HIGH-THROUGHPUT NUCLEOTIDE LIBRARY SEQUENCING
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 method of barcoding polynucleotides in at least a first vessel and a second vessel of a plurality of vessels,
wherein the first and second vessels each comprises (i) a single cell from a plurality of cells, (ii) a plurality of molecular-barcoded oligonucleotide molecules each comprising a unique molecular barcode, and (ii) a vessel-barcoded oligonucleotide comprising a vessel barcode, wherein the vessel barcode in the first vessel is different from the vessel barcode in the second vessel; and wherein the molecular-barcoded oligonucleotide molecules of the plurality and the vessel-barcoded oligonucleotide are separate nucleic acids,
the method comprising:
(a) producing within the first and second vessels (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;
(b) forming a first molecular-barcoded polynucleotide and a second molecular-barcoded polynucleotide, wherein the first molecular-barcoded polynucleotide comprises the molecular barcode of a first molecular-barcoded oligonucleotide molecule of the plurality or an amplified product thereof and the first complementary polynucleotide or an amplified product thereof, and the second molecular-barcoded polynucleotide comprises the molecular barcode of a second molecular-barcoded oligonucleotide molecule of the plurality or an amplified product thereof and the second complementary polynucleotide or an amplified product thereof, and
(c) forming a first vessel-molecular-barcoded polynucleotide and a second vessel-molecular-barcoded polynucleotide, wherein the first vessel-molecular-barcoded polynucleotide comprises the vessel barcode of the vessel-barcoded oligonucleotide or an amplified product thereof and the first molecular-barcoded polynucleotide or an amplified product thereof, and the second vessel-molecular-barcoded polynucleotide comprises the vessel barcode of the vessel-barcoded oligonucleotide or an amplified product thereof and the second molecular-barcoded polynucleotide or an amplified product thereof,
wherein the first and the second vessel-molecular-barcoded polynucleotides in a single vessel of the plurality comprise different molecular barcodes, and the vessel barcode of the first and the second vessel-molecular-barcoded polynucleotides in a single vessel of the plurality are the same.
527 . The method 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 method of claim 526 , further comprising amplifying the first and the second vessel-molecular-barcoded polynucleotides.
529 . The method of claim 528 , further comprising sequencing the first and the second vessel-molecular-barcoded polynucleotide or amplified products thereof.
530 . The method of claim 527 , wherein the single cell is a B-cell or a T-cell.
531 . The method of claim 526 , wherein the vessel-barcoded oligonucleotides in two or more different vessels of the plurality of vessels comprise a first common-vessel sequence upstream of their vessel barcode, a second common-vessel sequence downstream of their vessel barcode, or both.
532 . The method of claim 526 , wherein each of the molecular-barcoded oligonucleotide molecules in a same vessel of the plurality of vessels comprises a first common-molecular sequence upstream of its molecular barcode, a second common-molecular sequence downstream of its molecular barcode, or both.
533 . The method of claim 526 , wherein the vessel-barcoded oligonucleotide or a complement thereof comprises a sequence complementary to a 3′ end region of a complement of each of the first and the second molecular-barcoded polynucleotides.
534 . The method of claim 526 , wherein:
(i) the first and the second molecular-barcoded oligonucleotide molecules are not fused together,
(ii) the first and the second molecular-barcoded polynucleotides are not fused together, and/or
(iii) the first and the second vessel-molecular-barcoded polynucleotides are not fused together.
535 . The method of claim 526 , wherein (a) comprises extending a first target primer hybridized to a common-sequence of the first cell polynucleotide and extending a second target primer hybridized to a common-sequence of the second cell polynucleotide, wherein the first and the second target primers are extended by a reverse transcriptase comprising a non-template terminal transferase activity, wherein 3 or more identical non-template nucleotides are added to a 3′ end of the first and the second complementary polynucleotides.
536 . The method of claim 535 , wherein the first target primer and/or the second target primer is not attached to a solid support.
537 . The method of claim 526 , wherein the forming of (b) comprises hybridizing a 3′ end region of the first and the second molecular-barcoded oligonucleotide molecules of the plurality to 3 or more non-template nucleotides present on a 3′ end of the first and the second complementary polynucleotides, respectively.
538 . The method of claim 537 , wherein the forming of (b) further comprises extending the 3′ end of the first and the second complementary polynucleotides.
539 . The method of claim 526 , wherein the vessels of the plurality are emulsions.
540 . The method of claim 526 , wherein the vessel-barcoded oligonucleotide and/or the plurality of molecular-barcoded oligonucleotide molecules is not attached to a solid support.
541 . The method of claim 526 , wherein the first and the second cell polynucleotides are RNA.
542 . The method of claim 526 , further comprising determining at least one of:
(a) a number of starting molecules with a sequence of the first or second cell polynucleotide, or both, based on the unique molecular barcode;
(b) a variance of a sequence of the first cell polynucleotide from a germ line sequence, a variance of a sequence of the second cell polynucleotide from a germ line sequence, or both;
(c) a total number of unique first cell polynucleotide sequences;
(d) a total number of unique second cell polynucleotide sequences;
(e) a total number of unique pairs of the first cell polynucleotide sequence and the second cell polynucleotide sequence;
(f) a frequency of a sequence of the first cell polynucleotide;
(g) a frequency of a sequence of the second cell polynucleotide; and
(h) a frequency of a sequence of the first cell polynucleotide and a sequence of the second cell polynucleotide that are paired.
543 . The method of claim 526 , wherein the single cell is a lysed single cell.
544 . The method of claim 526 , wherein (a)-(c) are performed in a same single vessel of the plurality.
545 . A composition comprising a plurality of vessels comprising at least a first vessel and a second vessel, wherein the first and second vessels each comprises:
(a) a single cell from a plurality of cells;
(b) a plurality of molecular-barcoded oligonucleotide molecules each comprising a unique molecular barcode;
(c) a vessel-barcoded oligonucleotide comprising a vessel barcode, wherein the vessel barcode of the vessel-barcoded oligonucleotide in the first vessel is different from the vessel barcode of the vessel-barcoded oligonucleotide in the second vessel;
wherein the molecular-barcoded oligonucleotide molecules of the plurality and the vessel-barcoded oligonucleotide are separate nucleic acids;
and wherein, within each of the first and second vessels,
(d) a first polynucleotide comprising (i) a first unique molecular barcode or a complement thereof, (ii) the vessel barcode or a complement thereof, and (iii) a region complementary to a first cell polynucleotide from the single cell is produced; and
(e) a second polynucleotide comprising (i) a second unique molecular barcode or a complement thereof, (ii) the vessel barcode sequence or a complement thereof, and (iii) a region complementary to a second cell polynucleotide from the single cell is produced.
546 . A method of barcoding polynucleotides in at least a first vessel and a second vessel of a plurality of vessels,
wherein the first and second vessels each comprises (i) a single cell from a plurality of cells, (ii) a plurality of molecular-barcoded oligonucleotide molecules each comprising a unique molecular barcode, and (ii) a vessel-barcoded oligonucleotide comprising a vessel barcode, wherein the vessel barcode in the first vessel is different from the vessel barcode in the second vessel and wherein the vessel-barcoded oligonucleotide in each of the first and second vessels is present as a single molecule,
the method comprising:
(a) producing within the first and second vessels (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;
(b) forming a first molecular-barcoded polynucleotide and a second molecular-barcoded polynucleotide;
(c) amplifying the vessel-barcoded oligonucleotide to generate one or more copies of the vessel-barcoded oligonucleotide;
(d) 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
(e) forming a first vessel-molecular-barcoded polynucleotide and a second vessel-molecular-barcoded polynucleotide.
547 . The method of claim 546 , wherein
the hybridizing of (d) comprises hybridizing a region of the first copy of the vessel-barcoded oligonucleotide or a complement thereof to a 3′ end of the first molecular-barcoded polynucleotide and hybridizing a region of the second copy of the vessel-barcoded oligonucleotide or a complement thereof to a 3′ end of the second molecular-barcoded polynucleotide; and/or
the forming of (e) comprises extending the 3′ end of the first molecular-barcoded polynucleotide and extending the 3′ end of the second molecular-barcoded polynucleotide.