METHODS AND COMPOSITIONS FOR WHOLE TRANSCRIPTOME AMPLIFICATION

• US 20160312276A1
• Filed: 04/21/2016
• Published: 10/27/2016
• Est. Priority Date: 04/23/2015
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Betts, Craig, Lam, Gretchen Yinbon, Fu, Glenn, Fan, Christina
• Granted Patent: US 11,390,914 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/327 RNAse, e.g. RNAseH
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The disclosure provides for methods, compositions, systems, devices, and kits for whole transcriptome amplification using stochastic barcodes.

Claims

1. A method for labeling a plurality of targets from a sample, comprising:

hybridizing the plurality of targets from the sample with a plurality of nucleic acids each comprising a first universal label;

extending the plurality of nucleic acids to generate a plurality of first strand polynucleotides;

synthesizing a plurality of second strand polynucleotides using the plurality of first strand polynucleotides as templates to generate a plurality of double-stranded polynucleotides;

ligating an adaptor to the plurality of double-stranded polynucleotides, wherein said adaptor comprises a second universal label; and

amplifying the plurality of double-stranded polynucleotides using the first universal label and the second universal label, thereby generating a plurality of amplicons comprising the plurality of targets.

2. The method of claim 1, wherein each of the plurality of nucleic acids comprises a stochastic barcode.

3. The method of claim 2, wherein said stochastic barcode comprises a molecular label, a cellular label, a target-specific region, or any combination thereof.

4. The method of claim 1, wherein the plurality of targets are mRNAs.

5. The method of claim 4, wherein synthesizing the plurality of second strand polynucleotides comprises nicking the plurality of mRNAs with an RNase, thereby generating one or more mRNA primers.

6. The method of claim 4, wherein the plurality of targets are nucleic acids from a single cell.

7. The method of claim 4, wherein the plurality of amplicons comprises a whole transcriptome amplification (WTA) product.

8. The method of claim 1, wherein each of the plurality of nucleic acids is immobilized on a solid support.

9. The method of claim 8, wherein said solid support is a bead.

10. The method of claim 8, wherein at least two of said plurality of nucleic acids immobilized on a single solid support comprises different molecular labels.

11. The method of claim 8, wherein said plurality of nucleic acids attached to a solid support comprises the same cellular label.

12. The method of claim 1, wherein the sample comprises a single cell.

13. The method of claim 1, wherein the first universal label and the second universal label are different.

14. A method for labeling a plurality of targets from a sample comprising:

hybridizing the plurality of targets from the sample with a plurality of nucleic acids each comprising a first universal label;

extending the plurality of nucleic acids to generate a plurality of first strand polynucleotides, wherein the plurality of first stand polynucleotides and the plurality of targets form a plurality of double-stranded polynucleotides;

fragmenting the plurality of double-stranded polynucleotides using a first transposome to generate a plurality of double stranded polynucleotides that are ligated with a first adaptor, wherein said first adaptor comprises a second universal label, and wherein the first transposome comprises a first transposase and the first adaptor; and

amplifying the plurality of double-stranded polynucleotides that are ligated with the first adaptor using the first universal label and the second universal label, thereby generating a plurality of amplicons comprising the plurality of targets.

15. The method of claim 14, further comprising synthesizing a plurality of second strand polynucleotides using the plurality of first strand polynucleotides as templates.

16. The method of claim 14, wherein said first adaptor comprises a stochastic barcode.

17. The method of claim 14, wherein said second universal label is a transposome sequence.

18. The method of claim 14, wherein each of the plurality of nucleic acids is immobilized on a solid support.

19. The method of claim 18, wherein said solid support is a bead.

20. The method of claim 19, comprising purifying double-stranded polynucleotides that are immobilized on beads, wherein the double-stranded polynucleotides are double-stranded polynucleotides that are ligated with the first adaptor, the second adaptor, or both.

21. A kit comprising:

a plurality of solid supports, wherein each of the solid support comprises a plurality of nucleic acids each comprising a first universal label sequence;

an nucleic acid adaptor comprising a second universal label sequence; and

an enzyme, wherein the enzyme is a ligase or a transposase.

22. The kit of claim 21, wherein said enzyme is a ligase.

23. The kit of claim 21, wherein said enzyme is a transposase.

24. The kit of claim 21, wherein said plurality of nucleic acids comprises different molecular labels.

25. The kit of claim 21, wherein said plurality of nucleic acids comprises the same cellular label.

26. A method for labeling a plurality of target sequences from a single cell, comprising:

providing the single cell to a partition comprising a solid support immobilized with a plurality of nucleic acids each comprising a first universal label;

lysing the single cell to release the plurality of target sequences;

hybridizing the plurality of target sequences from the single cell with the plurality of nucleic acids;

extending the plurality of nucleic acids to generate a plurality of first strand polynucleotides;

adding an adaptor sequence to the plurality of first strand polynucleotides, wherein said adaptor sequence comprises a second universal label; and

amplifying the plurality of first strand polynucleotides using the first universal label and the second universal label, thereby generating a plurality of amplicons comprising the plurality of target sequences.

27. The method of claim 26, wherein said adaptor sequence is added by a transposome.

28. The method of claim 26, wherein said adaptor is added by a ligation step.

29. A system for generating a whole transcriptome amplification (WTA) product from a plurality of single cells comprising:

a substrate comprising a plurality of partitions each comprising a single cell and a solid support immobilized with a plurality of nucleic acids, wherein each of the plurality of nucleic acids comprises;

a first universal label;

a cellular label; and

a molecular label;

an nucleic acid adaptor comprising a second universal label sequence; and

an enzyme, wherein the enzyme is a ligase or a transposase.

30. The system of claim 29, wherein the substrate is a microwell array.

31. A composition comprising:

a quasi-symmetric stochastically barcoded nucleic acid comprising;

a stochastic barcode sequence comprising a first universal label;

a second strand synthesis primer sequence comprising a second universal label,

wherein said second universal label is at most 99% identical to said first universal label.

32. The composition of claim 31, wherein said quasi-symmetric stochastically barcoded nucleic acid is capable of undergoing suppression PCR.

33. The composition of claim 31, wherein said strand synthesis primer sequence further comprises a restriction site.

34. The composition of claim 31, wherein said second universal label comprises a mismatch compared to said first universal label.

35. The composition of claim 31, wherein said second universal label hybridizes to at least 80% of said first universal label.

36. The composition of claim 31, wherein said second universal label is at most 99% identical to said first universal label over at least 90% of the length of said first universal label.