Single cell bar-coding for antibody discovery
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 first vessels each comprising: (i) a single cell, and (ii) a single solid support; (b) copying onto the single solid support: (i) a first copy of a first cell polynucleotide from the single cell, and (ii) a second copy of a second cell polynucleotide from the single cell; (c) forming a plurality of second vessels each comprising (i) a single solid support from the plurality of first vessels, and (ii) a barcoded polynucleotide; and (d) amplifying (i) the first copy and the second copy with a first primer set, and (ii) the barcode with a second primer set, wherein a primer of the first primer set is complementary to a primer of the second set; and (e) forming first and second single cell barcoded sequences.
1. A method comprising, in at least one vessel of a plurality of vessels:
(A) extending a first cell cDNA of a first cell polynucleotide from a single cell, wherein the first cell cDNA is hybridized to a first barcoded polynucleotide comprising a first primer binding site sequence, a first barcode sequence, and a first cDNA annealing sequence, or an amplicon thereof; and
(B) extending a second cell cDNA of a second cell polynucleotide from the single cell, wherein the second cell cDNA is hybridized to a second barcoded polynucleotide comprising a second primer binding site sequence, a second barcode sequence, and a second 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;
wherein the first barcode sequence and the second barcode sequence comprise the same barcode sequence.
2. The method of claim 1 , wherein the primer set does not comprise the first barcode sequence or second barcode sequence.
3. The method of claim 1 , wherein the at least one vessel of the plurality of vessels comprises the single cell.
4. The method of claim 3 , wherein the single cell is a lysed single cell.
5. The method of claim 1 , wherein the method comprises reverse transcribing the first cell polynucleotide and the second cell polynucleotide.
6. The method of claim 5 , wherein the at least one vessel of the plurality of vessels comprises the single cell and the method comprises lysing the single cell before reverse transcribing the first cell polynucleotide and the second cell polynucleotide.
7. The method of claim 5 , wherein the first cell polynucleotide and second cell polynucleotide are RNA.
8. The method of claim 5 , wherein the first cell polynucleotide and second cell polynucleotide are DNA.
9. The method of claim 5 , 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.
10. 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.
11. The method of claim 10 , 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.
12. The method of claim 11 , 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.
13. The method of claim 11 , wherein the first cell cDNA and the second cell cDNA each comprise a 3′ end with a sequence complementary to the template switch oligonucleotide.
14. The method of claim 11 , wherein the three or more identical non-template nucleotides are 3-riboguanine or 3-guanine.
15. 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 (i) the first barcoded polynucleotide or an amplified product thereof or (ii) the second barcoded polynucleotide or an amplified product thereof.
16. The method of claim 1 , wherein the first primer binding site sequence and the second primer binding site sequence comprise the same primer binding sequence and a primer of the primer set comprises a sequence complementary to the primer binding site sequence.
17. 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.
18. 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.
19. The method of claim 1 , wherein the single cell is a B-cell or a T-cell.
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.