in a cell originating barcode (COB).
11. The method of claim 9 , wherein one amplification primer is capable of hybridizing to a common sequence in the nucleic acids containing the cell origination barcode.
12. The method of claim 1 , further comprising sequencing nucleic acids comprising a cell originating barcode (COB) and at least a part of a unique binding agent (UBA).
13. The method of claim 1 , further comprising, prior to step a), fixing the cells.
14. The method of claim 1 , further comprising, prior to step a), permeabilizing the cells.
15. The method of claim 1 , wherein the APS oligonucleotides are linked by ligation.
16. The method of claim 1 , wherein the APS oligonucleotides are linked by Click chemistry.
17. A method comprising:
assembling cell originating barcodes (COBs) onto unique binding agents (UBAs) that are hybridized to mRNA in a plurality of cells by adding one or more assayable polymer subunit (APS) oligonucleotides to the hybridized UBAs in an ordered manner during successive rounds of split pool synthesis to create unique codes that represent the identities of at least some of the individual cells in which the UBAs are hybridized, wherein the method does not include a step of isolating each cell in the plurality of cells.