Kit for split-pool barcoding target molecules that are in or on cells or cell organelles
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 kit for split-pool barcoding target molecules that are in or on cells or cell organelles, comprising:
(i) at least two sets of assayable polymer subunit (APS) oligonucleotides, wherein each set comprises at least 10 APS oligonucleotides that each comprise:
a 5′ end sequence that is common to the APS oligonucleotides in the set,
a 3′ end sequence that is common to the APS oligonucleotides in the set, and
an internal sequence that distinguishes the APS oligonucleotides in the set from one another, and
(ii) one or more linking oligonucleotides, wherein a linking oligonucleotide is complementary to the 5′ end sequence of the APS oligonucleotides of one set and the 3′ end sequence of the APS oligonucleotides of another set,
wherein the APS oligonucleotides from different sets are configured to link together in an ordered fashion in the presence of the one or more linking oligonucleotides to form all or part of a cell or organelle origination barcode.
2. The kit of claim 1 , wherein the different APS oligonucleotides of a set are in different wells of a multi-well plate.
3. The kit of claim 1 , wherein the kit comprises at least three sets of APS oligonucleotides, each set comprising at least 10 of the APS oligonucleotides.
4. The kit of claim 1 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides are capable of functioning as primers.
5. The kit of claim 1 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides comprise a unique binding agent (UBA).
6. The kit of claim 1 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides additionally comprise a random sequence between the 5′ end sequence and the 3′ end sequence.
7. The kit of claim 1 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides additionally comprise an affinity tag.
8. The kit of claim 1 , wherein the kit comprises:
(a) a first set of at least 10 APS oligonucleotides that are in separate containers, each comprising:
(i) a 5′ end sequence that is common to the APS oligonucleotides in the first set;
(ii) an internal sequence that distinguishes the APS oligonucleotides in the first set from one another; and
(iii) a 3′ end sequence that is common to the APS oligonucleotides in the first set;
(b) a second set of at least 10 APS oligonucleotides that are in separate containers, each comprising:
(i) a 5′ end sequence that is common to the APS oligonucleotides in the second set;
(ii) an internal sequence that distinguishes the APS oligonucleotides in the second set from one another; and
(iii) a 3′ end sequence that is common to the APS oligonucleotides in the second set; and
(c) a first linking oligonucleotide that comprises:
(i) a first sequence that is complementary to the 5′ end sequence of the APS oligonucleotides of (a); and
(ii) a second sequence that is adjacent to the first sequence and complementary to the 3′ end sequence of APS oligonucleotides of (b);
wherein the first linking oligonucleotide is configured to bring the 5′ end an APS oligonucleotide of (a) into ligatable proximity with the 3′ end of an APS oligonucleotide of (b).
9. The kit of claim 8 , wherein the separate containers are wells in a multi-well plate.
10. The kit of claim 8 , wherein the APS oligonucleotides of (a) or (b) are hybridized to the first linking oligonucleotide.
11. The kit of claim 8 , wherein the first linking oligonucleotide of (c):
(iii) does not comprise a sequence that is complementary to any of the internal sequences of the APS oligonucleotides of (a) and (b).
12. The kit of claim 8 , wherein the APS oligonucleotides of the first set of APS oligonucleotides are capable of functioning as primers.
13. The kit of claim 8 , wherein the APS oligonucleotides of the first or second sets of APS oligonucleotides comprise a unique binding agent (UBA).
14. The kit of claim 8 , wherein the first set of APS oligonucleotides of (a) comprises more than 20 APS oligonucleotides and the second set of APS oligonucleotides of (b) comprises more than 20 APS oligonucleotides.
15. The kit of claim 8 , further comprising:
(d) a third set of at least 10 APS oligonucleotides that are in separate containers, each comprising:
(i) a 5′ end sequence that is common to the APS oligonucleotides in the third set;
(ii) an internal sequence that distinguishes the APS oligonucleotides in the third set from one another; and
(iii) a 3′ end sequence that is common to the APS oligonucleotides in the third set; and
(e) a second linking oligonucleotide that comprises:
(i) a first sequence that is complementary to the 5′ end sequence of the APS oligonucleotides of (b); and
(ii) a second sequence that is adjacent to the first sequence and complementary to the 3′ end sequence of APS oligonucleotides of (d);
wherein the second linking oligonucleotide is configured to bring the 5′ end of an APS oligonucleotide of (b) into ligatable proximity with the 3′ end of an APS oligonucleotide of (d).
16. The kit of claim 15 , wherein the second linking oligonucleotide of (e):
(iii) does not comprise a sequence that is complementary to any of the internal sequences of the APS oligonucleotides of (b) and (d).
17. The kit of claim 15 , wherein the APS oligonucleotides of (b) or (d) are hybridized to the second linking oligonucleotide.
18. The kit of claim 15 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides comprises an additional random internal sequence.
19. The kit of claim 15 , wherein the kit further comprises a primer, and wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides comprise a binding site for the primer, or the complement thereof.
20. The kit of claim 15 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides additionally comprise an affinity tag.
21. The kit of claim 15 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides comprise a unique binding agent (UBA).
22. The kit of claim 15 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides are capable of functioning as primers.
23. The kit of claim 1 , wherein the kit comprises:
(a) a first set of at least 10 APS oligonucleotides that are in separate containers, each comprising:
(i) a 5′ end sequence that is common to the APS oligonucleotides in the first set;
(ii) an internal sequence that distinguishes the APS oligonucleotides in the first set from one another; and
(iii) a 3′ end sequence that is common to the APS oligonucleotides in the first set;
(b) a second set of at least 10 APS oligonucleotides that are in separate containers, each comprising:
(i) a 5′ end sequence that is common to the APS oligonucleotides in the second set;
(ii) an internal sequence that is distinguishes the APS oligonucleotides in the second set from one another; and
(iii) a 3′ end sequence that is common the APS oligonucleotides in the second set; and
(c) a first linking oligonucleotide that comprises:
(i) a first sequence that is complementary to the 3′ end sequence of the APS oligonucleotides of (a); and
(ii) a second sequence that is adjacent to the first sequence and complementary to the 5′ end sequence of APS oligonucleotides of (b);
wherein the first linking oligonucleotide is configured to bring the 3′ end of an APS oligonucleotide of (a) into ligatable proximity with the 5′ end of an APS oligonucleotide of (b).
24. The kit of claim 23 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides comprises a unique binding agent (UBA).
25. The kit of claim 23 , wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides are capable of functioning as primers.
26. The kit of claim 23 , wherein the kit further comprises a primer, and wherein the APS oligonucleotides of at least one of the sets of APS oligonucleotides comprise a binding site for the primer, or the complement thereof.