Sample indexing for single cells

• US 10,676,779 B2
• Filed: 03/27/2018
• Issued: 06/09/2020
• Est. Priority Date: 06/05/2017
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Chang, Christina, Fan, Christina, Shum, Eleen, Martin, Jody, Bansal, Nidhanjali, Ghadiali, James, Lazaruk, Katherine, Lam, Gretchen Yinbon
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US15/937,713
• Publication Number:
  US 20180346970A1
• Time in Patent Office: 805 Days
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals

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

1. A method, comprising:

(a) contacting a first sample comprising a first plurality of cells and a second sample comprising a second plurality of cells with a first sample indexing composition and a second sample indexing composition, respectively, to form a first labeled sample and a second labeled sample, respectively, each comprising cells associated with the first sample indexing composition and the second sample indexing composition, respectively,

wherein the first sample indexing composition comprises a first cellular component binding reagent associated with a first sample indexing oligonucleotide, wherein the first cellular component binding reagent is capable of specifically binding to a cellular component target of each of the first plurality of cells,

wherein the second sample indexing composition comprises a second cellular component binding reagent associated with a second sample indexing oligonucleotide, wherein the second cellular component binding reagent is capable of specifically binding to a cellular component target of each of the second plurality of cells,

wherein the first and second sample indexing oligonucleotides comprise a first sample indexing sequence and a second sample indexing sequence, respectively, and wherein the first sample indexing sequence and the second sample indexing sequence comprise different sequences, and

wherein the cellular component target of each of the first plurality of cells and the cellular component target of each of the second plurality of cells are the same;

(b) pooling the first labeled sample and the second labeled sample to form a combined labeled sample comprising cells associated with the first sample indexing composition and cells associated with the second sample indexing composition, respectively;

(c) partitioning the cells associated with the first sample indexing composition and the cells associated with the second sample indexing composition of the combined labeled sample to a plurality of partitions, wherein the partitions of the plurality of partitions each comprises a single cell of the combined labeled sample;

(d) subjecting the first sample indexing oligonucleotide or the second sample indexing oligonucleotide to one or more reactions to generate a first barcoded sample indexing oligonucleotide comprising the first sample indexing sequence, or a complement thereof, or a second barcoded sample indexing oligonucleotide comprising the second sample indexing sequence, or a complement thereof;

(e) obtaining sequencing information of the first barcoded sample indexing oligonucleotide or a second barcoded sample indexing oligonucleotide, or a portion thereof; and

(f) determining cells in the combined labeled sample is from the first sample or the second sample based on the first sample indexing sequence or the second sample indexing sequence, respectively, a complementary sequence thereof, or a portion thereof, in the sequence information obtained in (e).

2. The method of claim 1, wherein the cellular component binding reagent comprises a protein binding reagent, wherein the protein binding reagent is associated with the sample indexing oligonucleotide associated with the corresponding cellular component binding reagent, and wherein the cellular component target comprises a protein target.

3. The method of claim 1, wherein subjecting the first sample indexing oligonucleotide or the second sample indexing oligonucleotide to one or more reactions to generate a first barcoded sample indexing oligonucleotide comprising the first sample indexing sequence, or complement thereof, or a second barcoded sample indexing oligonucleotide comprising the second sample indexing sequence, or a complementary sequence thereof, in (d) comprises:

barcoding the first sample indexing oligonucleotide or the second sample oligonucleotide using a plurality of barcodes to generate the first barcoded sample indexing oligonucleotide or the second barcoded sample indexing oligonucleotide.

4. The method of claim 1, wherein determining the cells in the combined labeled sample is from the first sample or the second sample comprises identifying the presence or absence of the first sample indexing sequence, the second sample index sequence, a complementary sequence thereof, or a portion thereof, in the barcoded sample indexing compositions.

5. The method of claim 1, wherein at least one of the first sample indexing sequence and the second sample indexing sequence is 6-60 nucleotides in length.

6. The method of claim 1, wherein at least one of the cellular component targets of each of the first plurality of cells and the cellular component target of each of the second plurality of cells is on a cell surface.

7. The method of claim 1, comprising removing the first and second sample indexing compositions that are not associated with the first plurality and the second plurality of cells.

8. The method of claim 1, wherein the first sample indexing oligonucleotide is configured to be non-detachable from the first cellular component binding reagent, or the second sample indexing oligonucleotide is configured to be non-detachable from the second component binding reagent, or both.

9. The method of claim 1, comprising detaching the first sample indexing oligonucleotide from the first cellular component binding reagent, detaching the second sample indexing oligonucleotide from the second cellular component binding reagent, or both.

10. The method of claim 3, wherein the first and/or second sample indexing oligonucleotide comprises a sequence complementary to a capture sequence configured to capture the sequence of the first and/or second sample indexing oligonucleotide, and wherein each of the barcodes comprises a target-binding region which comprises the capture sequence.

11. The method of claim 10, wherein the target-binding region comprises a poly(dT) region, and wherein the sequence of the sample indexing oligonucleotide complementary to the capture sequence comprises a poly(dA) region.

12. The method of claim 1, wherein at least one of the first sample indexing oligonucleotide and the second sample indexing oligonucleotide comprises a molecular label sequence, a binding site for a universal primer, or both.

13. The method of claim 1, wherein the cellular component target of each of the first plurality of cells, the cellular component target of each of the second plurality of cells, or both comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell-surface protein, a cell marker, a B-cell receptor, a T-cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

14. The method of claim 3, wherein the plurality of barcodes is associated with a particle.

15. The method of claim 14, wherein at least one barcode of the plurality of barcodes is immobilized on the particle, partially immobilized on the particle, enclosed in the particle, partially enclosed in the particle, or a combination thereof.

16. The method of claim 14, wherein the particle is a disruptable bead or a disruptable hydrogel bead.

17. The method of claim 14, wherein barcoding the first sample indexing oligonucleotide or the second sample indexing oligonucleotide using the plurality of barcodes comprises:

contacting the plurality of barcodes with the first sample indexing oligonucleotide or the second sample indexing oligonucleotide to allow barcodes to hybridize to the first sample indexing oligonucleotide or the second sample indexing oligonucleotide, wherein each of the barcodes comprises a target binding region capable of specifically binding to the first sample indexing oligonucleotide or the second sample indexing oligonucleotide, or both; and

extending the barcodes hybridized to the first sample indexing oligonucleotide or the second sample indexing oligonucleotide to generate the first barcoded sample indexing oligonucleotide or the second barcoded sample indexing oligonucleotide.

18. The method of claim 14, wherein each of the plurality of barcodes comprises an identical barcode sequence.

19. The method of claim 1, wherein at least one of the first sample indexing oligonucleotide and the second sample indexing oligonucleotide is 50-500 nucleotides in length.

20. The method of claim 1, wherein at least one of the first cellular component binding reagent and the second cellular component binding reagent comprises an antibody, a tetramer, an aptamer, a protein scaffold, or a combination thereof.

21. The method of claim 1, wherein the first sample indexing oligonucleotide is conjugated to the first cellular component binding reagent through a linker, or the second sample indexing oligonucleotide is conjugated to the second cellular component binding reagent through a linker, or both.

22. The method of claim 1, wherein the first cellular component binding reagent and the second cellular component binding reagent are the same.

23. The method of claim 1, wherein at least one of the cellular component target of each of the first plurality of cells and the cellular component target of each of the second plurality of cells comprises a cell surface protein, an intracellular protein, a cell marker, a B-cell receptor, a T-cell receptor, an antibody, a major histocompatibility complex, a tumor antigen, a receptor, or a combination thereof.

24. The method of claim 1, wherein the plurality of partitions comprises a well or a droplet.

25. The method of claim 1, wherein the first plurality of cells, the second plurality of cells, or both comprise T cells, B cells, tumor cells, myeloid cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof.

26. The method of claim 3, wherein each of the plurality of barcodes comprises a cell label, a binding site for a universal primer, an amplification adaptor, a sequencing adaptor, or a combination thereof.

27. The method of claim 14, wherein partitioning the first plurality of cells associated with the first sample indexing composition and the second plurality of cells associated with the second sample indexing composition of the combined barcoded sample to a plurality of partitions in (c) comprises partitioning the first plurality of cells associated with the first sample indexing composition, the second plurality of cells associated with the second sample indexing composition of the combined barcoded sample, and a plurality of particles comprising the particle that comprises the plurality of barcodes.

28. The method of claim 14, comprising releasing one or more of the plurality of barcodes from the particle in the partition comprising the single cell prior to (d).

29. The method of claim 1, wherein subjecting the first sample indexing oligonucleotide or the second sample indexing oligonucleotide to one or more reactions comprises in the partitions of the plurality of partitions, subjecting the first sample indexing oligonucleotide or the second sample indexing oligonucleotide to one or more reactions to generate a first barcoded sample indexing oligonucleotide comprising the first sample indexing sequence, or a complement thereof, or a second barcoded sample indexing oligonucleotide comprising the second sample indexing sequence, or a complement thereof.