IP Library › Granted Patent US 12,371,729
Granted Patent B2
US 12,371,729 · App. 16/789,311 · Granted Jul 29, 2025

Sample indexing for single cells

Inventors: Christina Chang (San Jose, CA); Christina Fan (San Jose, CA); Eleen Shum (San Jose, CA)
Assignee: BECTON, DICKINSON AND COMPANY
C12Q1/6804C12Q1/6806C12Q1/6809C12Q1/6811C12Q1/6816C12Q1/6855C12Q1/6865C12Q1/6869C12Q1/6876G01N33/532B82Y5/00C12Q1/6834C12Q1/6837C12Q2563/149C12Q2563/179C12Q2563/185C12Q2600/16G01N2458/10
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Quick Facts
Patent No.
US 12,371,729
App. No.
16/789,311
Granted
Jul 29, 2025
Kind
B2
Abstract

Disclosed herein include systems, methods, compositions, and kits for sample identification. A sample indexing composition can comprise, for example, a protein binding reagent associated with a sample indexing oligonucleotide. Different sample indexing compositions can include sample indexing oligonucleotides with different sequences. Sample origin of cells can be identified based on the sequences of the sample indexing oligonucleotides. Sample indexing oligonucleotides can be barcoded using barcoded and lengthened using daisy-chaining primers.

Claims (47)

1. A composition, comprising a first oligonucleotide-associated antibody and a second oligonucleotide-associated antibody, wherein

the first oligonucleotide-associated antibody comprises a first antibody capable of specifically binding to a first protein target and is associated with a first sample indexing oligonucleotide, wherein said first sample indexing oligonucleotide comprises a binding site for a first target sequence, a first universal primer sequence, and a first sample indexing sequence, and

the second oligonucleotide-associated antibody comprises a second antibody capable of specifically binding to a second protein target and is associated with a second sample indexing oligonucleotide, wherein said second sample indexing oligonucleotide comprises a binding site for a second target sequence, a second universal primer sequence, and a second sample indexing sequence, and

wherein the first sample indexing sequence and the second sample indexing sequence are not homologous to the genomic sequences of a mammalian cell, wherein the first sample indexing sequence and the second sample indexing sequence are the same, wherein the first protein target and the second protein target are different, wherein the first protein target is a major histocompatibility complex protein and the second protein target is CD298, and wherein the first antibody and the second antibody are different antibodies.

2. The composition of claim 1 , wherein (1) in the first sample indexing oligonucleotide, the first sample indexing sequence is located between the binding site for the first target sequence and the first universal primer sequence, (2) in the second sample indexing oligonucleotide, the second sample indexing sequence is located between the binding site for the second target sequence and the second universal primer sequence, or both (1) and (2).

3. The composition of claim 1 , wherein the first target sequence, the second target sequence, or both, comprises a non-specific target nucleic acid sequence.

4. The composition of claim 1 , wherein the first target sequence, the second target sequence, or both, comprises an oligonucleotide probe, a poly(T) region, or a combination thereof.

5. The composition of claim 1 , wherein the binding site for the first target sequence, the binding site for the second target sequence, or both, comprises a poly(A) sequence.

6. The composition of claim 1 , wherein the binding site for the first target sequence is at the 3′ region of the first sample indexing oligonucleotide, the binding site for the second target sequence is at the 3′ region of the second sample indexing oligonucleotide, or both.

7. The composition of claim 1 , wherein the first sample indexing oligonucleotide is reversibly associated with the first antibody, the second sample indexing oligonucleotide is reversibly associated with the second antibody, or both.

8. The composition of claim 1 , wherein the first sample indexing oligonucleotide and the second sample indexing oligonucleotide is associated with the first antibody and the second antibody, respectively, through a chemical group selected from the group consisting of a UV photocleavable group, a streptavidin, a biotin, an amine, and a combination thereof.

9. The composition of claim 1 , wherein the first sample indexing oligonucleotide, the second sample indexing oligonucleotide, or both, is 50-500 nucleotides in length.

10. The composition of claim 1 , wherein the first sample indexing sequence, the second sample indexing sequence, or both, is 6-60 nucleotides in length.

11. The composition of claim 1 , wherein the first sample indexing oligonucleotide, the second sample indexing oligonucleotide, or both, comprises a barcode sequence, a poly(A) sequence, a molecular label sequence, a cell label sequence, an amplification adaptor, a sequencing adaptor, a random multimer sequence, or a combination thereof.

12. The composition of claim 1 , further comprising one or more additional antibodies which are not associated with an oligonucleotide.

13. The composition of claim 12 , wherein the one or more additional antibodies which are not associated with an oligonucleotide are the same as the first antibody or the second antibody.

14. The composition of claim 1 , wherein the first universal primer sequence, the second universal primer sequence, or both, comprises the sequence of a sequencing primer associated with high-throughput sequencing platforms.

15. The composition of claim 1 , wherein the first universal primer sequence, the second universal primer sequence, or both, is a PCR primer sequence.

16. The composition of claim 1 , wherein at least one of the first antibody and the second antibody is a human antibody or a mouse antibody.

17. The composition of claim 1 , further comprising a third oligonucleotide-associated antibody which comprises a third antibody capable of specifically binding to a third protein target, and is associated with an oligonucleotide comprising a binding site for a third target sequence, a third universal primer sequence, and an identifier sequence.

18. The composition of claim 17 , wherein the third protein target is different from the first protein target and the second protein target.

19. The composition of claim 17 , wherein the third antibody is different from at least one of the first antibody and the second antibody.

20. The composition of claim 17 , wherein the identifier sequence is a sample indexing oligonucleotide sequence.

21. The composition of claim 17 , wherein the identifier sequence is a unique identifier sequence for the antibody that it is associated therewith.

22. A system for sample indexing, comprising

a composition, comprising a first oligonucleotide-associated antibody and a second oligonucleotide-associated antibody, wherein

the first oligonucleotide-associated antibody comprises a first antibody capable of specifically binding to a first protein target and is associated with a first sample indexing oligonucleotide, wherein said first sample indexing oligonucleotide comprises a binding site for a first target sequence, a first universal primer sequence, and a first sample indexing sequence, and

the second oligonucleotide-associated antibody comprises a second antibody capable of specifically binding to a second protein target and is associated with a second sample indexing oligonucleotide, wherein said second sample indexing oligonucleotide comprises a binding site for a second target sequence, a second universal primer sequence, and a second sample indexing sequence, and

wherein the first sample indexing sequence and the second sample indexing sequence are not homologous to the genomic sequences of a mammalian cell, wherein the first sample indexing sequence and the second sample indexing sequence are the same, wherein the first protein target and the second protein target are different, wherein the first protein target is a major histocompatibility complex protein and the second protein target is CD298, and wherein the first antibody and the second antibody are different antibodies; and

a plurality of barcoding particles each comprising a plurality of oligonucleotide probes each comprising a target binding region and a barcode sequence selected from a diverse set of unique barcode sequences.

23. The system of claim 22 , wherein the plurality of barcoding particles comprises sepharose beads, streptavidin beads, agarose beads, magnetic beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorochrome microbeads, hydrogel beads, or a combination thereof.

24. The system of claim 22 , further comprising a plurality of partitions configured to accommodate a single cell and one of the plurality of barcoding particles.

25. The composition of claim 1 , comprising a third oligonucleotide-associated antibody and a fourth oligonucleotide-associated antibody, wherein

the third oligonucleotide-associated antibody comprises a third antibody capable of specifically binding to a third protein target and is associated with a third sample indexing oligonucleotide, wherein the third sample indexing oligonucleotide comprises a binding site for a third target sequence, a third universal primer sequence, and a third sample indexing sequence, and

the fourth oligonucleotide-associated antibody comprises a fourth antibody capable of specifically binding to a fourth protein target and is associated with a fourth sample indexing oligonucleotide, wherein the fourth sample indexing oligonucleotide comprises a binding site for a fourth target sequence, a fourth universal primer sequence, and a fourth sample indexing sequence, and

wherein the third sample indexing sequence and the fourth sample indexing sequence are the same, and the third protein target and the fourth protein target are different.

26. The composition of claim 25 , wherein the first sample indexing sequence and the third sample indexing sequence are different.

27. The composition of claim 25 , wherein the first antibody and the third antibody are the same, or the second antibody and the fourth antibody are the same, or both.

28. The composition of claim 25 , wherein the first protein target and the third protein target are the same, or the second protein target and the fourth protein target are the same, or both.

29. The system of claim 22 , comprising a third oligonucleotide-associated antibody and a fourth oligonucleotide-associated antibody, wherein

the third oligonucleotide-associated antibody comprises a third antibody capable of specifically binding to a third protein target and is associated with a third sample indexing oligonucleotide, wherein the third sample indexing oligonucleotide comprises a binding site for a third target sequence, a third universal primer sequence, and a third sample indexing sequence, and

the fourth oligonucleotide-associated antibody comprises a fourth antibody capable of specifically binding to a fourth protein target and is associated with a fourth sample indexing oligonucleotide, wherein the fourth sample indexing oligonucleotide comprises a binding site for a fourth target sequence, a fourth universal primer sequence, and a fourth sample indexing sequence, and

wherein the third sample indexing sequence and the fourth sample indexing sequence comprise the same sequences, and the third protein target and the fourth protein target are different.

30. The composition of claim 29 , wherein the first sample indexing sequence and the third sample indexing sequence are different.

31. The composition of claim 29 , wherein the first antibody and the third antibody are the same, or the second antibody and the fourth antibody are the same, or both.

32. The composition of claim 29 , wherein the first protein target and the third first protein target are the same, or the second first protein target and the fourth first protein target are the same, or both.

33. The system of claim 22 , wherein at least one of the first antibody and the second antibody is a human antibody or a mouse antibody.

Assignments (6)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 22, 2025
From: CHANG, CHRISTINA; FAN, CHRISTINA; SHUM, ELEEN
To: CELLULAR RESEARCH, INC.
Reel/Frame 071475/0591 →
MERGER Recorded Jun 22, 2025
From: TUCSON ACQUISITION CORP.
To: CELLULAR RESEARCH, INC.
Reel/Frame 071475/0652 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 22, 2025
From: CELLULAR RESEARCH, INC.
To: BECTON, DICKINSON AND COMPANY
Reel/Frame 071475/0690 →
MERGER Recorded Feb 14, 2020
From: TUCSON ACQUISITION CORP.
To: CELLULAR RESEARCH, INC.
Reel/Frame 051827/0079 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 14, 2020
From: CHANG, CHRISTINA; FAN, CHRISTINA; SHUM, ELEEN
To: CELLULAR RESEARCH, INC.
Reel/Frame 051827/0093 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 14, 2020
From: CELLULAR RESEARCH, INC.
To: BECTON, DICKINSON AND COMPANY
Reel/Frame 051827/0099 →
Continuity (9)
Continuation 15937713 · Mar 27, 2018
Provisional Application 62645703 · Mar 20, 2018
Provisional Application 62578957 · Oct 30, 2017
Provisional Application 62554425 · Sep 5, 2017
Provisional Application 62532949 · Jul 14, 2017
Provisional Application 62532905 · Jul 14, 2017
Provisional Application 62532971 · Jul 14, 2017
Provisional Application 62515285 · Jun 5, 2017
Related Publication 20200172957A1 · Jun 4, 2020
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