Single-particle analysis of particle populations

• US 9,840,732 B2
• Filed: 05/21/2013
• Issued: 12/12/2017
• Est. Priority Date: 05/21/2012
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Anderson, Megan, Chen, Peilin, Fowler, Brian, Kaper, Fiona, Lebofsky, Ronald, May, Andrew
• Primary Examiner(s): Lu, Frank
• Application Number:
  US13/899,397
• Publication Number:
  US 20130323732A1
• Time in Patent Office: 1,666 Days
• Field of Search: 435 61, 435 611, 435 612, 435 911, 435 912, 435 9151, 435183, 435 94, 435501, 4352871, 4352872, 536 231, 536 243, 536 2433, 536 253
• CPC Class Codes: B01D 15/08 Selective adsorption, e.g. chromatography
• 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 1/6813 Hybridisation assays
• 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 2565/629 being a microfluidic device

Abstract

In certain embodiments, the invention provides methods and devices for assaying single particles in a population of particles, wherein at least two parameters are measured for each particle. One or more parameters can be measured while the particles are in the separate reaction volumes. Alternatively or in addition, one or more parameters can be measured in a later analytic step, e.g., where reactions are carried out in the separate reaction volumes and the reaction products are recovered and analyzed. In particular embodiments, one or more parameter measurements are carried out “in parallel,” i.e., essentially simultaneously in the separate reaction volumes.

Claims

1. A method of producing a data set comprising a plurality of parameters for single particles in a plurality of particles, wherein each of said single particles comprises one or more target nucleic acids, said method comprising:

capturing particles of said plurality of particles in separate compartments of a microfluidic device, thereby producing a plurality of separate reaction volumes in the separate compartments of the microfluidic device such that at least 35% of said separate reaction volumes comprise only a single particle of said plurality of particles, the separate compartments being arranged as an array defined by rows and columns;

determining which of said separate reaction volumes comprise only a single particle of said plurality of particles by microscopy and/or using a stain, dye, or label;

performing a plurality of reactions in each of said separate reaction volumes, thereby producing a plurality of reaction products in each of said separate reaction volumes, wherein at least one reaction of the plurality of reactions in each of said separate reaction volumes comprises a nucleic acid amplification reaction that produces at least one amplicon in each of said separate reaction volumes,

wherein the at least one amplicon in each of said separate reaction volumes is produced using amplification primers, wherein each of at least two of the amplification primers comprises a barcode nucleotide sequence, the at least one amplicon comprising a combination of the barcode nucleotide sequence from each of the at least two of the amplification primers,

wherein the combination of the barcode nucleotide sequence from each of the at least two amplification primers in the at least one amplicon in each of said separate reaction volumes identifies the row and column of a compartment of the separate compartments of the microfluidic device and thereby identifies the source of the at least one amplicon;

recovering the reaction products, comprising the at least one amplicon from each of said separate reaction volumes by pooling the reaction products from each of the separate reaction compartments, thereby producing pooled reaction products;

obtaining analysis results by analyzing the pooled reaction products;

generating analysis results for the single particles in the plurality of particles by disregarding the analysis results from said separate reaction volumes that contain no particle of said plurality of particles or contain more than one single particle of said plurality of particles; and

producing the data set comprising the plurality of parameters for the single particles in the plurality of particles by associating the analysis results for the single particles in the plurality of particles with each of said separate reaction volumes that comprise only a single particle of the plurality of particles.

2. The method of claim 1, wherein said at least 35% of said separate reaction volumes that comprise only a single particle of said plurality of particles is at least 50% of the total number of said separate reaction volumes.

3. The method of claim 1, wherein said at least 35% of said separate reaction volumes that comprise only a single particle of said plurality of particles is at least 65% of the total number of said separate reaction volumes.

4. The method of claim 1, wherein said at least 35% of said separate reaction volumes that comprise only a single particle of said plurality of particles is at least 85% of the total number of said separate reaction volumes.

5. The method of claim 1, wherein the particles are captured in said separate reaction volumes prior to adding one or more reagents for performing said plurality of reactions in each of said separate reaction volumes.

6. The method of claim 1, wherein the particles are cells.

7. The method of claim 1, wherein the particles are nucleic acids.

8. The method of claim 1, wherein fewer than 40,000 of said particles are employed in the method.

9. The method of claim 1, wherein fewer than 10,000 of said particles are employed in the method.

10. The method of claim 1, wherein the number of said particles distributed in said separate reaction volumes is greater than 100.

11. The method of claim 1, wherein a limiting dilution of said particles is carried out before said capturing particles of said plurality of particles in separate compartments of the microfluidic device.

12. The method of claim 11, wherein said limiting dilution of said particles is carried out by:

preparing a series of dilutions of a suspension of said plurality of particles;

distributing the particles from each dilution of said series of dilutions into the separate compartments of a microfluidic device;

determining which of said separate compartments comprise only a single particle of said plurality of particles; and

selecting the dilution that produces the highest number of said separate compartments that comprise only a single particle of said plurality of particles for use in said capturing single particles of said plurality of particles in separate compartments of the microfluidic device.

13. The method of claim 12, wherein the number of particles in each of said separate compartments is determined by brightfield microscopy or by fluorescence microscopy when the particles are labeled with a fluorescent dye or label.

14. The method of claim 12, wherein a stain, dye, or label is employed for determining which of said separate compartments comprise only a single particle of said plurality of particles.

15. The method of claim 14, wherein the particles are cells, and the stain, dye, or label is a membrane-permeant stain, dye, or label.

16. The method of claim 14, wherein the particles are cells, and said determining which of said separate compartments comprise only a single particle of said plurality of particles is by using a cell membrane-permeant nucleic acid dye.

17. The method of claim 14, wherein the particles are cells, and the stain, dye, or label is a cell-surface stain, dye, or label.

18. The method of claim 14, wherein the particles are cells, and said determining which of said separate compartments comprise only a single particle of said plurality of particles is by using a labeled antibody specific for a cell-surface marker.

19. The method of claim 1, wherein said capturing particles of said plurality of particles in separate compartments of the microfluidic device comprises mechanically capturing the particles of said plurality of particles at a plurality of capture sites in the separate compartments of the microfluidic device.

20. The method of claim 19, wherein each of said plurality of capture sites comprises:

a capture feature with a size holding only one single particle of said plurality of particles;

and a drain feature, wherein, when the capture feature is not occupied by the single particle, the drain feature permits a flow of fluid to pass through the capture site of said plurality of capture sites.

21. The method of claim 19, wherein the microfluidic device comprises a focusing feature to focus said plurality of particles to each of said plurality of capture sites.

22. The method of claim 19, wherein said capturing particles of said plurality of particles in separate compartments of the microfluidic device comprises passing a solution comprising said plurality of particles through the separate compartments of the microfluidic device.

23. The method of claim 1, wherein said capturing particles of said plurality of particles in separate compartments of the microfluidic device further comprises affinity-based capturing of said plurality of particles and employs a binding partner that binds a particle of said plurality of particles.

24. The method of claim 23, wherein the binding partner is affixed to a discrete region of the separate compartments of the microfluidic device, wherein the discrete region permits binding of only one particle of said plurality of particles.

25. The method of claim 23, wherein said affinity-based capturing of said plurality of particles comprises:

capturing supports comprising the binding partner at a plurality of capture sites in the separate compartments of the microfluidic device and producing immobilized supports at each of said plurality of capture sites, wherein the binding partner on each of the immobilized supports binds only one particle of said plurality of particles; and

binding one of said plurality of particles to the binding partner on each of the immobilized supports.

26. The method of claim 25, wherein the support is captured by mechanical capture.

27. The method of claim 25, wherein each of said plurality of capture sites comprises:

a capture feature with a size holding only one of the supports; and

a drain feature, wherein, when the capture feature is not occupied by the only one support, the drain feature permits a flow of fluid to pass through the capture site of said plurality of capture sites.

28. The method of claim 25, wherein the microfluidic device comprises a focusing feature to focus the supports and/or said plurality of particles to each of said plurality of capture sites.

29. The method of claim 1, wherein the one or more target nucleic acids comprise DNA or RNA and the reactions comprise one or more reactions selected from amplification of the DNA, digestion of the DNA with a nuclease, ligation of the DNA, ligation of an adaptor sequence onto the DNA, transposase-mediated incorporation of a transposon into the DNA, sequencing of the DNA, reverse transcription of the RNA, amplification of the RNA, and digestion of the RNA with an RNase.

30. The method of claim 1, wherein the particles comprise cells and the nucleic acid amplification reaction is whole genome amplification or whole transcriptome amplification.

31. The method of claim 1, wherein the amplification primers comprise a pair of inner primers and a pair of outer primers, wherein:

the inner primers comprise;

a forward, inner primer comprising a first nucleotide tag sequence, a first barcode nucleotide sequence, and a target-specific portion; and

a reverse, inner primer comprising a target-specific portion, the first barcode nucleotide sequence, and a second nucleotide tag sequence; and

the outer primers comprise;

a forward, outer primer comprising a second barcode nucleotide sequence and a first nucleotide tag-specific portion; and

a reverse, outer primer comprising a second nucleotide tag-specific portion and the second barcode nucleotide sequence;

wherein the outer primers are in excess of the inner primers; and

wherein an amplicon of the at least one amplicon comprises, from 5′

to 3′

, the second barcode nucleotide sequence, the first nucleotide tag sequence, the first barcode nucleotide sequence, a target nucleotide sequence from the one or more target nucleic acids, the first barcode nucleotide sequence, the second nucleotide tag sequence, and the second barcode nucleotide sequence.

32. The method of claim 1, wherein the amplification primers comprise a pair of inner primers, a pair of stuffer primers, and a pair of outer primers, wherein:

the inner primers comprise;

a forward, inner primer comprising a first nucleotide tag sequence and a target-specific portion; and

a reverse, inner primer comprising a target-specific portion and a second nucleotide tag sequence;

the stuffer primers comprise;

a forward, stuffer primer comprising a third nucleotide tag sequence, a first barcode nucleotide sequence, and a first nucleotide tag-specific portion; and

a reverse, stuffer primer comprising a second nucleotide tag-specific portion, the first barcode nucleotide sequence, a fourth nucleotide tag sequence; and

the outer primers comprise;

a forward, outer primer comprising a second barcode nucleotide sequence and a third nucleotide tag-specific portion; and

a reverse, outer primer comprising a fourth nucleotide tag-specific portion and the second barcode nucleotide sequence;

wherein the outer primers are in excess of the stuffer primers, which are in excess of the inner primers; and

wherein the amplicon of the at least one amplicon comprises, from 5′

to 3′

, the second barcode nucleotide sequence, the third nucleotide tag sequence, the first barcode nucleotide sequence, the first nucleotide tag sequence, a target nucleotide sequence from the one or more target nucleic acids, the second nucleotide tag sequence, the first barcode nucleotide sequence, the fourth nucleotide tag sequence, and the second barcode nucleotide sequence.

33. The method of claim 31, wherein the outer primers additionally comprise first and second DNA sequencing primer binding sites, and wherein an amplicon of the at least one amplicon comprises, from 5′

to 3′

, the first DNA sequencing primer binding site, the second barcode nucleotide sequence, the first nucleotide tag sequence, the first barcode nucleotide sequence, the target nucleotide sequence from the one or more target nucleic acids, the first barcode nucleotide sequence, the second nucleotide tag sequence, the second barcode nucleotide sequence, and the second DNA sequencing primer binding site.

34. The method of claim 33, the method further comprises sequencing the at least one amplicon.

35. The method of claim 1, the method additionally comprises disrupting said particles prior to performing the nucleic acid amplification, when the particles are cells.

36. The method of claim 35, wherein said particles comprise cells, and the cells are disrupted by lysis prior to performing the nucleic acid amplification.

37. The method of claim 1, wherein the particles are treated with an agent that elicits a biological response prior to performing the plurality of reactions when the particles are cells.

38. The method of claim 1, wherein the method further comprises determining the presence or amount of said one or more target nucleic acids.

39. The method of claim 1, wherein the method further comprises determining the copy numbers of one or more DNA molecules in each of said particles when the one or more target nucleic acids are the one or more DNA molecules.

40. The method of claim 1, wherein the method further comprises determining the genotypes at one or more loci in genomic DNA in each of said particles when the one or more target nucleic acids are a part of the genomic DNA.

41. The method of claim 1, wherein the method further comprises determining a haplotype for a plurality of loci in genomic DNA in each of said particles when the one or more target nucleic acids are a part of the genomic DNA.

42. The method of claim 1, wherein the method further comprises determining the expression levels of one or more RNA molecules in each of said particles when the one or more target nucleic acids are the one or more RNA molecules.

43. The method of claim 1, wherein the method further comprises determining the nucleotide sequences of one or more RNA molecules in each of said particles when the one or more target nucleic acids are the one or more RNA molecules.

44. The method of claim 1, wherein the method further comprises determining the expression levels of one or more proteins in each of said particles when the particles are cells.

45. The method of claim 1, wherein said recovering the reaction products, comprising the at least one amplicon, from each of said separate reaction volumes comprises pooling the reaction products from each of the separate reaction compartments in a row or in a column.

46. The method of claim 1, wherein said recovering the reaction products, comprising the at least one amplicon, from each of said separate reaction volumes comprises pooling the reaction products from each of the separate reaction compartments in the microfluidic device.