METHODS FOR IDENTIFYING MULTIPLE EPITOPES IN SELECTED SUB-POPULATIONS OF CELLS

• US 20180320241A1
• Filed: 12/21/2015
• Published: 11/08/2018
• Est. Priority Date: 12/19/2014
• Status: Abandoned Application

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Inventors: Nolan, Garry P.
• Application Number:
  US15/525,876
• Publication Number:
  US 20180320241A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

A method for identifying a sub-population within a mixed population of cells is disclosed. The method involves contacting the mixed population of cells with at least one unique binding agent, wherein the at least one unique binding agent is designed to bind to a target molecule present in the sub-population, and wherein the at least one unique binding agent is attached to an epitope specific barcode that represents the identity of the target molecule. The method further involves sequentially attaching two or more assayable polymer subunits to the epitope specific barcode to create unique cell origination barcodes that represent the identities of individual cells to which the at least one unique binding agent has bound; and decoding the epitope specific barcode and cell origination barcodes, thereby identifying the sub-population within the mixed population of cells.

Claims

1. A method for identifying a sub-population within a mixed population of cells, the method comprising:

a) contacting the mixed population of cells with at least one unique binding agent, wherein the at least one unique binding agent is designed to bind to a target molecule present in the sub-population, and wherein the at least one unique binding agent is attached to an epitope specific barcode that represents the identity of the target molecule;

b) sequentially attaching two or more assayable polymer subunits to the epitope specific barcode to create unique cell origination barcodes that represent the identities of individual cells to which the at least one unique binding agent has bound; and

c) decoding the epitope specific barcode and cell origination barcodes, thereby identifying the sub-population within the mixed population of cells.

2. The method of claim 1, wherein the epitope specific barcode and the assayable polymer subunits comprise oligonucleotide sequences.

3. The method of claim 1, wherein the two or more assayable polymer subunits are attached to the epitope specific barcode using a split-pool combinatorial synthesis approach.

4. The method of claim 2, wherein each occurrence of the epitope specific barcode and the two or more assayable polymer subunits of an associated cell origination barcode are linked to form a conjugate that can be amplified and sequenced.

5. The method of claim 4, further comprising amplification of epitope specific barcode—

cell origination barcode conjugates.

6. The method of claim 5, wherein the decoding step comprises sequencing all or a portion of the amplified epitope specific barcode—

cell origination barcode conjugates.

7. The method of claim 6, wherein a ratio of the number of cell origination barcodes associated with the sub-population to the total number of cells in the mixed population provides a measure of the fraction of cells within the mixed population that contain the target molecule.

8. The method of claim 6, wherein two or more unique binding agents are used to identify the subpopulation.

9. The method of claim 6, wherein two or more unique binding agents are used to identify two or more subpopulations.

10. The method of claim 8, wherein at least one of the unique binding agents is designed to bind to a target molecule selected from the group consisting of DNA, histones, and housekeeping proteins.

11. The method of claim 10, wherein the at least one unique binding agent is an antibody or antibody fragment directed towards a target molecule selected from the group consisting of DNA, histones, and housekeeping proteins.

12. The method of claim 10, wherein the at least one unique binding agent is a DNA intercalating molecule selected from the group consisting of berberine, ethidium bromide, proflavine, daunomycin, dactinomycin, doxorubicin, daunorubicin, and thalidomide.

13. The method of claim 10, wherein the at least one unique binding agent comprises an amine-reactive probe selected from the group consisting of succinimidyl esters, sulfosuccinimidyl esters, tetrafluorophenyl esters, sulfodichlorophenol esters, isothiocyanates, and sulfonyl chlorides.

14. A method for detecting one or more target molecules in a sub-population of cells, the method comprising:

a) contacting a sample comprising a complex mixture of cells with two or more unique binding agents, wherein the two or more unique binding agents are designed to bind to different target molecules, and wherein the two or more unique binding agents are attached to epitope specific barcodes that represent the identities of the target molecules;

b) sequentially attaching two or more assayable polymer subunits to the epitope specific barcodes to create unique cell origination barcodes that represent the identities of individual cells to which one or more unique binding agents have bound;

c) selectively amplifying and sequencing the epitope specific barcode and cell origination barcodes associated with at least a first unique binding agent to identify a sub-population of cells; and

d) selectively amplifying and sequencing the epitope specific barcode(s) associated with at least a second unique binding agent that are attached to cell origination barcodes matching those identified in step (c) to detect the presence of at least a second target molecule in individual cells of the specified sub-population of cells.

15. The method of claim 14, wherein at least one unique binding agent comprises a nucleic acid sequence that is capable of hybridizing to an intracellular nucleic acid sequence.

16. The method of claim 15, wherein the at least one unique binding agent comprises a nucleic acid sequence that is capable of hybridizing to a viral genome nucleic acid sequence.

17. The method of claim 16, wherein the at least one unique binding agent comprises a nucleic acid sequence that is capable of hybridizing to an HIV viral genome nucleic acid sequence.

18. The method of claim 14, wherein at least one unique binding agent comprises an antibody or antibody fragment directed towards a cell surface marker.

19. The method of claim 14, wherein the selective amplification of step (d) comprises performing two or more successive rounds of multiplexed, nested amplification reactions.

20. The method of claim 19, wherein each successive round of amplification utilizes a set of primers designed to hybridize to the assayable polymer subunits located at a different position in the sequence of two or more assayable polymer subunit positions that constitute the cell origination barcodes identified in step (c).