Single cell bar-coding for antibody discovery

• US 10,119,134 B2
• Filed: 03/14/2014
• Issued: 11/06/2018
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Abvitro LLC (Bristol-Myers Squibb Company)
• Original Assignee: Abvitro LLC (Bristol-Myers Squibb Company)
• Inventors: Vigneault, Francois, Briggs, Adrian Wrangham
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US14/776,788
• Publication Number:
  US 20160032282A1
• Time in Patent Office: 1,698 Days
• Field of Search: 435 612, 435 912
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/54 F(ab')2
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1041 Ribosome/Polysome display, e.g. SPERT, ARM
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• 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: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• 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 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays
• CPC Class Codes: G01N 33/6854 : Immunoglobulins

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

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.

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

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

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

5. 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.

6. 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.

7. 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.

8. 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.

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

10. 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.

11. 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.

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

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

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

15. 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.

16. 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.

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

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

19. 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.

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

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

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

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