# US 2014/0155274 A1 · Single Cell Nucleic Acid Detection and Analysis

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- Number: US 2014/0155274 A1
- Document ID: US20140155274A1
- Status: Published application
- Kind: A1
- Application number: 14/006,971
- Filed: 2012-03-22
- Published: 2014-06-05
- Earliest claimed: 2011-03-24
- Assignees: President and Fellows of Harvard College
- Inventors: Xiaoliang Sunney Xie; Katsuyuki Shiroguchi; Peter A. Sims; Tony Z Jia
- Web page: https://patentgrep.com/patent/US20140155274A1

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6853: using modified primers or templates
- C12N 2320/10: in screening processes
- 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

Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.

## Claims

### Claim 1

A method of determining copy number of a nucleic acid molecule in a sample including a plurality of nucleic acid molecules comprising
attaching a unique barcode sequence to substantially each of the plurality of nucleic acid molecules in the sample to produce a plurality of barcoded nucleic acid molecules,
amplifying the plurality of barcoded nucleic acid molecules in the sample to produce amplicons of the plurality of barcoded nucleic acid molecules,
sequencing each amplicon to identify an associated nucleic acid sequence and an associated barcode sequence,
selecting a first target nucleic acid sequence and determining the number of unique associated barcode sequences for the first target nucleic acid sequence, wherein the number of unique associated barcode sequences is the copy number of the first target nucleic acid sequence.

### Claim 2 (depends on 1)

The method of claim 1 wherein the nucleic acid molecules are DNA or RNA.

### Claim 3 (depends on 1)

The method of claim 1 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded RNA molecules and further including the steps of reverse transcribing the plurality of barcoded RNA molecules to produce barcoded cDNA molecules and amplifying the barcoded cDNA molecules to produce amplicons of the barcoded cDNA molecules.

### Claim 4 (depends on 3)

The method of claim 3 comprising repeatedly reverse transcribing the plurality of barcoded RNA molecules to produce linear pre-amplified barcoded cDNA molecules and amplifying the linear pre-amplified barcoded cDNA molecules to produce amplicons of the linear pre-amplified barcoded cDNA molecules.

### Claim 5 (depends on 4)

The method of claim 4 wherein the step of repeatedly reverse transcribing the plurality of barcoded RNA molecules includes using reverse transcriptase and a nicking enzyme.

### Claim 6 (depends on 1)

The method of claim 1 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded DNA molecules and further including the steps of repeated replication of the plurality of barcoded DNA molecules to produce a plurality of pre-amplified barcoded DNA molecules and amplifying the plurality of pre-amplified barcoded DNA molecules to produce amplicons of the plurality of pre-amplified barcoded DNA molecules.

### Claim 7 (depends on 6)

The method of claim 6 wherein the step of repeated replication of the plurality of barcoded DNA molecules includes using DNA polymerase and a nicking enzyme.

### Claim 8 (depends on 1)

The method of claim 1 wherein the sample is obtained from one or more cells of a first cell type and wherein amplification includes use of a primer generated from genomic DNA of the first cell type.

### Claim 9

A method of counting nucleic acid molecules in a sample including a plurality of nucleic acid molecules comprising
attaching a unique barcode sequence to substantially each of the plurality of nucleic acid molecules in the sample to produce a plurality of barcoded nucleic acid molecules,
amplifying the plurality of barcoded nucleic acid molecules in the sample to produce amplicons of the plurality of barcoded nucleic acid molecules,
sequencing each amplicon to identify an associated barcode sequence, and
counting the number of unique associated barcode sequences as a measure of the number of nucleic acid molecules in the sample.

### Claim 10 (depends on 9)

The method of claim 9 wherein the nucleic acid molecules are DNA or RNA.

### Claim 11 (depends on 9)

The method of claim 9 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded RNA molecules and further including the steps of reverse transcribing the plurality of barcoded RNA molecules to produce barcoded cDNA molecules and amplifying the plurality of barcoded cDNA molecules to produce amplicons of the plurality of barcoded cDNA molecules.

### Claim 12 (depends on 11)

The method of claim 11 comprising repeatedly reverse transcribing the plurality of barcoded RNA molecules to produce linear pre-amplified barcoded cDNA molecules and amplifying the linear pre-amplified barcoded cDNA molecules to produce amplicons of the linear pre-amplified barcoded cDNA molecules.

### Claim 13 (depends on 12)

The method of claim 12 wherein the step of repeatedly reverse transcribing the plurality of barcoded RNA molecules includes using reverse transcriptase and a nicking enzyme.

### Claim 14 (depends on 9)

The method of claim 9 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded DNA molecules and further including the steps of repeated replication of the plurality of barcoded DNA molecules to produce a plurality of pre-amplified barcoded DNA molecules and amplifying the plurality of pre-amplified barcoded DNA molecules to produce amplicons of the plurality of pre-amplified barcoded DNA molecules.

### Claim 15 (depends on 14)

The method of claim 14 wherein the step of repeated replication of the plurality of barcoded DNA molecules includes using DNA polymerase and a nicking enzyme.

### Claim 16 (depends on 9)

The method of claim 9 wherein the sample is obtained from one or more cells of a first cell type and wherein amplification includes use of a primer generated from genomic DNA of the first cell type.

### Claim 17

A method of determining copy numbers of nucleic acid molecules in a sample comprising
attaching a unique barcode sequence to substantially each of the nucleic acid molecules in the sample to produce a plurality of barcoded nucleic acid molecules,
amplifying the plurality of barcoded nucleic acid molecules in the sample to produce amplicons of the plurality of barcoded nucleic acid molecules,
massively parallel sequencing the amplicons of the plurality of barcoded nucleic acid molecules to identify for each amplicon an associated nucleic acid sequence and an associated barcode sequence, and
determining the number of unique associated barcode sequences for each nucleic acid sequence in the sample.

### Claim 18 (depends on 17)

The method of claim 17 wherein the nucleic acid molecules are DNA or RNA.

### Claim 19 (depends on 17)

The method of claim 17 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded RNA molecules and further including the steps of reverse transcribing the plurality of barcoded RNA molecules to produce barcoded cDNA molecules and amplifying the plurality of barcoded cDNA molecules to produce amplicons of the plurality of barcoded cDNA molecules.

### Claim 20 (depends on 19)

The method of claim 19 comprising repeatedly reverse transcribing the plurality of barcoded RNA molecules to produce linear pre-amplified barcoded cDNA molecules and amplifying the linear pre-amplified barcoded cDNA molecules to produce amplicons of the linear pre-amplified barcoded cDNA molecules.

### Claim 21 (depends on 20)

The method of claim 20 wherein the step of repeatedly reverse transcribing the plurality of barcoded RNA molecules includes using reverse transcriptase and a nicking enzyme.

### Claim 22 (depends on 17)

The method of claim 17 wherein the plurality of barcoded nucleic acid molecules is a plurality of barcoded DNA molecules and further including the steps of repeated replication of the plurality of barcoded DNA molecules to produce a plurality of pre-amplified barcoded DNA molecules and amplifying the plurality of pre-amplified barcoded DNA molecules to produce amplicons of the plurality of pre-amplified barcoded DNA molecules.

### Claim 23 (depends on 22)

The method of claim 22 wherein the step of repeated replication of the plurality of barcoded DNA molecules includes using DNA polymerase and a nicking enzyme.

### Claim 24 (depends on 17)

The method of claim 17 wherein the sample is obtained from one or more cells of a first cell type and wherein amplification includes use of a primer generated from genomic DNA of the first cell type.