# US 2025/0084401 A1 · Methods for making libraries for nucleic acid sequencing

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- Number: US 2025/0084401 A1
- Document ID: US20250084401A1
- Status: Published application
- Kind: A1
- Application number: 18/558,035
- Filed: 2022-05-12
- Published: 2025-03-13
- Earliest claimed: 2021-05-14
- Assignees: Becton, Dickinson and Company
- Applicants: Becton, Dickinson and Company
- Inventors: Alvaro GODINEZ
- Web page: https://patentgrep.com/patent/US20250084401A1

## CPC

- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/1068: {Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

Disclosed herein include methods, compositions, and kits suitable for use in generating libraries for nucleic acid sequencing. There are provided, in some embodiments, a plurality of protein complexes. Each protein complex can comprise a transposome and a programmable DNA binding unit capable of specifically binding to a user-selected binding site on a target double-stranded DNA (dsDNA). The binding site for each of the plurality of protein complexes can be different from each other. The transposome can comprise a transposase, a first adaptor, and a second adaptor. The first adaptor, the second adaptor, or both, can be a sequencing adaptor.

## Claims

### Claim 1

A composition, comprising a plurality of protein complexes, wherein each of the plurality of protein complexes comprises a transposome and a programmable DNA binding unit capable of specifically binding to a binding site on a target double-stranded DNA (dsDNA), wherein the transposome comprises a transposase, a first adaptor and a second adaptor, and wherein the binding site for each of the plurality of protein complexes is different from each other.

### Claim 2 (depends on 1)

The composition of claim 1, wherein at least two of the plurality of protein complexes comprise the same transposome or wherein all of the plurality of protein complexes comprise the same transposome.

### Claim 3

(canceled)

### Claim 4

(canceled)

### Claim 5 (depends on 1)

The composition of claim 1, wherein the first adaptor and the second adaptor in the same transposome are the same; wherein the first adaptor, the second adaptor, or both, in different transposome are different, or any combination thereof.

### Claim 6

(canceled)

### Claim 7

(canceled)

### Claim 8

(canceled)

### Claim 9 (depends on 1)

The composition of claim 1, wherein the first adaptor, the second adaptor, or both, is a sequencing adaptor.

### Claim 10

(canceled)

### Claim 11 (depends on 1)

The composition of claim 1, wherein the binding sites of at least two of the plurality of protein complexes are on the same target dsDNA, wherein:
the binding sites of at least two of the plurality of protein complexes are about 1-50000 nucleotides apart on the same target dsDNA;
the distance between the binding sites of a pair of the plurality of protein complexes is substantially the same as the distance between the binding sites of another pair of the plurality of protein complexes;
the distance between the binding sites of a pair of the plurality of protein complexes is different as the distance between the binding sites of another pair of the plurality of protein complexes; or any combination thereof.

### Claim at position 12

-15. (canceled)

### Claim 16 (depends on 1)

The composition of claim 1, wherein at least two of the plurality of protein complexes are capable of specifically binding to different target dsDNA.

### Claim 17 (depends on 1)

The composition of claim 1, wherein the plurality of protein complexes are capable of specifically binding to about 2-5000 target dsDNA.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the transposase is Tn5 transposase, Tn7 transposase, mariner Tc1-like transposase, Himar1C9 transposase, or Sleeping Beauty transposase.

### Claim 19

(canceled)

### Claim 20 (depends on 1)

The composition of claim 1, wherein the programmable DNA binding unit comprises a nuclease-deficient CRISPR associated protein (dCAS protein) and a guide RNA (gRNA) capable of specifically binding to the binding site of the target dsDNA.

### Claim 21 (depends on 20)

The composition of claim 20, wherein the transposome is associated with the programmable DNA binding unit via a linker connecting the transposase and the dCAS protein.

### Claim at position 19

-32. (canceled)

### Claim 33

A reaction mixture, comprising
a composition comprising a plurality of protein complexes, wherein each of the plurality of protein complexes comprises a transposome and a programmable DNA binding unit capable of specifically binding to a binding site on a target double-stranded DNA (dsDNA), wherein the transposome comprises a transposase, a first adaptor and a second adaptor, and wherein the binding site for each of the plurality of protein complexes is different from each other; and
sample nucleic acids suspected of comprising one or more target dsDNA.

### Claim 34 (depends on 33)

The reaction mixture of claim 33, further comprising a DNA polymerase, dNTPs, or a combination thereof and a plurality of dsDNA fragments each comprising the first adaptor and the second adaptor of one of the plurality of protein complexes at each terminus respectively.

### Claim 35

(canceled)

### Claim 36

(canceled)

### Claim 37 (depends on 33)

The reaction mixture of claim 33, wherein the sample nucleic acids comprise eukaryotic DNA, bacterial DNA, viral DNA, fungal DNA, protozoa DNA, or a combination thereof; wherein the target dsDNA is genomic DNA, mitochondrial DNA, plasmid DNA, or a combination thereof; and
wherein the sample nucleic acids are from a biological sample, a clinical sample, an environmental sample, or a combination thereof, wherein the biological sample comprises stool, sputum, peripheral blood, plasma, serum, lymph nodes, respiratory tissue, exudates, bodily fluid, or a combination thereof.

### Claim 38

(canceled)

### Claim 39

(canceled)

### Claim 40

(canceled)

### Claim 41

(canceled)

### Claim 42

A method for generating a sequencing library, comprising:
contacting a composition with a sample suspected of comprising a plurality of target double-stranded DNA (dsDNA) to form a reaction mixture, wherein the composition comprises a plurality of protein complexes, wherein each of the plurality of protein complexes comprises a transposome and a programmable DNA binding unit capable of specifically binding to a binding site on a target double-stranded DNA (dsDNA), wherein the transposome comprises a transposase, a first adaptor and a second adaptor, and wherein the binding site for each of the plurality of protein complexes is different from each other;
incubating the reaction mixture to generate a plurality of dsDNA fragments each comprising the first adaptor and the second adaptor of one of the plurality of protein complexes at each terminus respectively; and
amplifying the plurality of dsDNA fragments with primers capable of binding to the adaptors at the termini of the dsDNA fragments to generate a sequencing library.

### Claim 43 (depends on 42)

The method of claim 42, wherein each of the primers is about 5-80 nucleotides in length.

### Claim 44 (depends on 42)

The method of claim 42, wherein amplifying the plurality of dsDNA fragments with the primers is carried out using polymerase chain reaction (PCR).

### Claim 45

(canceled)

### Claim 46

(canceled)

### Claim 47 (depends on 42)

The method of claim 42, wherein:
the sample comprises eukaryotic DNA, bacterial DNA, viral DNA, fungal DNA, protozoa DNA, or a combination thereof;
wherein the plurality of target dsDNA comprises genomic DNA, mitochondrial DNA, plasmid DNA, or a combination thereof; and
wherein the sample is, or is derived from, a biological sample, a clinical sample, an environmental sample, or a combination thereof.

### Claim 48

(canceled)

### Claim 49

(canceled)

### Claim 50

(canceled)

### Claim 51

(canceled)

### Claim 52 (depends on 42)

The method of claim 42, further comprising generating the plurality of target dsDNA from a plurality of target RNA with a reverse transcriptase or wherein the plurality of target dsDNA comprises target dsDNA generated from target RNA with a reverse transcriptase.

### Claim at position 40

-64. (canceled)

### Claim 65 (depends on 42)

The method of claim 42, wherein the plurality of protein complexes and the plurality of target dsDNA are present in the reaction mixture at a molecular ratio of about 2:1 to about 2,000:1.

### Claim 66

(canceled)

### Claim 67 (depends on 42)

The method of claim 42, further comprising labeling one or both ends of one or more of the plurality of dsDNA fragments.

### Claim at position 44

-70. (canceled)