# US 10,246,703 B2 · High-throughput single cell barcoding

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- Number: US 10,246,703 B2
- Document ID: US10246703B2
- Status: Granted
- Kind: B2
- Application number: 15/874,137
- Filed: 2018-01-18
- Published: 2018-05-31
- Granted: 2019-04-02
- Earliest claimed: 2010-10-08
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: George M. Church; Francois Vigneault
- Web page: https://patentgrep.com/patent/US10246703B2

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C40B 50/16: involving encoding steps
- B01J 2219/00547: Bar codes
- B01J 2219/00576: fluorophore
- B01J 2219/00722: Nucleotides
- C12Q 2600/172: Haplotypes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS

## Abstract

Methods and compositions for high-throughput, single cell analyzes are provided. The methods and compositions can be used for analysis of genomes and transcriptomes, as well as antibody discovery, HLA typing, haplotyping and drug discovery.

## Claims

### Claim 1

A method comprising sequestering a single cell and one bead into a vessel;
wherein the one bead comprises a first polynucleotide attached thereto,
wherein the first polynucleotide comprises a sequencing primer sequence, a barcode sequence, a universal primer sequence and an annealing primer sequence.

### Claim 2 (depends on 1)

The method of claim 1, wherein the method further comprises lysing the single cell.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method comprises sequestering a plurality of cells, with each cell of the plurality being a single cell sequestered with one bead.

### Claim 4 (depends on 1)

The method of claim 1, wherein the method comprises sequestering a plurality of cells, with each cell of the plurality being a single cell sequestered with one bead having a unique barcode.

### Claim 5 (depends on 1)

The method of claim 1, wherein the method further comprises attaching a target polynucleotide from the single cell to the annealing primer sequence.

### Claim 6 (depends on 1)

The method of claim 1, wherein the method further comprises correlating a sequence of a barcoded target polynucleotide to a single cell.

### Claim 7 (depends on 1)

The method of claim 1, wherein the vessel is a vessel of an oil in water emulsion.

### Claim 8 (depends on 1)

The method of claim 1, wherein the annealing primer sequence comprises a universal binding sequence.

### Claim 9 (depends on 1)

The method of claim 1, wherein the annealing primer sequence comprises an oligo-d(t) sequence.

### Claim 10 (depends on 1)

The method of claim 1, wherein the single cell is an immune cell, a cell from a tumor, or a cell from a subject with a tumor.

### Claim 11 (depends on 1)

The method of claim 1, wherein the single cell is a B cell or a T cell.

### Claim 12 (depends on 1)

The method of claim 1, wherein the single cell is a bacteria cell.

### Claim 13 (depends on 1)

The method of claim 1, wherein the barcode comprises from 4 nucleotides to 36 nucleotides.

### Claim 14

A bead in an oil-water emulsion comprising
(a) a first polynucleotide,
(b) a second polynucleotide,
(c) a barcode sequence, and
(d) a copy of the barcode sequence;
wherein the first polynucleotide comprises a sequencing primer sequence, the barcode sequence, and an annealing primer sequence which targets a nucleic acid of interest, and
wherein the second polynucleotide comprises a copy of the sequencing primer sequence, the copy of the barcode sequence, and a copy of the annealing primer sequence.

### Claim 15 (depends on 1)

The method of claim 1, further comprising a second polynucleotide attached to the bead, wherein the second polynucleotide comprises the sequencing primer sequence of the first polynucleotide, the barcode sequence of the first polynucleotide, the universal primer sequence of the first polynucleotide, and a second annealing primer sequence.

### Claim 16 (depends on 15)

The method of claim 15, wherein the method further comprises
(i) attaching a first target polynucleotide from the single cell to the first annealing primer sequence of the first polynucleotide and
(ii) attaching a second target polynucleotide from the single cell to the second annealing primer sequence of the second polynucleotide.

### Claim 17 (depends on 15)

The method of claim 15, wherein the first polynucleotide comprises a sequence complementary to a heavy chain polynucleotide sequence, and the second polynucleotide comprises a sequence complementary to a light chain polynucleotide sequence.

### Claim 18 (depends on 16)

The method of claim 16, wherein the method further comprises performing a reverse transcription reaction on the bead to form a first bar-coded target polynucleotide and a second bar-coded target polynucleotide.

### Claim 19 (depends on 16)

The method of claim 16, wherein the method further comprises amplifying (i) the first target polynucleotide, or (ii) the second target polynucleotide before attaching the first and second target polynucleotide.

### Claim 20 (depends on 18)

The method of claim 18, wherein the method further comprises amplifying (i) the first bar-coded polynucleotide, or (ii) the second bar-coded polynucleotide.

### Claim 21 (depends on 20)

The method of claim 20, wherein the method further comprises sequencing (i) the first bar-coded polynucleotide or an amplified product thereof, or (ii) the second bar-coded polynucleotide or an amplified product thereof.

### Claim 22 (depends on 18)

The method of claim 18, wherein the method further comprises sequencing (i) the first bar-coded target polynucleotide or an amplified product thereof, or (ii) the second bar-coded target polynucleotide or an amplified product thereof.

### Claim 23 (depends on 20)

The method of claim 20, wherein the method further comprises sequencing the first barcoded target polynucleotides and the second barcoded target polynucleotides from a plurality of individual cells, or amplified products thereof, the plurality comprising at least one thousand cells.

### Claim 24 (depends on 23)

The method of claim 23, wherein the barcode sequence of the target polynucleotides is unique for each cell.

### Claim 25 (depends on 16)

The method of claim 16, wherein the first target polynucleotide and/or the second target polynucleotide comprises a polynucleotide selected from the group consisting of a polynucleotide having an immune cell nucleic acid sequence, a polynucleotide encoding a T cell receptor component, a polynucleotide encoding an HLA component, a polynucleotide comprising a sequence indicative of haplotype, a polynucleotide encoding a B cell receptor component, a heavy chain polynucleotide, and a light chain polynucleotide.

### Claim 26 (depends on 16)

The method of claim 16, wherein the first target polynucleotide comprises a heavy chain polynucleotide sequence and the second polynucleotide comprises a light chain polynucleotide sequence.

### Claim 27 (depends on 15)

The method of claim 15, wherein the first polynucleotide and/or the second polynucleotide comprises a sequence complementary to a polynucleotide selected from the group consisting of a polynucleotide having an immune cell nucleic acid sequence, a polynucleotide encoding a T cell receptor component, a polynucleotide encoding an HLA component, a polynucleotide comprising a sequence indicative of haplotype, a polynucleotide encoding a B cell receptor component, a heavy chain polynucleotide, and a light chain polynucleotide.