Capsule Array Devices and Methods of Use

• US 20140287963A1
• Filed: 04/10/2014
• Published: 09/25/2014
• Est. Priority Date: 08/14/2012
• Status: Abandoned Application

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10x Technology Inc.
• Inventors: Hindson, Benjamin, Saxonov, Serge
• Application Number:
  US14/249,673
• Publication Number:
  US 20140287963A1
• Time in Patent Office: Days
• US Class Current: 506/32
• 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: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/508 rigid containers not provided for above
• CPC Class Codes: B01L 3/523 with means for closing or opening
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 2535/122 Massive parallel sequencing
• 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

Abstract

This disclosure provides microwell capsule array devices. The microwell capsule array devices are generally capable of performing one or more sample preparation operations. Such sample preparation operations may be used as a prelude to one more or more analysis operations. For example, a device of this disclosure can achieve physical partitioning and discrete mixing of samples with unique molecular identifiers within a single unit in preparation for various analysis operations. The device may be useful in a variety of applications and most notably nucleic-acid-based sequencing, detection and quantification of gene expression and single-cell analysis.

Claims

1. A method for delivering a reagent to a partition, comprising:

providing the reagent releasably coupled to a microcapsule;

separating the microcapsule into a partition; and

releasing the reagent into the partition.

2. The method of claim 1, wherein the releasing step comprises applying a stimulus to the microcapsule to release the reagent into the partition.

3. The method of claim 2, wherein the stimulus is selected from a chemical stimulus, an electrical stimulus, a thermal stimulus, a magnetic stimulus, and a photostimulus.

4. The method of claim 3, wherein the stimulus comprises a chemical stimulus.

5. The method of claim 4, wherein the microcapsule comprises chemical cross-linking and the chemical stimulus cleaves the chemical cross-linking to release the reagent into the partition.

6. The method of claim 4, wherein the reagent is coupled to the microcapsule through a chemically cleavable linkage, and the chemical stimulus cleaves the chemically cleavable linkage to release the reagent into the partition.

7. The method of claim 5, wherein the chemical cross-linking comprises disulfide bonds.

8. The method of claim 7, wherein the chemical stimulus comprises a reducing agent.

9. The method of claim 8, wherein the reducing agent is selected from dithiothreitol and tris(2-carboxyethyl)phosphine

10. The method of claim 6, wherein the chemically cleavable linkage comprises a disulfide bond.

11. The method of claim 10, wherein the chemical stimulus comprises a reducing agent.

12. The method of claim 11, wherein the reducing agent is selected from dithiothreitol and tris(2-carboxyethyl)phosphine

13. The method of claim 3, wherein the stimulus comprises a thermal stimulus.

14. The method of claim 13, wherein the thermal stimulus comprises heating the microcapsule to release the reagent into the partition.

15. The method of claim 1, wherein the reagent comprises a unique identifier reagent, and the partition comprises a sample reagent to be assigned with the unique identifier reagent.

16. The method of claim 15, wherein the unique identifier reagent comprises a nucleic acid barcode.

17. The method of claim 15, wherein the reagent to be assigned the unique identifier reagent comprises a target nucleic acid.

18. The method of claim 16, wherein the microcapsule comprises a population of nucleic acid barcode sequences releasably coupled thereto, wherein the barcode sequences substantially all comprise the same barcode nucleotide sequence.

19. The method of claim 10, wherein the unique identifier reagent comprises a nucleic acid barcode, and further comprising coupling the barcode to the target nucleic acid.

20. The method of claim 1, wherein the microcapsule comprises a crosslinked polymer microcapsule.

21. The method of claim 20, wherein the crosslinked polymer microcapsule comprises a polyacrylamide polymer.

22. The method of claim 1, wherein the partition is selected from a well, a microwell, and a droplet in an emulsion.

23. The method of claim 1, comprising:

separating a plurality of microcapsules into separate partitions, each microcapsule having a reagent releasably coupled thereto; and

releasing the reagents from the plurality of microcapsules into the different partitions.

24. The method of claim 23, wherein the plurality of microcapsules comprises at least 1,000 microcapsules, and the releasing step comprises releasing the reagents from the at least 1000 microcapsules into separate partitions.

25. The method of claim 23, wherein the plurality of microcapsules comprises at least 1,000 different microcapsules, each of the at least 1,000 different microcapsules comprises a different reagent, and the releasing step comprises releasing each of the different reagents into a different partition.

26. The method of claim 23, wherein each of the at least 1000 microcapsules comprises a different reagent, and the releasing step comprises releasing a different reagent into each of at least 1000 separate partitions.

27. The method of claim 23, wherein the plurality of microcapsules comprises at least 5,000 different microcapsules, each of the at least 5,000 different microcapsules comprises a different reagent, and the releasing step comprises releasing each of the different reagents into a different partition.

28. The method of claim 23, wherein the plurality of microcapsules comprises at least 10,000 different microcapsules, each of the at least 10,000 different microcapsules comprises a different reagent, and the releasing step comprises releasing each of the different reagents into a different partition.

29. The method of claim 23, wherein the plurality of microcapsules comprises at least 100,000 different microcapsules, each of the at least 100,000 different microcapsules comprises a different reagent, and the releasing step comprises releasing each of the different reagents into a different partition.

30. The method of claim 26, wherein each different reagent comprises a different barcode sequence.

31. The method of claim 27, wherein each different reagent comprises a different barcode sequence.

32. The method of claim 28, wherein each different reagent comprises a different barcode sequence.

33. The method of claim 29, wherein each different reagent comprises a different barcode sequence.

34. The method of claim 23, wherein the reagent coupled to each of the plurality of microcapsules comprises at least 10,000 nucleic acid barcodes having the same barcode sequence releasably coupled thereto.

35. The method of claim 23, wherein the reagent coupled to each of the plurality of microcapsules comprises at least 100,000 nucleic acid barcodes having the same barcode sequence releasably coupled thereto.

36. The method of claim 23, wherein the reagent coupled to each of the plurality of microcapsules comprises at least 1,000,000 nucleic acid barcodes having the same barcode sequence releasably coupled thereto.