SINGLE CELL ANALYSIS USING SEQUENCE TAGS

• US 20150247182A1
• Filed: 07/22/2013
• Published: 09/03/2015
• Est. Priority Date: 07/24/2012
• Status: Abandoned Application

• Current Assignee: Adaptive Biotechnologies Corp.
• Original Assignee: Adaptive Biotechnologies Corp.
• Inventors: Faham, Malek, Willis, Thomas, Zheng, Jianbiao
• Application Number:
  US14/425,036
• Publication Number:
  US 20150247182A1
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2527/101 Temperature
• 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

Abstract

The invention provides a method of making measurements on individual cells of a population by forming reactors containing single cells and a predetermined number, usually one, homogeneous sequence tag. In one aspect, the invention provides a method of making multiparameter measurements on individual cells of such a population by carrying out a polymerase cycling assembly (PCA) reaction to link their identifying nucleic acid sequences, such as sequence tag copies derived from a homogeneous sequence tag, to other cellular nucleic acids of interest, thereby forming fusion products. The fusion products of such PCA reactions are then sequenced and tabulated to generate multiparameter data for cells of the population.

Claims

1. A method of analyzing a plurality of target nucleic acids of single cells of a population, the method comprising the steps of:

providing multiple reactors each containing a single cell of the population and a single homogeneous sequence tag in an amplification mixture, the amplification mixture comprising a pair of primers for amplifying each target nucleic acid of the plurality;

providing amplifiable sequence tags from the homogeneous sequence tags;

amplifying the target nucleic acids and amplifiable sequence tags to form amplicons comprising sequence tags; and

sequencing the amplicons from the reactors to identify the target nucleic acids of each cell from the population by the sequence tags incorporated into the amplicons.

2. The method of claim 1 wherein said step of amplifying is carried out by a polymerase chain reaction.

7. The method of claim 1 wherein said step of providing said amplifiable sequence tags comprising generating said amplifiable sequence tags by an EXPAR.

8. The method of claim 1 wherein said homogeneous sequence tag is a rolling circle amplicon comprising a plurality of said sequence tagged primers.

9. The method of claim 1 wherein said homogeneous sequence tag is a bead having a plurality of sequence tagged primers attached thereto.

10. The method of claim 1 wherein said reactors are micelles of an emulsion.

11. The method of claim 10 wherein said micelles are generated in a microfluidics device.

12. The method of claim 10 wherein said micelles have a distribution of volumes with a coefficient of variation of thirty percent or less.

13. The method of claim 1 wherein said population of said single cells are from the same sample.

14. The method of claim 1 further including a step of lysing said single cells in each of said reactors prior to said step of amplifying.

15. The method of claim 1 wherein said homogeneous sequence tag comprises a random genomic segment.

16. The method of claim 1 wherein said homogeneous sequence tag comprises a random transcriptome segment.

3. The method of 1 wherein said step of providing said amplifiable sequence tags comprises releasing said amplifiable sequence tags from said homogeneous sequence tag.

4. The method of claim 3 wherein said step of releasing said amplifiable sequence tags is carried out by cleaving said amplifiable sequence tags from said homogeneous sequence tag by a thermostable restriction endonuclease.

5. The method of claim 4 wherein each of said amplifiable sequence tags is a sequence tagged primer.

6. The method of claim 4 wherein each of said amplifiable sequence tags is a sequence tag flanked by primer binding sites and wherein said amplification mixture further comprises a pair of primers capable of amplifying said amplifiable sequence tag in a PCR.

17. A method of analyzing a plurality target nucleic acids of each cell of a population, the method comprising the steps of:

providing multiple reactors each containing a single cell and a single homogeneous sequence tag in a polymerase cycling assembly (PCA) reaction mixture, the homogeneous sequence tag comprising at least one sequence tagged primer, and the PCA reaction mixture comprising a pair of outer primers and one or more pairs of linking primers specific for the plurality of target nucleic acids, wherein at least one of the outer primers or linking primers is a sequence tagged primer of the homogeneous sequence tag;

performing a PCA reaction in the reactors so that homogeneous sequence tags release or produce sequence tagged primers and so that fusion products of the target nucleic acids and sequence tagged primers are formed in the reactors; and

sequencing the fusion products from the reactors to identify the target nucleic acids of each cell in the population.

18. The method of claim 17 wherein said multiple reactors are aqueous micelles of a water-in-oil emulsion.

19. The method of claim 18 wherein said water-in-oil emulsion is generated by a microfluidics device.

20. The method of claim 17 wherein said target nucleic acids are transcripts of a transcriptome.

21. The method of claim 17 wherein said homogeneous sequence tag is a bead having a plurality of sequence tagged primers attached thereto.

22. The method of claim 17 further including a step of lysing said single cells in each of said reactors prior to said step of amplifying.

23. A method of analyzing a plurality of target nucleic acids of single cells of a population, the method comprising the steps of:

providing multiple reactors each containing a single cell of the population, a first homogeneous sequence tag and a second homogeneous sequence tag in an amplification mixture, the amplification mixture comprising a pair of primers for amplifying each target nucleic acid of the plurality;

providing amplifiable sequence tags from the homogeneous sequence tags in the presence of helper oligonucleotides so that flap structures form at 5′

ends of strands of the target nucleic acids;

cleaving the flap structures with a flap endonuclease to provide 5′

ends on the strands of target nucleic acids that are ligatable to amplifiable sequence tags;

ligating the amplifiable sequence tags to the ligatable 5′

ends of the strands of target nucleic acids;

amplifying the strands of each target nucleic acid and amplifiable sequence tags to form amplicons comprising sequence tags; and

sequencing the amplicons from the reactors to identify the target nucleic acids of each cell from the population by the sequence tags incorporated into the amplicons.

24. The method of claim 23 wherein said multiple reactors are aqueous micelles of a water-in-oil emulsion.

25. The method of claim 24 wherein said water-in-oil emulsion is generated by a microfluidics device.

26. The method of claim 23 wherein said target nucleic acids are transcripts of a transcriptome.

27. The method of claim 23 wherein said homogeneous sequence tag is a bead having a plurality of sequence tagged primers attached thereto.

28. The method of claim 23 further including a step of lysing said single cells in each of said reactors prior to said step of amplifying.