Single cell bar-coding for antibody discovery
First Claim
1. A method comprising:
- (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, wherein the first primer set comprises a first primer complementary to a 3′
portion of the first cell polynucleotide and a 3′
portion of the second cell polynucleotide, and a second primer complementary to a 5′
portion of the first cell polynucleotide and a 5′
portion of the second cell polynucleotide, and(ii) the barcoded polynucleotide with a second primer set, wherein a primer of the second primer set comprises a third primer complementary to a forward primer binding sequence of the barcoded polynucleotide, and a fourth primer complementary to a reverse primer binding sequence of the barcoded polynucleotide,wherein the first primer and the fourth primer are complementary, the second primer and third primer are complementary, the first primer and the third primer are complementary, or the second primer and the fourth primer are complementary; and
wherein the amplifying comprises extending the first copy when hybridized to the barcoded polynucleotide or an amplicon thereof, and extending the second copy when hybridized to the barcoded polynucleotide or an amplicon thereof;
thereby forming first and second single cell barcoded sequences.
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Accused Products
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 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.
203 Citations
38 Claims
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1. A method comprising:
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(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, wherein the first primer set comprises a first primer complementary to a 3′
portion of the first cell polynucleotide and a 3′
portion of the second cell polynucleotide, and a second primer complementary to a 5′
portion of the first cell polynucleotide and a 5′
portion of the second cell polynucleotide, and(ii) the barcoded polynucleotide with a second primer set, wherein a primer of the second primer set comprises a third primer complementary to a forward primer binding sequence of the barcoded polynucleotide, and a fourth primer complementary to a reverse primer binding sequence of the barcoded polynucleotide, wherein the first primer and the fourth primer are complementary, the second primer and third primer are complementary, the first primer and the third primer are complementary, or the second primer and the fourth primer are complementary; and wherein the amplifying comprises extending the first copy when hybridized to the barcoded polynucleotide or an amplicon thereof, and extending the second copy when hybridized to the barcoded polynucleotide or an amplicon thereof; thereby forming first and second single cell barcoded sequences. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A method comprising:
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(a) forming a plurality of vessels each comprising a single cell; (b) extending (i) a first extension primer hybridized to a first cell polynucleotide from the single cell, thereby forming a first cell cDNA, and (ii) a second extension primer hybridized to a second cell polynucleotide from the single cell, thereby forming a second cell cDNA; and (c) amplifying (i) the first cell cDNA and the second cell cDNA with a first primer set, wherein the first primer set comprises a first primer complementary to a 3′
portion of the first cell cDNA and a 3′
portion of the second cDNA, and a second primer complementary to a 5′
portion of the first cell cDNA and a 5′
portion of the second cDNA, and(ii) a barcode of a barcoded polynucleotide with a second primer set, wherein a primer of the second primer set comprises a third primer complementary to a forward primer binding sequence of the barcoded polynucleotide and a fourth primer complementary to a reverse primer binding sequence of the barcoded polynucleotide, wherein the first primer and the fourth primer are complementary, the second primer and third primer are complementary, the first primer and the third primer are complementary, or the second primer and the fourth primer are complementary; and wherein the amplifying the first cell cDNA and the second cell cDNA comprises extending the first cell cDNA when hybridized to the barcoded polynucleotide or an amplicon thereof and extending the second cell cDNA when hybridized to the barcoded polynucleotide or an amplicon thereof; thereby forming first and second single cell barcoded sequences. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
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Specification