Sample indexing for single cells

• US 10,669,570 B2
• Filed: 06/19/2018
• Issued: 06/02/2020
• 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
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US16/012,635
• Publication Number:
  US 20180346969A1
• Time in Patent Office: 714 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 oligonucleotide-conjugated antibody and a second oligonucleotide-conjugated antibody, respectively, to form a first barcoded sample and a second barcoded sample, respectively, each comprising cells associated with the first oligonucleotide-conjugated antibody and the second oligonucleotide-conjugated antibody, respectively,

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

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

wherein the first sample indexing sequence and the second sample indexing sequence comprise different sequences and

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

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

(c) partitioning the first plurality of cells associated with the first oligonucleotide-conjugated antibody and the second plurality of cells associated with the second oligonucleotide-conjugated antibody of the combined barcoded sample to a plurality of partitions, wherein partitions of the plurality of partitions each comprises a single cell of the combined barcoded sample;

(d) in each of the partitions of the plurality of partitions, contacting a barcoding particle with the first sample indexing oligonucleotide or the second 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 of the barcoding particle;

(e) extending each oligonucleotide probe hybridized to the first sample indexing oligonucleotide or the second indexing oligonucleotide via the hybridization between the poly(A) tail of the first sample indexing oligonucleotide or the second sample indexing oligonucleotide and the poly(T) region of the oligonucleotide probe to produce a first labeled nucleic acid or a second labeled nucleic acid, wherein the first labeled nucleic acid and the second labeled nucleic acid comprises a first sample indexing sequence and a second sample indexing sequence, respectively, or a complementary sequence thereof, and the barcode sequence; and

(f) obtaining sequencing information of the first labeled nucleic acid comprising the first sample indexing sequence or the second labeled nucleic acid comprising the second sample indexing sequence, or a portion thereof; and

(g) determining each single cell in the partitions of the plurality of partitions of the combined barcoded 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.

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

3. The method of claim 1, wherein the first antibody capable of specifically binding to the protein target of each of the first plurality of cells and the second antibody capable of specifically binding to the protein target of each of the second plurality of cells are the same.

4. The method of claim 1, comprising detaching the first sample indexing oligonucleotide and the second sample indexing oligonucleotide from the first oligonucleotide-conjugated antibody and the second oligonucleotide-conjugated antibody, respectively.

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 first sample indexing oligonucleotide and the second sample indexing oligonucleotide is 50-500 nucleotides in length.

7. The method of claim 1, wherein the at least one of the protein target of each of the first plurality of cells and the protein target of each of the second plurality of cells 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.

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

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

10. 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.

11. The method of claim 1, wherein at least one oligonucleotide probe of the plurality of oligonucleotide probes is immobilized on the barcoding particle, partially immobilized on the particle, enclosed in the particle, partially enclosed in the particle, or a combination thereof.

12. The method of claim 1, wherein step (d) comprises releasing the plurality of oligonucleotide probes from the barcoding particle in the partition comprising the single cell.

13. The method of claim 1, wherein the barcoding 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.

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

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

16. The method of claim 1, wherein a single cell of one of the partitions of the plurality of partitions comprises a plurality of proteins of interest, and wherein:

step (a) further comprises contacting a third plurality of oligonucleotide-conjugated antibodies with the single cell, wherein each of the third 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 first plurality of labeled nucleic acids, wherein each of the first plurality of labeled nucleic acids comprises a unique identifier, or a complementary sequence thereof, and a barcode sequence; and

step (f) further comprises obtaining sequence information of the first 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 the single cell.

17. The method of claim 16, 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.

18. The method of claim 16, wherein a protein of interest of the plurality of proteins 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.

19. The method of claim 16, wherein the single cell comprises a plurality of mRNA target molecules, and wherein:

step (d) further comprises hybridizing mRNA target molecules from the single 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 probes;

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 second plurality of labeled nucleic acids, wherein each of the second plurality of labeled nucleic acids 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 second 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 the single cell.