# US 2017/0283864 A1 · Use of transposase and y adapters to fragment and tag dna

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- Number: US 2017/0283864 A1
- Document ID: US20170283864A1
- Status: Published application
- Kind: A1
- Application number: 15/407,998
- Filed: 2017-01-17
- Published: 2017-10-05
- Earliest claimed: 2016-03-31
- Applicants: Agilent Technologies, Inc.
- Inventors: Robert A. Ach; Nicholas M. Sampas; Brian Jon Peter
- Web page: https://patentgrep.com/patent/US20170283864A1

## CPC

- C12Q 1/6855: Ligating adaptors
- 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 1/6869: Methods for sequencing
- 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

## Abstract

Described herein, among other things, is an adapter comprising a population of first oligonucleotides, a second oligonucleotide and a third oligonucleotide, wherein the first oligonucleotides, the second oligonucleotide and the third oligonucleotide are hybridized together to produce a complex that comprises: (i) a first end comprising a transposase recognition sequence, (ii) a central single-stranded region of variable sequence and (iii) a second end comprising sequences that are non-complementary. A method, as well as a kit for practicing the method, are also provided.

## Claims

### Claim 1

An adapter comprising a population of first oligonucleotides, a second oligonucleotide and a third oligonucleotide, wherein the first oligonucleotides, the second oligonucleotide and the third oligonucleotide are hybridized together to produce a complex that comprises: (i) a first end comprising a transposase recognition sequence, (ii) a central single-stranded region of variable sequence and (iii) a second end comprising sequences that are non-complementary.

### Claim 2 (depends on 1)

The adaptor of claim 1, wherein:
a) the population of first oligonucleotides comprise a 5′ region, a 3′ region, and a region of variable sequence between the 5′ region and the 3′ region;
b) the second oligonucleotide is complementary to and hybridizes with the 3′ region of the first oligonucleotides to form the transposase recognition sequence; and
c) the third oligonucleotide comprises 5′ end that is complementary to and is hybridized with the 3′ end of the 3′ region of the first oligonucleotide and comprises a 3′ tail that is non-complementary to the 5′ end of the 3′ region of the first oligonucleotide.

### Claim 3 (depends on 1)

The adaptor of claim 1, wherein the 5′ end of the first oligonucleotide and the 3′ tail of the third oligonucleotide are in a single molecule and joined together by a cleavable region.

### Claim 4 (depends on 1)

The adaptor of claim 1, wherein the variable sequence has a complexity of at least 1,000.

### Claim 5 (depends on 1)

The adaptor of claim 1, wherein the first, second, and third oligonucleotides are formed by allowing self-annealing of a single oligonucleotide molecule of greater than 60 nucleotides, followed by cleavage of cleavable sites located between the sequences between the first and third oligonucleotides and between the sequences of the second and third oligonucleotide.

### Claim 6 (depends on 1)

The adaptor of claim 1, wherein the transposase recognition sequence is the recognition sequence for a Vibhar transposase or variant thereof.

### Claim 7 (depends on 1)

The adaptor of claim 1, wherein the 3′ tail of the third oligonucleotide comprises a modification rendering the 3′ end resistant to digestion by a 3′-5′ exonuclease activity.

### Claim 8 (depends on 1)

A method for tagmenting a sample, comprising:
contacting a sample comprising double-stranded DNA with a transposase loaded with the adaptor of claim 1; and
filling in and sealing the central single-stranded region of the adaptor using a polymerase and ligase, thereby producing a population of DNA fragments that are tagged at both ends by a Y adaptor each comprising the variable sequence of a first oligonucleotide on both strands.

### Claim 9 (depends on 8)

The method of claim 8, wherein the method is done by combining, in a single reaction vessel, the sample, the transposase loaded with the adaptor, the polymerase and the ligase.

### Claim 10 (depends on 8)

The method of claim 8 wherein the filling in is done by Sulfolobus DNA polymerase IV, at a temperature less than 50 degrees Celsius.

### Claim 11 (depends on 8)

The method of claim 8, wherein the method further comprises amplifying the population of DNA fragments using primers that target the arms of the Y adaptor.

### Claim 12 (depends on 11)

The method of claim 11, wherein the amplifying is done in solution.

### Claim 13 (depends on 11)

The method of claim 11, wherein the amplifying is done by bridge PCR.

### Claim 14 (depends on 8)

The method of claim 8, further comprising sequencing at least some of the tagged DNA fragments.

### Claim 15 (depends on 1)

A kit comprising:
a transposase;
an adaptor of claim 1; and
a polymerase.

### Claim 16 (depends on 15)

The kit of claim 15, wherein the transposase is loaded with the adaptor.

### Claim 17 (depends on 16)

The kit of claim 16, wherein the loaded transposase, polymerase, and ligase are in a mix.

### Claim 18 (depends on 15)

The kit of claim 15, wherein the kit further comprises a pair of primers that are complementary to or the same as the non-complementary sequences at the second end of the adaptor.