Back to results
US 10,590,483 B2Granted
High-throughput nucleotide library sequencing
AssigneeAbVitro LLC
InventorsFrancois Vigneault, Adrian Wrangham Briggs, Christopher Ryan Clouser, Stephen Jacob Goldfless, Sonia Timberlake
Life of this patent
Estimating the term…
Abstract
Provided herein are methods and composition for immune repertoire sequencing and single cell barcoding. The methods and compositions can be used to pair any two sequences originating from a single cell, such as heavy and light chain antibody sequences, alpha and beta chain T-cell receptor sequences, or gamma and delta chain T-cell receptor sequences, for antibody and T-cell receptor discovery, disease and immune diagnostics, and low error sequencing.
Claims39
A method comprising:
- (a) forming a plurality of vessels each vessel of the plurality comprising a single cell from a sample comprising a plurality of cells, a plurality of molecular-barcoded oligonucleotide molecules each comprising a unique molecular barcode, and a vessel-barcoded oligonucleotide comprising a vessel barcode;
- (b) producing within one or more vessels of the plurality (i) a first complementary polynucleotide that is complementary to a first cell polynucleotide from the single cell and (ii) a second complementary polynucleotide that is complementary to a second cell polynucleotide from the single cell;
- and subsequently
- (c) forming a first molecular-barcoded polynucleotide and a second molecular-barcoded polynucleotide;
- and
- (d) forming a first vessel-molecular-barcoded polynucleotide and a second vessel-molecular-barcoded polynucleotide;
- wherein the molecular-barcoded oligonucleotide molecules of the plurality and the vessel barcoded oligonucleotide in (a) are separate nucleic acid molecules.
4 more elements
A method of barcoding polynucleotides comprising:
- (a) forming a plurality of vessels, each vessel of the plurality comprising a single cell from a sample comprising a plurality of cells, a plurality of molecular-barcoded oligonucleotide molecules each comprising a unique molecular barcode, and a vessel-barcoded oligonucleotide comprising a vessel barcode;
- (b) producing within one or more vessels of the plurality (i) a first complementary polynucleotide that is complementary to a first cell polynucleotide from the single cell and (ii) a second complementary polynucleotide that is complementary to a second cell polynucleotide from the single cell;
- and subsequently
- (c) forming a first molecular-barcoded polynucleotide and a second molecular-barcoded polynucleotide;
- (d) amplifying a vessel-barcoded oligonucleotide to generate one or more copies of the vessel-barcoded oligonucleotide;
- (e) hybridizing a first copy of the one or more copies of the vessel-barcoded oligonucleotide to the first molecular-barcoded polynucleotide and hybridizing a second copy of the one or more copies of the vessel-barcoded oligonucleotide to the second molecular-barcoded polynucleotide;
- and
- (f) forming a first vessel-molecular-barcoded polynucleotide and a second vessel-molecular-barcoded polynucleotide.
5 more elements
Dependencies are inferred from the claim text.