Compositions and methods for molecular labeling
The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.
1. A method for analyzing proteins of a cell, the method comprising:
segregating a single cell and barcoded binders, each barcoded binder comprising a binder linked to an oligonucleotide comprising a binder-identifying barcode, into a first droplet in an immiscible carrier fluid within a microfluidic channel, wherein the binder-identifying barcodes are sticky-ended barcodes made by a process that includes rounds of successive addition of sticky-ended partial barcodes all performed by microfluidic droplet merger;
binding the barcoded binders to proteins of the single cell;
providing a second droplet from a universal barcode droplet library into the microfluidic channel, where the second droplet includes a plurality of copies of a droplet-identifying barcode;
merging the second droplet into the first droplet;
attaching the copies of the droplet-identifying barcode to the binder-identifying barcodes of the barcoded binders bound to the proteins of the single cell to form composite barcodes;
sequencing the composite barcodes; and
identifying the binders from the sequences of the composite barcodes, thereby identifying the proteins of the single cell.
2. The method of claim 1 , wherein a number of composite barcodes sequenced quantitates the proteins of the single cell.
3. The method of claim 1 , wherein a plurality of different barcoded binders are provided to the first droplet, wherein each different barcoded binder comprises a different binder-identifying barcode and each different barcoded binder binds to a different protein of the single cell.
4. The method of claim 1 , wherein the oligonucleotide comprises a sequencing adaptor and the binder-identifying barcode.
5. The method of claim 4 , wherein the oligonucleotide further comprises a restriction site.
6. The method of claim 1 , wherein the binders of the barcoded binders are antibodies.
7. The method of claim 1 , wherein the proteins are on the surface of the cell.
8. The method of claim 1 , wherein the proteins are intracellular.
9. The method of claim 1 , wherein prior to binding the barcoded binders to the proteins of the single cell, the cell is lysed, thereby releasing at least one protein.
10. The method of claim 9 , wherein a capture-tagged binder is provided, the method further comprising binding a barcoded binder to the at least one released protein and binding the capture-tagged binder to the at least one released protein, wherein said capture-tagged binder is attached to a solid surface.
11. The method of claim 10 , wherein the capture-tagged binder is biotinylated and attached to a streptavidin-containing solid surface.
12. The method of claim 10 , wherein the solid surface is a bead.
13. The method of claim 10 , wherein the binder of the capture-tagged binder and the binder of the barcoded binder are antibodies and the released protein is an antigen.
14. The method of claim 1 , wherein each barcoded binder comprises a cleavable linker that links the binder-identifying barcode to the binder.
15. The method of claim 1 , wherein the sequencing step includes amplifying the composite barcodes using solid-phase amplification.
16. The method of claim 15 , wherein the solid-phase amplification uses forward and reverse amplification primers immobilized on a solid surface.
17. The method of claim 1 , wherein the barcoded binders bind specifically to proteins involved in cancer signaling pathways.
18. The method of claim 17 , wherein the proteins include one or more of Akt, EGF, Src, TNFRI/II, PSA, RANKL, CEA, AFP, CA125, and beta2 microglobin.