# US 12,760,360 B2 · Transposase-based genomic analysis

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- Number: US 12,760,360 B2
- Document ID: US12760360B2
- Status: Granted
- Kind: B2
- Application number: 18/499,894
- Filed: 2023-11-01
- Published: 2024-05-16
- Granted: 2026-10-13
- Earliest claimed: 2017-11-02
- Assignees: Bio-Rad Laboratories, Inc.
- Applicants: Bio-Rad Laboratories, Inc.
- Inventors: Ronald Lebofsky; Jennifer Chew
- Web page: https://patentgrep.com/patent/US12760360B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6855: Ligating adaptors
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6862: Ligase chain reaction [LCR]
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 2563/185: Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
- C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
- C12Q 2521/301: Endonuclease
- C12Q 2521/501: Ligase
- C12Q 2525/191: incorporating an adaptor
- C12Q 2535/122: Massive parallel sequencing
- C12Q 2563/149: Particles, e.g. beads
- C12Q 2563/159: Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
- C12Q 2563/179: the label being a nucleic acid
- C12Q 2565/514: characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
- 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)
- C12Y: ENZYMES

## Abstract

The methods and reagents are provided for barcoding and analysis of DNA samples using partition (e.g., droplet) technology while avoiding performing amplification in droplets.

## Claims

### Claim 1

A method of barcoding DNA, the method comprising
introducing oligonucleotide adaptors randomly into DNA comprising DNA-bound proteins by contacting the DNA with a transposase loaded with the oligonucleotide adaptors,
wherein the oligonucleotide adaptors comprise a single stranded portion and a double stranded portion, and
wherein the transposase introduces double-stranded breaks into the DNA, wherein each double-stranded break forms two DNA ends and the transposase ligates the first oligonucleotide to one strand of each DNA end, to form DNA fragments comprising the oligonucleotide adaptors at both ends;
forming partitions, wherein the partitions contain the DNA fragments and a first oligonucleotide primer having a bead-specific barcode sequence, wherein the first oligonucleotide primer is linked to a bead and comprises a free 3′ end;
in the partitions, gap filling 5′ overhangs with a polymerase that has strand displacing activity or 5′ to 3′ exonuclease activity to create a double-stranded sequence by extending 3′ ends;
hybridizing the 3′ end of the first oligonucleotide primer to a 3′ single stranded portion of the oligonucleotide adaptor, thereby forming barcoded DNA fragments;
combining contents of the partitions to form a reaction mixture.

### Claim 2 (depends on 1)

The method of claim 1, further comprising amplifying the barcoded fragments.

### Claim 3 (depends on 2)

The method of claim 2, wherein the amplifying comprises polymerase chain reaction.

### Claim 4 (depends on 1)

The method of claim 1, comprising stripping the transposase from the DNA before the hybridizing.

### Claim 5 (depends on 4)

The method of claim 4, wherein the stripping occurs in the droplets.

### Claim 6 (depends on 4)

The method of claim 4, wherein the DNA is in a nucleus and the stripping occurs before the forming of the droplets.

### Claim 7 (depends on 1)

The method of claim 1, comprising cleaving the oligonucleotide primer from beads before the hybridizing.

### Claim 8 (depends on 1)

The method of claim 1, wherein the transposase is loaded with two different adaptor oligonucleotides having the same double stranded portion and different single stranded portions.

### Claim 9 (depends on 1)

The method of claim 1, wherein the transposase is loaded with two identical adaptor oligonucleotides.

### Claim 10 (depends on 1)

The method of claim 1, wherein the first oligonucleotide primer comprises a 5′ PCR handle sequence.

### Claim 11 (depends on 10)

The method of claim 10, wherein the droplets further comprise the second oligonucleotide primer and wherein the second oligonucleotide primer comprises a 5′ PCR handle.

### Claim 12 (depends on 1)

The method of claim 1, wherein the single-stranded portion of the second oligonucleotide comprises:
i. a 3′ end sequence less than 50% complementary to the first oligonucleotide primer; and
ii. a middle sequence that is at least 50% complementary to the free 3′ end of the first oligonucleotide primer.

### Claim 13 (depends on 1)

The method of claim 1, further comprising removing the DNA-bound proteins from the DNA following the combining.

### Claim 14 (depends on 1)

The method of claim 1, further comprising removing the DNA-bound proteins from the DNA before the combining.

### Claim 15 (depends on 1)

The method of claim 1, wherein the forming maintains contiguity of the DNA fragments compared to the DNA.

### Claim 16 (depends on 1)

The method of claim 1, wherein the DNA is purified following the combining and before the contacting.

### Claim 17 (depends on 1)

The method of claim 1, further comprising during the combining, mixing the contents of the droplets with a competitor oligonucleotide comprising the single-stranded portion, which hybridizes to 3′ ends of unbound copies of the first oligonucleotide primer, thereby preventing de novo binding of unbound DNA fragments after the combining.

### Claim 18 (depends on 1)

The method of claim 1, further comprising during the combining, mixing the contents of the droplets with a competitor oligonucleotide comprising the single-stranded portion, which hybridizes to 3′ ends of unbound copies of the oligonucleotide adaptors, thereby preventing de novo binding of unbound DNA fragments after the combining.

### Claim 19 (depends on 18)

The method of claim 18, wherein the competitor oligonucleotides comprise 3′ ends that are not extendable by a polymerase.

### Claim 20 (depends on 1)

The method of claim 1, wherein the introducing comprises introducing the transposase loaded with the oligonucleotide adaptors into nuclei or fixed and permeabilized cells such that the transposase cleaves the DNA as chromatin structure in the DNA allows.

### Claim 21 (depends on 1)

The method of claim 1, wherein the partitions are droplets or microwells.