# US 10,119,134 B2 · Single cell bar-coding for antibody discovery

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- Number: US 10,119,134 B2
- Document ID: US10119134B2
- Status: Granted
- Kind: B2
- Application number: 14/776,788
- Filed: 2014-03-14
- Published: 2016-02-04
- Granted: 2018-11-06
- Earliest claimed: 2013-03-15
- Assignees: AbVitro LLC
- Applicants: AbVitro LLC
- Inventors: Francois Vigneault; Adrian Wrangham Briggs
- Web page: https://patentgrep.com/patent/US10119134B2

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C12N 15/1041: {Ribosome/Polysome display, e.g. SPERT, ARM}
- C12N 15/1055: {Protein x Protein interaction, e.g. two hybrid selection}
- C12N 15/1062: {mRNA-Display, e.g. polypeptide and encoding template are connected covalently}
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6853: using modified primers or templates
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6883: for diseases caused by alterations of genetic material
- G01N 33/6854: {Immunoglobulins}
- C07K 2317/10: characterized by their source of isolation or production
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/54: F(ab')2
- C07K 2317/55: Fab or Fab'
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/622: Single chain antibody (scFv)
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided herein are methods and composition for immune repertoire sequencing and single cell barcoding. In some aspects, such methods may comprise steps of: (a) forming a plurality of vessels each comprising a first cell cDNA of a first cell polynucleotide from a single cell and a second cell cDNA of a second cell polynucleotide from the single cell; (b) extending the first cell cDNA that is hybridized to a barcoded polynucleotide comprising a primer binding site sequence and a barcode sequence and a cDNA annealing sequence, or an amplicon thereof; and extending the second cell cDNA that is hybridized to a barcoded polynucleotide comprising a primer binding site sequence and the barcode sequence and a cDNA annealing sequence, or an amplicon thereof; and (c) amplifying the extended first cell cDNA and the extended second cell cDNA with a primer set.

## Claims

### Claim 1

A method comprising:
(a) forming a plurality of vessels each comprising a first cell cDNA of a first cell polynucleotide from a single cell and a second cell cDNA of a second cell polynucleotide from the single cell;
(b) extending the first cell cDNA, wherein the first cell cDNA is hybridized to a first barcoded polynucleotide comprising a primer binding site sequence, a barcode sequence, and a cDNA annealing sequence, or an amplicon thereof; and extending the second cell cDNA, wherein the second cell cDNA is hybridized to a second barcoded polynucleotide comprising a primer binding site sequence, the barcode sequence, and a cDNA annealing sequence, or an amplicon thereof; and
(c) amplifying the extended first cell cDNA and the extended second cell cDNA with a primer set.

### Claim 2 (depends on 1)

The method of claim 1, wherein the extended first cell cDNA and the extended second cell cDNA comprise a same barcode sequence.

### Claim 3 (depends on 1)

The method of claim 1, wherein the primer set does not comprise the barcode sequence.

### Claim 4 (depends on 1)

The method of claim 1, wherein the method comprises reverse transcribing the first cell polynucleotide and the second cell polynucleotide.

### Claim 5 (depends on 1)

The method of claim 1, wherein the extending adds three or more identical non-template nucleotides to a 3′ end of the first cell cDNA and the second cell cDNA.

### Claim 6 (depends on 5)

The method of claim 5, wherein the method further comprises hybridizing a template switch oligonucleotide to the three or more identical non-template nucleotides of the first cell cDNA and the second cell cDNA.

### Claim 7 (depends on 6)

The method of claim 6, wherein the extending is after hybridizing the template switch oligonucleotide to the three or more identical non-template nucleotides of the first cell cDNA and the second cell cDNA.

### Claim 8 (depends on 6)

The method of claim 6, wherein the first cell cDNA and the second cell cDNA each comprise a 3′ end with a sequence complementary to the template switch oligonucleotide.

### Claim 9 (depends on 6)

The method of claim 6, wherein the three or more identical non-template nucleotides are 3-riboguanine or 3-guanine.

### Claim 10 (depends on 1)

The method of claim 1, wherein the first cell cDNA and the second cell cDNA each comprise a 3′ end with a sequence complementary to an end of the first barcoded polynucleotide or an amplified product thereof, or the second barcoded polynucleotide or an amplified product thereof.

### Claim 11 (depends on 4)

The method of claim 4, wherein the method comprises lysing the single cell before reverse transcribing the first cell polynucleotide and the second cell polynucleotide.

### Claim 12 (depends on 4)

The method of claim 4, wherein the first cell polynucleotide and second cell polynucleotide are RNA.

### Claim 13 (depends on 4)

The method of claim 4, wherein the first cell polynucleotide and second cell polynucleotide are DNA.

### Claim 14 (depends on 1)

The method of claim 1, wherein a primer of the primer set comprises a sequence complementary to the primer binding site sequence.

### Claim 15 (depends on 1)

The method of claim 1, further comprising sequencing the extended first cell cDNA or an amplified product thereof, the extended second cell cDNA or an amplified product thereof, or both.

### Claim 16 (depends on 15)

The method of claim 15, further comprising determining the first cell polynucleotide and the second cell polynucleotide to be from a same single cell when the barcode sequences of the extended first cell cDNA or an amplified product thereof and the extended second cell cDNA or an amplified product thereof are the same.

### Claim 17 (depends on 1)

The method of claim 1, wherein the first cell polynucleotide comprises a variable heavy chain (VH) sequence and the second cell polynucleotide comprises a variable light chain (VL) sequence.

### Claim 18 (depends on 1)

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

### Claim 19 (depends on 4)

The method of claim 4, wherein reverse transcribing comprises reverse transcribing the first cell polynucleotide and the second cell polynucleotide with a first extension primer and a second extension primer attached to a solid support.

### Claim 20 (depends on 1)

The method of claim 1, wherein the first barcoded polynucleotide, the second barcoded polynucleotide, or a combination thereof is attached to a solid support.

### Claim 21 (depends on 1)

The method of claim 1, wherein each vessel of the plurality comprises a single cell.

### Claim 22 (depends on 21)

The method of claim 21, wherein the single cell is a lysed single cell.

### Claim 23 (depends on 1)

The method of claim 1, wherein the first barcoded polynucleotide comprises a same barcode sequence as the second barcoded polynucleotide.