METHODS AND COMPOSITIONS FOR IDENTIFYING OR QUANTIFYING TARGETS IN A BIOLOGICAL SAMPLE

• US 20180251825A1
• Filed: 02/02/2018
• Published: 09/06/2018
• Est. Priority Date: 02/02/2017
• Status: Abandoned Application

• Current Assignee: New York Genome Center
• Original Assignee: New York Genome Center
• Inventors: Stoeckius, Marlon, Smibert, Peter, Houck-Loomis, Brian
• Application Number:
  US15/887,144
• Publication Number:
  US 20180251825A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Compositions, kits and methods are described that comprise one or more constructs, each construct comprising a ligand attached or conjugated to a polymer construct, e.g., an oligonucleotide sequence, by a linker, each ligand binding specifically to a single target located in or on the surface of a cell. The polymer construct comprises a) an Amplification Handle; b) a Barcode that specifically identifies a single ligand; c) an optional Unique Molecular Identifier that is positioned adjacent to the Barcode on its 5′ or 3′ end; and d) an Anchor for hybridizing to a complementary sequence, e.g., for generation of a double-stranded oligonucleotide. These compositions are used in methods, including high throughput methods, for detecting one or more targets or epitopes in a biological sample. These compositions are also used in a high throughput method for characterizing a cell by simultaneous detection of one or more epitopes located in or on the cell and its transcriptome.

Claims

1. A method for detecting a sample or target in a multiplex assay, the method comprising:

a) contacting a first sample with a first construct comprising a first ligand attached to a first oligonucleotide, wherein the first ligand binds specifically to a first target, and the first oligonucleotide comprises;

i) a first amplification handle,

ii) a first barcode that specifically identifies the first sample, and

iii) a first anchor.

2. The method of claim 1, further comprising:

b) contacting a second sample with a second construct comprising a second ligand attached to a second oligonucleotide, wherein the second ligand binds specifically to a second target, and the second oligonucleotide comprises;

i) a second amplification handle,

ii) a second barcode that specifically identifies the second sample, and

iii) a second anchor.

3. The method of claim 2, wherein the first target and the second target are the same target, and optionally, the first amplification handle and the second amplification handle are substantially identical, and optionally, the first anchor and the second anchor are substantially identical.

4. The method of claim 3, further comprising:

c) contacting the first and the second samples with a third construct comprising a third ligand attached to a third oligonucleotide, wherein the third ligand binds specifically to a third target, and the third oligonucleotide comprises;

(i) a third amplification handle,

(ii) a third barcode that specifically identifies the third ligand, and

(iii) a third anchor.

5. The method of claim 4, further comprising:

d) contacting the first and the second samples with a fourth construct comprising a fourth ligand attached to a fourth oligonucleotide, wherein the fourth ligand binds specifically to a fourth target, and the fourth oligonucleotide comprises;

i) a fourth amplification handle,

ii) a fourth barcode that specifically identifies the fourth ligand, and

iii) a fourth anchor.

6. The method of claim 5, wherein the third amplification handle and the fourth amplification handle are substantially identical and are different from the first amplification handle and the second amplification handle.

7. The method of claim 5, wherein the first anchor, the second anchor, the third anchor and the fourth anchor are substantially identical, and optionally comprise a polyA sequence of at least 10 nucleotides in length.

8. The method of claim 5, wherein the third target and the fourth target are different targets, and optionally, the third target is different than the first or second targets, and optionally, the fourth target is different than the first or second targets.

9. The method of claim 5, further comprises

e) contacting a third sample with a fifth construct comprising a fifth ligand that binds specifically to a fifth target, wherein the fifth target is optionally the same as the first target, and the fifth ligand is attached to a fifth oligonucleotide comprising:

i) a fifth amplification handle, optionally substantially the same as the first amplification handle,

ii) a fifth barcode that specifically identifies the third sample, and

iii) a fifth anchor, optionally substantially the same as the first anchor, and optionally comprising a polyA sequence.

10. The method of claim 9, further comprising

f) contacting the first and the second samples, and optionally additional samples with a sixth construct comprising a sixth ligand, wherein the sixth ligand binds specifically to a sixth target, and is attached to a sixth oligonucleotide comprising:

i) a sixth amplification handle, optionally substantially the same as the third amplification handle,

ii) a sixth barcode that specifically identifies the sixth target, and

iii) a sixth anchor, optionally the same as the third anchor, and optionally comprising a polyA sequence.

11. The method of claim 10, wherein the first and the second samples, and optionally one or more additional samples, comprise one or more cells, and the first, second, third, fourth, fifth and sixth targets are present in, or on the surface of, at least one of the one or more cells.

12. The method of claim 11, wherein the contacting of (a), (b), (c), (d), (e) or (f) comprises contacting the one or more cells of the first sample, the second sample, and optional additional samples with the first, second, third, fourth, fifth or sixth constructs.

13. The method of claim 10, wherein the first and the second samples, and optionally one or more additional samples, comprise one or more cell organelles, mitochondria, exosomes, liposomes, synthetic or naturally occurring vesicles, microvesicles, ectosomes, nuclei, bacteria, virus, beads, particles, microparticles, nanoparticles, macromolecules, and synthetic or naturally occurring lipid, phospholipid or membrane spheres, and the first, second, third, fourth, fifth and sixth targets are present in, or on the surface of, at least one of the one or more cell organelles, mitochondria, exosomes, liposomes, synthetic or naturally occurring vesicles, microvesicles, ectosomes, nuclei, bacteria, virus, beads, particles, microparticles, nanoparticles, macromolecules, and synthetic or naturally occurring lipid, phospholipid or membrane spheres.

14. The method of claim 13, wherein the contacting of (a), (b), (c), (d), (e) or (f) comprises contacting the one or more cell organelles, mitochondria, exosomes, liposomes, synthetic or naturally occurring vesicles, microvesicles, ectosomes, nuclei, bacteria, virus, beads, particles, microparticles, nanoparticles, macromolecules, and synthetic or naturally occurring lipid, phospholipid or membrane spheres of the first sample, the second sample, and optional additional samples with the first, second, third, fourth, fifth or sixth constructs.

15. The method of claim 4, wherein the first, second, or third ligands comprise an antibody, or antigen binding fragment thereof

16. The method of claim 4, wherein (i) the first, second, or third anchor is located 3′

of the first, second, or third amplification handle, respectively, and 3′

of the first, second, or third barcode, respectively; and

optionally, (ii) the first, second, or third amplification handle is located 5′

of the first, second, or third barcode, respectively, and 5′

of the first, second, or third anchor, respectively.

17. The method of claim 3, further comprising washing the first sample, the second sample, or a mixture of the first sample and the second sample after any one or more of steps (a), and (b) to remove unbound constructs.

18. The method of claim 11, further comprising, after (a), (b), (c), (d), (e), or (f), encapsulating a first single cell of one of the first, second, or third, samples in a first droplet comprising a first bead conjugated to a plurality of a first capture oligonucleotide comprising, from 5′

to 3′

, a seventh amplification handle, a seventh barcode identifying the first bead, and a sequence complementary to the first, second, third, fourth, fifth or sixth Anchor sequence, and optionally encapsulating a second single cell of one of the first, second, or third samples in a second droplet comprising a second bead conjugated to a plurality of a second capture oligonucleotide comprising, from 5′

to 3′

, the seventh amplification handle, an eighth barcode identifying the second bead, and a sequence complementary to the first, second, third, fourth, fifth or sixth Anchor sequence.

19. The method of claim 18, further comprising, (i) lysing the first and second single cells thereby providing a first lysate encapsulated in the first droplet and a second lysate encapsulated in the second droplet, wherein the first and second lysates comprise mRNA, (ii) contacting the lysate of the first and second cells with a polymerase, (iii) generating cDNA and double stranded oligonucleotide sequences of the first, second, third, fourth, fifth or sixth oligonucleotides, and (iv) amplifying or detecting the first, second, third, fourth, fifth or sixth barcode sequences, optionally wherein the amplifying or detecting is by a process comprising hybridization or nucleic acid sequencing.

20. The method of claim 19, wherein the amplifying or detecting comprises determining the presence, amount or absence of the first, second, and third samples, and optionally, the presence, amount or absence of the first, second, third, fourth, fifth or sixth targets, wherein the first, second, third, fourth, fifth and sixth targets are independently selected from a protein, an amino acid, a carbohydrate or polysaccharide, a lipid, a nucleic acid, a compound, a molecule, an antigen, a cell, a virus, a bacteria, the like, or a combination thereof

21. The method of claim 10 wherein (i) the first, second, third, fourth, fifth or sixth oligonucleotides, or the first or second capture oligonucleotides comprise a UMI, and (ii) the first, second, third, fourth, fifth and sixth anchors are substantially identical and optionally comprise a polyA sequence.

22. A method for detecting at least two targets in at least a first and a second sample, the method comprising:

a) contacting the first sample with a first construct comprising a first ligand attached to a first oligonucleotide, wherein the first ligand binds specifically to a first target, and the first oligonucleotide comprises;

i) a first amplification handle,

ii) a first barcode that specifically identifies the first sample, and

iii) an anchor comprising a polyA sequence;

b) contacting the second sample with a second construct comprising the first ligand attached to a second oligonucleotide, wherein the second oligonucleotide comprises;

i) the first amplification handle,

ii) a second barcode that specifically identifies the second sample, and

iii) the anchor;

c) contacting the first and the second samples with a third construct comprising a second ligand attached to a third oligonucleotide, wherein the second ligand binds specifically to a second target, and the third oligonucleotide comprises;

(i) a second amplification handle,

(ii) a third barcode that specifically identifies the second ligand, and

(iii) the anchor; and

d) contacting the first and the second samples with a fourth construct comprising a third ligand attached to a fourth oligonucleotide, wherein the third ligand binds specifically to a third target, and the fourth oligonucleotide comprises;

i) the second amplification handle,

ii) a fourth barcode that specifically identifies the third ligand, and

iii) the anchor.