Methods and compositions for tagging and analyzing samples

• US 10,722,858 B2
• Filed: 07/23/2018
• Issued: 07/28/2020
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Lineage Biosciences, Inc. (AbCellera Biologics Inc.)
• Original Assignee: Lineage Biosciences, Inc. (AbCellera Biologics Inc.)
• Inventors: Fan, Hei-Mun Christina, Hutchins, Edward A.
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US16/043,060
• Publication Number:
  US 20180326389A1
• Time in Patent Office: 736 Days
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• 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 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The invention relates to methods of tagging analytes in a sample.

Claims

1. A method of tagging target oligonucleotides comprising:

performing an in vitro transcription reaction on DNA molecules partitioned within a plurality of separate compartments, wherein each DNA molecule comprises an RNA polymerase promoter region, a randomer tag region, and a hybridization region capable of hybridizing to one or more target oligonucleotides, thereby obtaining compartments comprising RNA comprising the RNA polymerase promoter region, the randomer tag region and the hybridization region;

merging the interior of the compartments comprising RNA with the interior of a set of compartments comprising target oligonucleotides, wherein prior to the merging, the compartments comprising target oligonucleotides are physically separate from the compartments comprising RNA;

hybridizing the RNA to the target oligonucleotides; and

performing a reaction to attach a sequence corresponding to the RNA to the target oligonucleotides, thereby tagging the target oligonucleotides.

2. The method of claim 1, wherein the DNA is double stranded.

3. The method of claim 1, wherein the compartments are droplets within an oil-and-water emulsion.

4. The method of claim 1, wherein the randomer tag region comprises a random molecule tag such that each DNA molecule within a compartment comprises a different molecule tag, a cell tag such that the DNA molecules in each compartment includes a cell tag unique to the compartment, or a combination thereof.

5. The method of claim 1, wherein the target oligonucleotides are DNA.

6. The method of claim 4, wherein the randomer tag region comprises the random molecule tag and the cell tag.

8. The method of claim 1, wherein the target oligonucleotides are cellular oligonucleotides.

9. The method of claim 8, wherein the cellular oligonucleotides are cellular mRNA.

10. The method of claim 9, further comprising conducting a reverse transcription on the cellular mRNA to generate cellular cDNA.

11. The method of claim 10, wherein the target oligonucleotides are the cellular cDNA.

12. The method of claim 1, wherein the reaction is a Rapid Amplification of cDNA Ends (RACE) reaction.

13. The method of claim 10, wherein the reverse transcription reaction is performed with a primer specific for a region of the genome.

14. The method of claim 1, wherein the DNA is conjugated to a solid support.

15. The method of claim 14, wherein the solid support is a bead.

16. The method of claim 10, wherein the reverse transcription reaction is conducted within the set of compartments prior to the merging step.

17. The method of claim 10, wherein the reverse transcription reaction is conducted within the merged compartments.

18. The method of claim 13, wherein the region of the genome is an immunoglobulin gene or a T-cell receptor gene.

19. The method of claim 1, wherein the target oligonucleotides are cDNA generated from cellular mRNA.

20. The method of claim 1, wherein the hybridization region of the RNA comprises a homopolymeric tail.

21. The method of claim 19, wherein cDNA comprises overhanging C bases generated via the terminal transferase activity of a reverse transcriptase enzyme.

22. The method of claim 21, wherein the hybridization region of the RNA comprises a homopolymeric tail.

23. The method of claim 22, wherein the homopolymeric tail comprises a series of G bases.

24. The method of claim 23, wherein the hybridizing comprises hybridizing the series of G bases to the overhanging C bases.

25. The method of claim 1, wherein the RNA polymerase promoter sequence is the T7 RNA polymerase promoter sequence.

26. The method of claim 1, wherein the RNA polymerase promoter sequence is the T3 RNA polymerase promoter sequence.

27. The method of claim 4, wherein the random molecule tag is from 4 bases to 14 bases long.

28. The method of claim 4, wherein the cell tag is from 4 bases to 14 bases long.

7. The method of wherein the target oligonucleotides are obtained by lysing one or more cells within individual compartments of the set of compartments to liberate cellular oligonucleotides, wherein the cellular oligonucleotides are the target oligonucleotides.