High-throughput nucleotide library sequencing

• US 10,590,483 B2
• Filed: 09/15/2015
• Issued: 03/17/2020
• Est. Priority Date: 09/15/2014
• Status: Active Grant

• Current Assignee: Abvitro LLC (Bristol-Myers Squibb Company)
• Original Assignee: Abvitro LLC (Bristol-Myers Squibb Company)
• Inventors: Vigneault, Francois, Briggs, Adrian Wrangham, Clouser, Christopher Ryan, Goldfless, Stephen Jacob, Timberlake, Sonia
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/854,399
• Publication Number:
  US 20160244825A1
• Time in Patent Office: 1,645 Days
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

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.

Claims

1. 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.

2. The method of claim 1, 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.

3. The method of claim 1, further comprising amplifying the first and the second vessel-molecular-barcoded polynucleotides.

4. The method of claim 1, further comprising sequencing the first and the second vessel-molecular-barcoded polynucleotide or amplified products thereof.

5. The method of claim 1, wherein the single cell is a B-cell or a T-cell.

6. The method of claim 1, wherein the first and the second vessel-molecular-barcoded polynucleotides in a single vessel of the plurality comprise different molecular barcodes.

7. The method of claim 1, wherein the molecular-barcoded oligonucleotide molecules of the plurality are not amplified products.

8. The method of claim 1, wherein a molecular barcode of a molecular-barcoded oligonucleotide molecule of the plurality in a first vessel of the plurality is different than a molecular barcode of a molecular-barcoded oligonucleotide molecule of the plurality in a second vessel of the plurality.

9. The method of claim 1, wherein a vessel barcode of a vessel-barcoded oligonucleotide in a first vessel of the plurality is different from a vessel barcode of a vessel-barcoded oligonucleotide in a second vessel of the plurality.

10. The method of claim 1, wherein the vessel-barcoded oligonucleotide in each vessel of the plurality in (a) is present as a single molecule.

11. The method of claim 1, wherein the vessel barcode of the first and the second vessel-molecular-barcoded polynucleotides in a single vessel of the plurality are the same.

12. The method of claim 1, wherein the vessel-barcoded oligonucleotides in two or more different vessels of the plurality comprise a first common-vessel sequence upstream of their vessel barcode, a second common-vessel sequence downstream of their vessel barcode, or both.

13. The method of claim 1, 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 their molecular barcode, a second common-molecular sequence downstream of their molecular barcode, or both.

14. The method of claim 1, 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, a second common-molecular sequence downstream of the molecular barcode, or both.

15. The method of claim 1, 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.

16. The method of claim 1, wherein the vessel-barcoded oligonucleotide or a complement thereof comprises a sequence complementary to a 3′

end region of the first and the second molecular-barcoded polynucleotides.

17. The method of claim 1, wherein one or more of the following are not fused together:

(i) a first and a second molecular-barcoded oligonucleotide molecule of the plurality,

(ii) the first and the second molecular-barcoded polynucleotides, and

(iii) the first and the second vessel-molecular-barcoded polynucleotides.

18. The method of claim 1, wherein (b) 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.

19. The method of claim 1, wherein the forming of (c) 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.

20. The method of claim 19, wherein the forming of (c) further comprises extending the 3′

end of the first and the second complementary polynucleotides.

21. The method of claim 1, wherein the vessels of the plurality are emulsions.

22. The method of claim 1, wherein the single cell is lysed before (b).

23. The method of claim 1, 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.

24. The method of claim 1, wherein (b)-(d) are performed in a same single vessel of the plurality.

25. The method of claim 1, wherein the first and the second cell polynucleotides are RNA.

26. The method of claim 1, further comprising determining the first and the second cell polynucleotide to be from a same cell based on the vessel barcode.

27. The method of claim 1, further comprising determining a germ line sequence of the first cell polynucleotide, the second cell polynucleotide, or both.

28. The method of claim 27, further comprising determining a variance of a sequence of the first or second cell polynucleotide from the respective germ line sequence, or both.

29. The method of claim 1, further comprising determining at least one of:

(a) a total number of unique first cell polynucleotide sequences;

(b) a total number of unique second cell polynucleotide sequences;

(c) a total number of unique paired first and second cell polynucleotide sequences;

(d) a frequency of a sequence of a first cell polynucleotide sequence;

(e) a frequency of a sequence of a second cell polynucleotide; and

(f) a frequency of sequence of a first cell polynucleotide and a sequence of a second cell polynucleotide that are paired.

30. The method of claim 1, further comprising determining a number of starting molecules with a sequence of the first or second cell polynucleotide, or both, based on the unique molecular barcode.

31. The method of claim 1, 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.

32. The method of claim 1, 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.

33. The method of claim 1, wherein (b)-(d) are performed in two or more vessels of the plurality simultaneously.

34. The method of claim 1, wherein the forming of (c) comprises attaching a first molecular-barcoded oligonucleotide molecule of the plurality to the first complementary polynucleotide and attaching a second molecular-barcoded oligonucleotide molecule of the plurality to the second complementary polynucleotide.

35. The method of claim 1, wherein the forming of (d) comprises attaching a first vessel-barcoded oligonucleotide molecule to the first molecular-barcoded polynucleotide and attaching a second vessel-barcoded oligonucleotide molecule to the second molecular barcoded polynucleotide.

36. The method of claim 1, wherein the plurality of vessels comprises at least 1000 vessels.

37. 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.

38. The method of claim 37, wherein the hybridizing of (e) comprises (i) 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 (ii) 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.

39. The method of claim 38, wherein the forming of (c) comprises extending the 3′

end of the first molecular-barcoded polynucleotide and extending the 3′

end of the second molecular-barcoded polynucleotide.