High-throughput nucleotide library sequencing

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.

1 - 525 . (canceled) 
     
     
         526 . A composition comprising a plurality of vessels, wherein at least one vessel of the plurality comprises:
 (a) a single cell from a sample comprising a plurality of cells;   (b) a plurality of molecular-barcoded oligonucleotide molecules;   (c) a vessel-barcoded oligonucleotide;   (d) a first polynucleotide comprising (i) a first molecular barcode sequence from a first molecular-barcoded oligonucleotide molecule of a plurality or a complement thereof, (ii) a vessel barcode sequence from the vessel barcoded oligonucleotide or a complement thereof, and (iii) a region complementary to a first cell polynucleotide from the single cell; and   (e) a second polynucleotide comprising (i) a second molecular barcode sequence from a second molecular-barcoded oligonucleotide molecule of the plurality or a complement thereof, (ii) the vessel barcode sequence from the vessel barcoded oligonucleotide or a complement thereof, and (iii) a region complementary to a second cell polynucleotide from the single cell;   wherein the molecular-barcoded oligonucleotide molecules of the plurality and the vessel-barcoded oligonucleotide are separate nucleic acid molecules.   
     
     
         527 . The composition of  claim 526 , wherein the first cell polynucleotide is a heavy chain immunoglobulin (IgH) polynucleotide, a T-cell receptor alpha (TCRα) polynucleotide, or a T-cell receptor gamma (TCRγ) polynucleotide; and wherein the second cell polynucleotide is a light chain immunoglobulin (IgL) polynucleotide, a T-cell receptor beta (TCRβ) polynucleotide, or a T-cell receptor delta (TCRδ) polynucleotide. 
     
     
         528 . The composition of  claim 526 , wherein the single cell is a B-cell or a T-cell. 
     
     
         529 . The composition of  claim 526 , wherein the first and the second polynucleotides in the at least one vessel of the plurality comprise different molecular barcode sequences. 
     
     
         530 . The composition of  claim 526 , wherein the molecular-barcoded oligonucleotide molecules of the plurality are not amplified products. 
     
     
         531 . The composition of  claim 526 , wherein a molecular barcode sequence of a molecular-barcoded oligonucleotide molecule of the plurality in a first vessel of the plurality is different than a molecular barcode sequence of a molecular-barcoded oligonucleotide molecule of the plurality in a second vessel of the plurality. 
     
     
         532 . The composition of  claim 526 , wherein a vessel barcode sequence of a vessel-barcoded oligonucleotide in a first vessel of the plurality is different from a vessel barcode sequence of a vessel-barcoded oligonucleotide in a second vessel of the plurality. 
     
     
         533 . The composition of  claim 526 , wherein the vessel-barcoded oligonucleotide in the at least one vessel of the plurality is present as a single molecule. 
     
     
         534 . The composition of  claim 526 , wherein the vessel barcode sequences of the first and the second polynucleotides in the at least one vessel of the plurality are the same. 
     
     
         535 . The composition of  claim 526 , wherein the vessel-barcoded oligonucleotides in two or more different vessels of the plurality comprise a first common-vessel sequence upstream of the vessel barcode sequence, a second common-vessel sequence downstream of the vessel barcode sequence, or both. 
     
     
         536 . The composition of  claim 526 , wherein each of the molecular-barcoded oligonucleotide molecules of the plurality in a same vessel of the plurality comprises a first common-molecular sequence upstream of the molecular barcode sequence, a second common-molecular sequence downstream of the molecular barcode sequence, or both. 
     
     
         537 . The composition of  claim 526 , wherein each of the molecular-barcoded oligonucleotide molecules of the plurality in each vessel of the plurality comprises a first common-molecular sequence upstream of the molecular barcode sequence, a second common-molecular sequence downstream of the molecular barcode sequence, or both. 
     
     
         538 . The composition of  claim 526 , wherein the vessel-barcoded oligonucleotide or a complement thereof comprises a sequence complementary to a region of a complement of each molecular-barcoded oligonucleotide molecule of the plurality. 
     
     
         539 . The composition of  claim 526 , wherein the vessel-barcoded oligonucleotide or a complement thereof comprises a sequence complementary to a 3′ end region of a first and a second molecular-barcoded polynucleotide. 
     
     
         540 . The composition of  claim 526 , wherein the vessels of the plurality are emulsions. 
     
     
         541 . The composition of  claim 526 , wherein the single cell is lysed. 
     
     
         542 . The composition of  claim 526 , wherein a first target primer, a second target primer, the vessel-barcoded oligonucleotide, the plurality of molecular-barcoded oligonucleotide molecules, or any combination thereof, is not attached to a solid support. 
     
     
         543 . The composition of  claim 526 , wherein the first and the second cell polynucleotides are RNA. 
     
     
         544 . The composition of  claim 526 , further comprising a non-template terminal transferase, a reverse transcriptase, a polymerase, or any combination thereof. 
     
     
         545 . The composition of  claim 526 , wherein the vessel-barcoded oligonucleotide is attached to a solid support.