Barcoded beads and method for making the same by split-pool synthesis
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 population of beads, comprising:
(a) a first bead associated with a first nucleic acid tag that comprises:
(i) a first cell origination barcode; and
(ii) a degenerate sequence; and
(b) a second bead associated with a second nucleic acid tag that comprises:
(i) a second cell origination barcode, and
(ii) a degenerate sequence;
wherein the first and second cell origination barcodes are different.
2. The population of beads of claim 1 , wherein the population of beads comprises 80,000 or more beads that are each associated with a nucleic acid tag that comprises:
(i) a cell origination barcode that uniquely identifies the bead with which the nucleic acid tag is associated; and
(ii) a degenerate sequence.
3. The population of beads of claim 1 , wherein the population of beads comprises greater than 1 million beads that are each associated with a nucleic acid tag that comprises:
(i) a cell origination barcode that uniquely identifies the bead to which the nucleic acid tag is associated; and
(ii) a degenerate sequence.
4. The population of beads of claim 1 , wherein the degenerate sequence is at least 2 nucleotides in length.
5. The population of beads of claim 1 , wherein the first and second beads are porous.
6. The population of beads of claim 1 , wherein the first and second beads are magnetic.
7. The population of beads of claim 1 , wherein the first and second nucleic acid tags further comprise a common linker sequence.
8. The population of beads of claim 1 , wherein the first and second nucleic acid tags are capable of acting as a primer and/or contain a binding site for an amplification primer or complement thereof.
9. The population of beads of claim 1 , wherein the degenerate sequences are random sequences.
10. The population of beads of claim 1 , wherein the beads are free of non-nucleic acid molecules made by combinatorial synthesis.
11. A method for making a population of barcoded beads, comprising:
adding nucleic acid tags that comprise a cell origination barcode and a degenerate sequence onto beads by a split-pool barcoding process.
12. The method of claim 11 , wherein the split-pool barcoding process comprises multiple rounds of:
(i) splitting a pool of beads that comprise tethered nucleic acids into multiple reaction volumes;
(ii) appending assayable oligonucleotide subunits to the tethered nucleic acids in the multiple reaction volumes; and then
(iii) pooling the beads.
13. The method of claim 12 , wherein the assayable oligonucleotide subunits are appended by chemical ligation, enzymatic ligation, extension by a polymerase or a gap-fill ligation.
14. The method of claim 12 , wherein the split-pool barcoding process comprises at least 3 rounds of (i)-(iii).
15. The method of claim 12 , wherein the split-pool barcoding process comprises at least 5 rounds of (i)-(iii).
16. The method of claim 12 , wherein the method further comprises appending a degenerate sequence to the tethered nucleic acids.
17. The method of claim 16 , wherein the degenerate sequence is a random sequence.
18. The method of claim 11 , wherein the split-pool barcoding process comprises multiple rounds of:
(a) splitting a pool of beads to which nucleic acids are tethered into multiple reaction volumes, wherein at least some of the reaction volumes comprises multiple beads;
(b) appending assayable oligonucleotide subunits to the tethered nucleic acids in the multiple reaction volumes, wherein a different assayable oligonucleotide subunit is appended to the tethered nucleic acids in each reaction volume; and
(c) pooling the beads of (b) to produce a pool.
19. The method of claim 18 , wherein in step (a) the pool of beads is split into 10 or more reaction volumes.
20. The method of claim 18 , wherein the appended assayable oligonucleotide subunits become part of the cell origination barcode.
21. The method of claim 18 , wherein the beads comprise a common linker sequence and the assayable oligonucleotide subunits added in the first round of the split-pool barcoding process are added to the common linker sequence.
22. The method of claim 21 , wherein the common linker sequence comprises a degenerate sequence.
23. The method of claim 11 , wherein the beads are magnetic.
24. The method of claim 11 , wherein the beads are porous.
25. The method of claim 11 , wherein the nucleic acid tags are added to the beads without adding any non-nucleic acid molecules to the beads by combinatorial synthesis.
26. A population of beads made by the method of claim 11 .