CAPSULE ARRAY DEVICES AND METHODS OF USE
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.
1 . A method for generating a plurality of barcoded template nucleic acid molecules in a cell, comprising:
(a) providing a partition comprising said cell and a plurality of nucleic acid barcode molecules, wherein said cell comprises a plurality of template nucleic acid molecules, and wherein said cell is permeable to said plurality of nucleic acid barcode molecules;
(b) with said cell in said partition, permitting said plurality of nucleic acid barcode molecules to be directed into said cell; and
(c) using said plurality of template nucleic acid molecules and said plurality of barcode molecules to generate said plurality of barcoded template nucleic acid molecules in said cell.
2 . The method of claim 1 , wherein said plurality of nucleic acid barcode molecules comprise a sequence capable of hybridizing to said plurality of template nucleic acid molecules in said cell.
3 . The method of claim 2 , wherein said sequence capable of hybridizing to said plurality of template nucleic acid molecules in said cell is a random N-mer sequence.
4 . The method of claim 1 , wherein, in (c), said plurality of barcoded template nucleic acid molecules are generated by nucleic acid amplification.
5 . The method of claim 1 , wherein said plurality of template nucleic acid molecules in said cell comprise a plurality of template RNA molecules and wherein, in (c), said plurality of barcoded template nucleic acid molecules are generated by a reverse transcription reaction.
6 . The method of claim 1 , wherein, in (c), said plurality of barcoded template nucleic acid molecules are generated by ligation of said plurality of barcoded nucleic acid molecules to said plurality of template nucleic acid molecules in said cell.
7 . The method of claim 2 , wherein said plurality of template nucleic acid molecules in said cell comprise a plurality of template RNA molecules, and wherein, in (c), said plurality of barcoded template nucleic acid molecules are generated by direct ligation of said plurality of barcoded nucleic acid molecules to said plurality of template RNA molecules.
8 . The method of claim 1 , wherein said partition further comprises reagents for generating said plurality of barcoded template nucleic acid molecules in said cell.
9 . The method of claim 1 , further comprising releasing said plurality of barcoded template nucleic acid molecules or derivatives thereof from said cell.
10 . The method of claim 9 , wherein said plurality of barcoded template nucleic acid molecules or derivatives thereof are pooled and subjected to a nucleic acid amplification reaction.
11 . The method of claim 1 , wherein said partition comprises a single cell.
12 . The method of claim 1 , wherein said partition comprises a plurality of cells.
13 . The method of claim 1 , wherein said plurality of nucleic acid barcode molecules are attached to a bead.
14 . The method of claim 13 , wherein said plurality of nucleic acid barcode molecules is releasably attached to said bead.
15 . The method of claim 13 , wherein said bead is a gel bead.
16 . The method of claim 14 , further comprising releasing said plurality of nucleic acid barcode molecules from said bead.
17 . The method of claim 14 , wherein said plurality of nucleic acid barcode molecules is released from said bead upon application of a chemical stimulus.
18 . The method of claim 17 , wherein said chemical stimulus is a reducing agent.
19 . The method of claim 17 , wherein said partition further comprises said chemical stimulus.
20 . The method of claim 1 , wherein said partition is an aqueous droplet in an emulsion.
21 . The method of claim 1 , wherein said partition is a well.
22 . The method of claim 1 , further comprising sequencing said plurality of barcoded template nucleic acid molecules or derivatives thereof.
23 . The method of claim 1 , further comprising providing a plurality of partitions, including said partition, wherein a given partition of said plurality of partitions comprises a cell and a plurality of nucleic acid barcode molecules, wherein said cell comprises a plurality of template nucleic acid molecules, and wherein said cell is permeable to said plurality of nucleic acid barcode molecules;
(a) with said cell in said given partition, permitting said plurality of nucleic acid barcode molecules to be directed into said cell; and
(b) using said plurality of template nucleic acid molecules and said plurality of barcode molecules to generate said plurality of barcoded template nucleic acid molecules in said cell.
24 . The method of claim 23 , wherein said plurality of nucleic acid barcode molecules in said given partition comprises a common barcode sequence that is different from barcode sequences of nucleic acid barcode molecules present in other partitions of said plurality of partitions.
25 . The method of claim 23 , wherein said given partition comprises a single cell.
26 . The method of claim 23 , wherein said given partition comprises a plurality of cells.
27 . The method of claim 23 , further comprising pooling said plurality of barcoded template nucleic acid molecules or derivatives thereof from said plurality of partitions.
28 . The method of claim 27 , further comprising subjecting said pooled plurality of barcoded template nucleic acid molecules or derivatives thereof to a nucleic acid amplification reaction.
29 . The method of claim 27 , further comprising sequencing said pooled plurality of barcoded template nucleic acid molecules or derivatives thereof.