# US 2016/0258012 A2 · Massively parallel single cell ananlysis

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- Number: US 2016/0258012 A2
- Document ID: US20160258012A2
- Status: Published application
- Kind: A2
- Application number: 14/472,363
- Filed: 2014-08-28
- Published: 2016-09-08
- Earliest claimed: 2013-08-28
- Assignees: Cellular Research, Inc.
- Applicants: Cellular Research, Inc.
- Inventors: Christina Fan; Stephen P.A. Fodor; Glenn Fu; Geoffrey Richard Facer; Julie Wilhelmy
- Web page: https://patentgrep.com/patent/US20160258012A2

## CPC

- C12Q 1/6888: for detection or identification of organisms
- C12N 15/1075: {by coupling phenotype to genotype, not provided for in other groups of this subclass}
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

## Claims

### Claim at position 1

-102. (canceled)

### Claim 103

A method comprising:
(a) separating cells into individual wells on a substrate such that a plurality of said wells have a single cell, and wherein said substrate comprises at least 1,000 wells; and
(b) associating a single bead with a single well having said single cell.

### Claim 104 (depends on 103)

The method of claim 103, wherein the method further comprises diluting said cells.

### Claim 105 (depends on 104)

The method of claim 104, wherein said diluting comprises diluting said cells such that at most one cell is distributed to one well.

### Claim 106 (depends on 104)

The method of claim 104, wherein said diluting comprises diluting said cells such that the number of cells is about 10% of the number of wells on said substrate.

### Claim 107 (depends on 103)

The method of claim 103, wherein said separating comprises distributing a single cell to at least 10% of said wells.

### Claim 108 (depends on 103)

The method of claim 103, wherein said separating is random.

### Claim 109 (depends on 103)

The method of claim 103, wherein said cells comprise a cell selected from the group consisting of: a rare cell, a tumor cell, a cell from a human, a cell from a tissue, a cell from a tumor, and a cell infected with viral polynucleotides, or any combination thereof.

### Claim 110 (depends on 103)

The method of claim 103, wherein said cells comprise at least 2 different cell types.

### Claim 111 (depends on 103)

The method of claim 103, wherein said associating occurs prior to lysing said cell.

### Claim 112 (depends on 103)

The method of claim 103, wherein said associating occurs after lysing said cell.

### Claim 113 (depends on 103)

The method of claim 103, further comprising removing said bead from said well.

### Claim 114 (depends on 113)

The method of claim 113, wherein said removing comprises removing with a magnet.

### Claim 115 (depends on 103)

The method of claim 103, wherein said bead comprises a plurality of oligonucleotides.

### Claim 116 (depends on 115)

The method of claim 115, wherein said plurality of oligonucleotides comprises at least 100,000 oligonucleotides.

### Claim 117 (depends on 115)

The method of claim 115, wherein said plurality of oligonucleotides each comprise a universal label, a cellular label, a molecular label, and a target-binding region.

### Claim 118 (depends on 115)

The method of claim 115, wherein individual oligonucleotides of said plurality of oligonucleotides have different molecular labels.

### Claim 119 (depends on 115)

The method of claim 115, wherein individual oligonucleotides of said plurality of oligonucleotides have the same cellular label.

### Claim 120 (depends on 103)

The method of claim 103, wherein individual oligonucleotides of a plurality of oligonucleotides on beads in different wells of said plurality of wells have different cellular labels.

### Claim 121 (depends on 117)

The method of claim 117, wherein said target-binding region comprises a sequence selected from the group consisting of: an oligo-dT sequence, a gene-specific sequence, and a random multimer sequence.

### Claim 122 (depends on 103)

The method of claim 103, further comprising stochastically labelling targets from said single cell.

### Claim 123 (depends on 122)

The method of claim 122, wherein said labelling comprises hybridizing said targets to a plurality of oligonucleotides on said bead.

### Claim 124 (depends on 123)

The method of claim 123, further comprising reverse transcribing said targets, thereby generating labelled target cDNAs.

### Claim 125 (depends on 124)

The method of claim 124, wherein said reverse transcribing is done on pooled beads.

### Claim 126 (depends on 124)

The method of claim 124, wherein said labelled target cDNAs are attached to said bead.

### Claim 127 (depends on 126)

The method of claim 126, further comprising performing second strand synthesis on said labelled target cDNAs, thereby generating double-stranded labelled target polynucleotides.

### Claim 128 (depends on 127)

The method of claim 127, further comprising amplifying said double-stranded labelled target polynucleotides, thereby generating labelled target amplicons.

### Claim 129 (depends on 128)

The method of claim 128, wherein said amplifying is performed with a universal primer and a random multimer primer.

### Claim 130 (depends on 128)

The method of claim 128, wherein said amplifying is performed with a universal primer and a gene-specific primer.

### Claim 131 (depends on 128)

The method of claim 128, wherein said amplifying comprises conducting a nested peR reaction.

### Claim 132 (depends on 128)

The method of claim 128, wherein said amplifying comprises amplifying the whole transcriptome of said one or more cells.

### Claim 133 (depends on 128)

The method of claim 128, further comprising estimating the number of said targets.

### Claim 134 (depends on 133)

The method of claim 133, wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons.

### Claim 135 (depends on 133)

The method of claim 133, wherein said estimating comprises determining the sequence of at least a portion said double-stranded labelled target amplicons and at least a portion of said oligonucleotide.

### Claim 136 (depends on 133)

The method of claim 133, wherein said estimating comprises counting the number of unique oligonucleotides associated with a distinct target polynucleotide per cell.

### Claim 137

A composition comprising: a substrate comprising at least 1,000 wells wherein a plurality of wells comprise a single bead.

### Claim 138

A method of diagnosis comprising:
(a) separating cells into individual wells on a substrate such that a plurality of said wells have a single cell;
(b) stochastically labeling nucleic acid targets from said cells with a plurality of oligonucleotide labels, thereby generating labelled nucleic acids;
(c) detecting said labelled nucleic acids; and (d) estimating the number of labelled nucleic acid targets in each of said single cells.