# US 10,501,786 B2 · Nucleic acid encoding reactions

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- Number: US 10,501,786 B2
- Document ID: US10501786B2
- Status: Granted
- Kind: B2
- Application number: 14/723,872
- Filed: 2015-05-28
- Published: 2016-07-21
- Granted: 2019-12-10
- Earliest claimed: 2011-05-20
- Assignees: Fluidigm Corporation
- Applicants: Fluidigm Corporation
- Inventors: Megan Anderson; Peilin Chen; Brian Fowler; Robert C. Jones; Fiona Kaper; Ronald Lebofsky; Andrew May
- Web page: https://patentgrep.com/patent/US10501786B2

## CPC

- C12Q 1/6855: Ligating adaptors
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/65: using markers (enzymes used as markers C12N15/52)
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- 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

Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.

## Claims

### Claim 1

A method of analyzing nucleic acids in or associated with single particles, the method comprising:
a) capturing particles in separate reaction volumes, such that a plurality of reaction volumes each comprise a single particle, wherein the separate reaction volumes are droplets in an emulsion;
b) producing barcoded reaction products from nucleic acids in or associated with the single particle in each of the separate reaction volumes, wherein producing the reaction products comprises incorporating barcoded adaptor nucleotide sequences into the reaction products using a plurality of different single-stranded barcoded adaptor molecules for each separate reaction volume, wherein each adaptor molecule comprises: (i) a nucleotide sequence comprising a primer binding site, (ii) a degenerate tail sequence, and (iii) a barcode nucleotide sequence, wherein each plurality of different single-stranded barcoded adaptor molecules comprises the same barcode;
c) pooling the barcoded reaction products;
d) amplifying the barcoded reaction products using primers that bind the primer binding sites, to produce templates for bidirectional DNA sequencing; and
e) sequencing the barcoded reaction products.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

The method of claim 2, further comprising lysing the cells to release cell contents, after step a) and prior to step b).

### Claim 4 (depends on 2)

The method of claim 2, wherein the distribution of cells in the separate reaction volumes approximates a Poisson distribution.

### Claim 5 (depends on 4)

The method of claim 4, wherein the cells are diluted to produce a highest fraction of reaction volumes having only one cell.

### Claim 6 (depends on 2)

The method of claim 2, wherein step a) further comprises binding the cells to a solid support.

### Claim 7 (depends on 6)

The method of claim 6, wherein the solid support has a binding partner distributed across its surface.

### Claim 8 (depends on 7)

The method of claim 7, wherein the binding partner is an antibody.

### Claim 9 (depends on 6)

The method of claim 6, wherein the solid support is a bead.

### Claim 10 (depends on 1)

The method of claim 1, wherein the particles are disrupted to release interior components, after step a) and prior to step b).

### Claim 11 (depends on 1)

The method of claim 1, wherein the particles are beads.

### Claim 12 (depends on 11)

The method of claim 11, wherein the beads are primer-coated.

### Claim 13 (depends on 1)

The method of claim 1, wherein step b) of producing reaction products comprises reverse transcription.

### Claim 14 (depends on 1)

The method of claim 1, wherein step b) of producing reaction products comprises amplification.

### Claim 15 (depends on 13)

The method of claim 13, wherein step b) of producing reaction products comprises amplification.

### Claim 16 (depends on 1)

The method of claim 1, further comprising f) associating sequencing results obtained in e) with single particles.

### Claim 17 (depends on 1)

The method of claim 1, wherein the barcoded adaptor molecule is a non-specific degenerate adaptor.