SAMPLE INDEXING FOR SINGLE CELLS

• US 20180346969A1
• Filed: 06/19/2018
• Published: 12/06/2018
• Est. Priority Date: 06/05/2017
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Chang, Christina, Fan, Christina, Shum, Eleen
• Granted Patent: US 10,669,570 B2
• Time in Patent Office: 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 for sample identification, comprising:

(a) contacting each of a plurality of samples comprising one or more cells with a first oligonucleotide-conjugated antibody to form a plurality of barcoded samples, respectively, each comprising one or more cells associated with a first oligonucleotide-conjugated antibody,

wherein each of the one or more cells comprises a protein target for sample indexing,

wherein the first antibody is capable of specifically binding to the protein target for sample indexing, and is conjugated with a sample indexing oligonucleotide comprising a poly(A) tail and a sample indexing sequence, and

wherein the sample indexing sequences of the first oligonucleotide-conjugated antibodies associated with cells of different barcoded samples comprise different sequences;

(b) pooling the plurality of barcoded samples to form a combined barcoded sample comprising a plurality of cells associated with the first oligonucleotide-conjugated antibodies;

(c) partitioning the plurality of cells from the combined barcoded sample to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of cells associated with the first oligonucleotide-conjugated antibodies;

(d) in the partition comprising the single cell, contacting a barcoding particle with the sample indexing oligonucleotide, wherein the barcoding particle comprises a plurality of oligonucleotide probes each comprising a poly(T) region and a barcode sequence common to the plurality of oligonucleotide probes;

(e) extending the oligonucleotide probes hybridized to the sample indexing oligonucleotide via the hybridization between the poly(A) tails of the sample indexing oligonucleotides and the poly(T) region of the oligonucleotide probes to produce a first plurality of labeled nucleic acids, wherein each of the first plurality of labeled nucleic acid comprises a sample indexing sequence, or a complementary sequence thereof, and the barcode sequence; and

(f) obtaining sequencing information of the first plurality of labeled nucleic acids or a portion thereof to determine sample origins of one or more of the plurality of cells in the combined barcoded sample.

2. The method of claim 1, comprising after step (a) removing the first oligonucleotide-conjugated antibodies that are not associated with the plurality of cells.

3. The method of claim 1, wherein the protein targets for sample indexing in each of the plurality of samples are the same.

4. The method of claim 1, wherein the protein targets for sample indexing in two or more of the plurality of samples are different.

5. The method of claim 1, comprising detaching the sample indexing oligonucleotide from the first oligonucleotide-conjugated antibody.

6. The method of claim 1, wherein the sample indexing sequence is 6-60 nucleotides in length.

7. The method of claim 1, wherein the sample indexing oligonucleotide is 50-500 nucleotides in length.

8. The method of claim 1, wherein the protein target is 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.

9. The method of claim 1, wherein partitioning the plurality of cells from the combined barcoded sample in step (c) comprises partitioning the plurality of cells associated with the first oligonucleotide-conjugated antibodies and a plurality of barcoding particles comprising the barcoding particle to the plurality of partitions, wherein the partition of the plurality of partitions comprises the single cell associated with the first oligonucleotide-conjugated antibody and the barcoding particle.

10. The method of claim 1, wherein step (f) obtaining sequencing information of the first plurality of labeled nucleic acids or a portion thereof comprises subjecting the labeled nucleic acids to one or more reactions to generate a set of nucleic acids for nucleic acid sequencing.

11. The method of claim 1, wherein each of the oligonucleotide probes comprises a cell label, a binding site for a universal primer, an amplification adaptor, a sequencing adaptor, or a combination thereof.

12. The method of claim 1, wherein at least one barcode of the plurality of barcodes is immobilized on the barcoding particle, partially immobilized on the particle, enclosed in the particle, partially enclosed in the particle, or a combination thereof.

13. The method of claim 1, comprising releasing the plurality of oligonucleotide probes form the barcoding particle in the partition comprising the single cell prior to step (d).

14. The method of claim 1, wherein the particle is a Sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, a hydrogel bead, or a disruptable hydrogel bead.

15. The method of claim 1, wherein the partition is a well or a droplet.

16. The method of claim 1, wherein the one or more cells comprise T cells, B cells, tumor cells, myeloid cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof.

17. The method of claim 1, wherein each of the one or more cells comprises a plurality of proteins of interest, and wherein:

step (a) further comprises contacting a plurality of second oligonucleotide-conjugated antibodies with the one or more cells, wherein each of the second plurality of oligonucleotide-conjugated antibodies comprises an antibody conjugated with an antibody specific oligonucleotide comprising a unique identifier for the antibody conjugated therewith and a poly(A) tail, and wherein the antibody is capable of specifically binding to at least one of the plurality of proteins of interest;

step (e) further comprises extending the oligonucleotide probes hybridized to the antibody specific oligonucleotides via the hybridization between the poly(A) tails of the antibody specific oligonucleotides and the poly(T) regions of the oligonucleotide probes to produce a second plurality of labeled nucleic acids, wherein each of the second plurality of labeled nucleic acid comprises a unique identifier, or a complementary sequence thereof, and a barcode sequence; and

step (f) further comprises obtaining sequence information of the second plurality of labeled nucleic acids or a portion thereof to determine the quantity of one or more of the plurality of proteins of interest in at least one of the one or more cells.

18. The method of claim 17, wherein the antibody specific oligonucleotide comprises a molecular label, a cell label, a binding site for a universal primer, an amplification adaptor, a sequencing adaptor, or a combination thereof.

19. The method of claim 17, wherein the protein of interest is 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.

20. The method of claim 17, wherein the each of the one or more cells comprises a plurality of mRNA target molecules, and wherein:

step (d) further comprises hybridizing mRNA target molecules from the cell with oligonucleotide probes of the plurality of oligonucleotide probes between the poly(A) tails of the mRNA target molecules and the poly(T) regions of the oligonucleotide probe;

step (e) further comprises extending the oligonucleotide probes hybridized to the mRNA target molecules via the hybridization between the poly(A) tails of the mRNA target molecules and the poly(T) regions of the oligonucleotide probes to produce a third plurality of labeled nucleic acids, wherein each of the third plurality of labeled nucleic acid comprises a complementary sequence of one of the mRNA target molecules and a barcode sequence; and

step (f) further comprises obtaining sequence information of the third plurality of labeled nucleic acids or a portion thereof to determine the quantity of one or more of the plurality of mRNA target molecules in at least one of the one or more cells.