# US 11,111,519 B2 · Sequencing of nucleic acids via barcoding in discrete entities

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- Number: US 11,111,519 B2
- Document ID: US11111519B2
- Status: Granted
- Kind: B2
- Application number: 15/015,015
- Filed: 2016-02-03
- Published: 2017-01-12
- Granted: 2021-09-07
- Earliest claimed: 2015-02-04
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Adam R. Abate; John R. Haliburton; Freeman Lan; Adam R. Sciambi
- Web page: https://patentgrep.com/patent/US11111519B2

## CPC

- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- G01N 15/1459: {the analysis being performed on a sample stream}
- B01F 25/4338: {Mixers with a succession of converging-diverging cross-sections, i.e. undulating cross-section}
- B01F 33/3021: {the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets}
- B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics}
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/68: involving nucleic acids
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6855: Ligating adaptors
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G01N 15/0205: by optical means
- G01N 15/1404: Handling flow, e.g. hydrodynamic focusing
- G01N 15/1484: {microstructural devices}
- B01F 21/00: Dissolving (separating by dissolving B01D; dissolving to effect cooling F25D5/00)
- B01L 3/502707: {characterised by the manufacture of the container or its components}
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples}
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2200/0652: Sorting or classification of particles or molecules
- B01L 2300/021: Identification, e.g. bar codes
- B01L 2300/0645: Electrodes
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2300/123: Flexible; Elastomeric
- B01L 2400/0409: centrifugal forces
- B01L 2400/0415: electrical forces, e.g. electrokinetic
- B01L 2400/0424: Dielectrophoretic forces
- B01L 2400/043: magnetic forces
- G01N 15/149: specially adapted for sorting particles, e.g. by their size or optical properties
- G01N 2015/0288: {Sorting the particles}
- G01N 2015/0294: {Particle shape}
- G01N 2015/1006: {for cytology}
- G01N 2015/1493: {Particle size}
- G01N 2015/1497: {Particle shape}
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Microfluidic methods for barcoding nucleic acid target molecules to be analyzed, e.g., via nucleic acid sequencing techniques, are provided. Also provided are microfluidic, droplet-based methods of preparing nucleic acid barcodes for use in various barcoding applications. The methods described herein facilitate high-throughput sequencing of nucleic acid target molecules as well as single cell and single virus genomic, transcriptomic, and/or proteomic analysis/profiling. Systems and devices for practicing the subject methods are also provided.

## Claims

### Claim 1

A method of introducing multiple copies of a first nucleic acid barcode to nucleic acids of a discrete entity, the method comprising:
encapsulating a plurality of nucleic acid target molecules in a discrete entity;
flowing the discrete entity in a first channel to a junction;
flowing a continuous, unsegmented reagent flow comprising multiple copies of a first nucleic acid barcode and multiple copies of a second nucleic acid barcode in a second channel to the junction;
flowing a carrier fluid in a third channel to the junction, wherein the second channel comprising the continuous, unsegmented reagent flow is positioned between the first channel comprising the discrete entity and the third channel comprising the carrier fluid;
merging the discrete entity into the continuous, unsegmented reagent flow downstream of the junction; and
downstream of the junction, inducing the continuous, unsegmented reagent flow to break off one individual microdroplet comprising the plurality of nucleic acid target molecules of the discrete entity, multiple copies of the first nucleic acid barcode, and no copies of the second nucleic acid barcode, such that substantially all of the plurality of nucleic acid target molecules of the discrete entity are broken off into the one individual microdroplet.

### Claim 2 (depends on 1)

The method of claim 1, wherein encapsulating a plurality of nucleic acid target molecules in a discrete entity comprises lysing a cell in an intermediate droplet, thereby encapsulating nucleic acid target molecules of the cell in the intermediate droplet.

### Claim 3 (depends on 1)

The method of claim 1, wherein the multiple copies of a first nucleic acid barcode are anchored on at least one solid substrate comprising a bead.

### Claim 4 (depends on 3)

The method of claim 3, wherein the merging comprises using an electric field to merge the discrete entity to the continuous, unsegmented reagent flow.

### Claim 5 (depends on 1)

The method of claim 1, further comprising flowing the continuous, unsegmented reagent flow under drip-producing conditions while merging the discrete entity into the reagent flow.

### Claim 6 (depends on 1)

The method of claim 1, further comprising flowing the continuous, unsegmented reagent flow under jetting conditions while merging the discrete entity into the continuous, unsegmented reagent flow.

### Claim 7 (depends on 1)

The method of claim 1, further comprising enzymatically incorporating a copy of the first nucleic acid barcode into a nucleic acid target molecule within the individual microdroplet, and synthesizing a complement of the nucleic acid target molecule tagged with the first nucleic acid barcode.

### Claim 8 (depends on 7)

The method of claim 7, wherein synthesizing the complement comprises amplification.

### Claim 9 (depends on 7)

The method of claim 7, wherein synthesizing the complement comprises reverse transcription.

### Claim 10 (depends on 7)

The method of claim 7, wherein synthesizing comprises synthesizing within a microdroplet.

### Claim 11 (depends on 1)

The method of claim 1, wherein each of the multiple copies of first barcode sequence comprises a corresponding unique molecular identifier.

### Claim 12 (depends on 1)

The method of claim 1, further comprising tagging the plurality of nucleic acid target molecules with a unique molecular identifier prior to the encapsulating.

### Claim 13 (depends on 12)

The method of claim 12, wherein the first nucleic acid barcode comprises a longer sequence length than the unique molecular identifier.

### Claim 14 (depends on 12)

The method of claim 12, further comprising amplifying the plurality of nucleic acid target molecules comprising the unique molecular identifier prior to the encapsulating.

### Claim 15 (depends on 1)

The method of claim 1, further comprising:
at the junction, contacting the discrete entity from the first channel with a first side of the continuous, unsegmented reagent flow from the second channel, and contacting the carrier fluid from the third channel with the continuous, unsegmented reagent flow from the second channel.

### Claim 16 (depends on 1)

The method of claim 1, further comprising:
prior to merging the discrete entity into the continuous, unsegmented reagent flow, co-flowing the discrete entity in contact with the continuous, unsegmented reagent flow downstream of the junction.