High-throughput single cell barcoding

Patent metadata

Field	Value
Publication number	US-9902950-B2
Application number	US-201113878406-A
Country	US
Kind code	B2
Filing date	Oct 11, 2011
Priority date	Oct 8, 2010
Publication date	Feb 27, 2018
Grant date	Feb 27, 2018

Abstract

Methods and compositions for high-throughput, single cell analyzes are provided. The methods and compositions can be used for analysis of genomes and transcriptomes, as well as antibody discovery, HLA typing, haplotyping and drug discovery.

First claim

What is claimed is:

1. An oil and water emulsion comprising a single cell sequestered with one bead, wherein the one bead comprises a first polynucleotide attached thereto, wherein the polynucleotide comprises a sequencing primer region, a barcode, and a universal primer region, wherein the sequencing primer region is for sequencing the polynucleotide and wherein the oil and water emulsion further comprises a second polynucleotide, wherein the second polynucleotide comprises a sequence complementary to the universal primer region and an annealing primer region.

2. The oil and water emulsion of claim 1 wherein the one bead further comprises a plurality of first polynucleotides attached thereto wherein each first polynucleotide of the plurality of polynucleotides includes a sequencing primer region, a barcode, and a universal primer region, wherein the sequencing primer region is for sequencing the polynucleotide.

3. The oil and water emulsion of claim 2 wherein the barcode of each polynucleotide is a same barcode sequence.

4. The oil and water emulsion of claim 1 wherein the barcode is unique to the cell.

5. The oil and water emulsion of claim 2 wherein the barcode is unique to the cell.

6. The oil and water emulsion of claim 1 wherein the single cell is a lysed single cell.

7. The oil and water emulsion of claim 1 wherein the single cell is a lysed single cell and a target nucleic acid is annealed to the annealing primer region.

8. The oil and water emulsion of claim 2 wherein the single cell is a lysed single cell and a plurality of target nucleic acids are annealed to first annealing primer regions.

9. The oil and water emulsion of claim 8 wherein the plurality of target nucleic acids comprises a heavy chain nucleic acid and a light chain nucleic acid.

10. The oil and water emulsion of claim 8 wherein the plurality of target nucleic acids comprises a heavy chain cDNA and a light chain cDNA.

11. The oil and water emulsion of claim 8 wherein the plurality of target nucleic acids comprises mRNA, a polynucleotide encoding a T cell receptor component, a polynucleotide having an immune cell nucleic acid sequence, a polynucleotide encoding an HLA component, a polynucleotide comprising a sequence indicative of haplotype, a polynucleotide encoding a B cell receptor component, a heavy chain polynucleotide or a light chain polynucleotide.

12. The oil and water emulsion of claim 2 wherein the oil and water emulsion further comprises a plurality of second polynucleotides, wherein the plurality of second polynucleotides each comprise a sequence complementary to the universal primer region and an annealing primer region.

13. The oil and water emulsion of claim 1 wherein the sequencing primer region is 5′ to the barcode and the universal primer region is 3′ to the barcode.

14. The oil and water emulsion of claim 1 wherein the single cell is a B cell or T cell.

15. The oil and water emulsion of claim 1 wherein the single cell is a bacterial cell.

16. The oil and water emulsion of claim 1 wherein the single cell is an immune cell, a cell from a tumor, or a cell from a subject with a tumor.

17. The oil and water emulsion of claim 1 further comprising a plurality of cells, with each cell of the plurality being a single cell sequestered with one bead.

18. The oil and water emulsion of claim 1 further comprising a plurality of cells, with each cell of the plurality being a single cell sequestered with one bead having a unique barcode.

19. The oil and water emulsion of claim 1 wherein the barcode comprises from 4 nucleotides to 36 nucleotides.

20. A method comprising
creating an oil and water emulsion comprising a lysed single cell sequestered with one bead, wherein the one bead comprises a plurality of polynucleotides attached thereto wherein each polynucleotide of the plurality of polynucleotides includes a sequencing primer region, a barcode, and a universal primer region, wherein the sequencing primer region is for sequencing the polynucleotide, wherein the oil and water emulsion further comprises a second polynucleotide, wherein the second polynucleotide comprises a sequence complementary to the universal primer region and an annealing primer region, and wherein a plurality of target nucleic acids are annealed to annealing primer regions; and
reverse transcribing or extending the target nucleic acids to produce complementary strands including the barcode.

21. The method of claim 20 further comprising amplifying the complementary strands including the barcode.

22. The method of claim 20 further comprising amplifying and sequencing the complementary strands including the barcode.

23. The method of claim 20 further comprising amplifying and sequencing the complementary strands including the barcode and correlating at least one of the sequenced strands to a unique barcode.

24. The method of claim 20 wherein the oil and water emulsion comprises a plurality of lysed cells, with each lysed cell of the plurality being a single lysed cell sequestered with one bead, wherein the one bead comprises a plurality of polynucleotides attached thereto wherein each polynucleotide of the plurality of polynucleotides includes a sequencing primer region, a unique barcode, and a universal primer region, wherein the sequencing primer region is for sequencing the polynucleotide, wherein the oil and water emulsion further comprises a plurality of second polynucleotides, wherein the plurality of second polynucleotides comprises a sequence complementary to the universal primer region and an annealing primer region, wherein a plurality of target nucleic acids are annealed to annealing primer regions; and
reverse transcribing or extending the target nucleic acids to produce complementary strands including the unique barcode.

25. The method of claim 20 further comprising amplifying the complementary strands including the unique barcode.

26. The method of claim 20 further comprising amplifying and sequencing the complementary strands including the unique barcode.

27. The method of claim 20 further comprising amplifying and sequencing the complementary strands including the unique barcode and correlating at least one of the sequenced strands to the unique barcode.

28. The method of claim 20 wherein the plurality of target nucleic acids comprises a heavy chain nucleic acid and a light chain nucleic acid.

29. The method of claim 20 wherein the plurality of target nucleic acids comprises a heavy chain cDNA and a light chain cDNA.

30. The method of claim 20 wherein the plurality of target nucleic acids comprises mRNA, a polynucleotide encoding a T cell receptor component, a polynucleotide having an immune cell nucleic acid sequence, a polynucleotide encoding an HLA component, a polynucleotide comprising a sequence indicative of haplotype, a polynucleotide encoding a B cell receptor component, a heavy chain polynucleotide or a light chain polynucleotide.

31. The method of claim 20 wherein the sequencing primer region is 5′ to the barcode and the universal primer region is 3′ to the barcode.

32. The method of claim 20 wherein the lysed single cell is a B cell or T cell.

33. The method of claim 20 wherein the lysed single cell is a bacterial cell.

34. The method of claim 2

Assignees

• Church George M
• Vigneault Francois
• Harvard College

Inventors

• Church George M
• Vigneault Francois

Classifications

• C40B40/06 Primary

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• B01J2219/00547

  Bar codes · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C40B50/14

  Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support · CPC title