# US 11,299,780 B2 · Methods of producing nucleic acid libraries

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- Number: US 11,299,780 B2
- Document ID: US11299780B2
- Status: Granted
- Kind: B2
- Application number: 16/316,268
- Filed: 2017-07-13
- Published: 2020-05-14
- Granted: 2022-04-12
- Earliest claimed: 2016-07-15
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Richard E. Green
- Web page: https://patentgrep.com/patent/US11299780B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 2521/501: Ligase
- C12Q 2525/179: incorporating arbitrary or random nucleotide sequences
- C12Q 2525/191: incorporating an adaptor
- C12Q 2525/204: specific length of the oligonucleotides
- C12Q 2533/107: Probe or oligonucleotide ligation
- C12Q 2563/179: the label being a nucleic acid
- C12Q 2565/631: being a biochannel or pore
- 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

Provided are methods of producing nucleic acid libraries. In certain aspects, the methods include combining target nucleic acids (e.g., 5′ phosphorylated nucleic acids) and an oligonucleotide pool. Oligonucleotides of the oligonucleotide pool may include complementarity regions of varying length and nucleotide sequence, and a complementarity region identification sequence. In such aspects, the combining is under conditions in which oligonucleotides of the oligonucleotide pool hybridize to nucleic acids of the target nucleic acids (e.g., 5′ phosphorylated nucleic acids) having overhang regions that are complementary in sequence and have corresponding lengths with respect to the complementarity regions of the oligonucleotides. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure are also provided.

## Claims

### Claim 1

A method for producing a nucleic acid library, comprising:
combining:
a nucleic acid composition comprising target nucleic acids, wherein some or all of the target nucleic acids comprise an overhang; and
an oligonucleotide pool comprising oligonucleotides, wherein:
some or all of the oligonucleotides comprise (i) a complementarity region capable of hybridizing to an overhang in a target nucleic acid, and (ii) a complementarity region identification polynucleotide,
the oligonucleotides in the pool comprising complementarity regions include complementarity regions of different lengths, and
the complementarity region identification polynucleotide is specific to one or more features of the complementarity region, wherein the one or more features comprise length of the complementarity region;
under conditions in which complementarity regions in the oligonucleotides hybridize to overhangs in the target nucleic acids having a corresponding length, thereby forming hybridization products for a nucleic acid library.

### Claim 2 (depends on 1)

The method of claim 1, wherein the one or more features of the complementarity region further comprise (i) a 5′ overhang, (ii) a 3′ overhang, (iii) a particular sequence, (iv) a combination of (i) and (iii), or (v) a combination of (ii) and (iii).

### Claim 3 (depends on 1)

The method of claim 1, wherein:
some of the target nucleic acids comprise no overhang; and
some of the oligonucleotides comprise no complementarity region and comprise an identification polynucleotide specific to no complementarity region.

### Claim 4 (depends on 1)

The method of claim 1, wherein:
each target nucleic acid comprising an overhang comprises an overhang at one end or an overhang at both ends; and
an end, or both ends, of each target nucleic acid comprising an overhang independently comprises a 5′ overhang or a 3′ overhang.

### Claim 5 (depends on 1)

The method of claim 1, wherein the target nucleic acids are, or are produced from, cell-free nucleic acids.

### Claim 6 (depends on 5)

The method of claim 5, wherein the cell-free nucleic acids comprise cell-free fetal DNAs, circulating cancer DNAs, or host and non-host DNAs.

### Claim 7 (depends on 1)

The method of claim 1, wherein the overhangs in the target nucleic acids are native overhangs.

### Claim 8 (depends on 7)

The method of claim 7, wherein the native overhangs are not filled-in, cleaved, or introduced prior to combining with the pool.

### Claim 9 (depends on 7)

The method of claim 7, wherein the target nucleic acids are not modified in length prior to combining with the pool.

### Claim 10 (depends on 1)

The method of claim 1, wherein:
the oligonucleotide pool comprises oligonucleotides that comprise an overhang;
each of the oligonucleotides comprising an overhang comprise an overhang at one end or an overhang at both ends;
an end, or both ends, of each of the oligonucleotides comprising an overhang independently comprise a 5′ overhang or a 3′ overhang; and
at an end, the overhang comprises the complementarity region.

### Claim 11 (depends on 10)

The method of claim 10, wherein the oligonucleotide pool comprises oligonucleotides that comprise different overhang polynucleotides for a particular overhang length.

### Claim 12 (depends on 10)

The method of claim 10, wherein the oligonucleotide pool comprises oligonucleotides that comprise all possible overhang polynucleotide combinations for a particular overhang length.

### Claim 13 (depends on 10)

The method of claim 10, wherein the oligonucleotide pool comprises oligonucleotides that comprise all possible overhang polynucleotide combinations for each overhang length.

### Claim 14 (depends on 10)

The method of claim 10, wherein the overhang polynucleotides in the oligonucleotides of the pool are random.

### Claim 15 (depends on 10)

The method of claim 10, wherein the oligonucleotide pool comprises oligonucleotides that comprise different overhang polynucleotides for a particular overhang length, and the overhang polynucleotides in the oligonucleotides of the pool are random.

### Claim 16 (depends on 10)

The method of claim 10, wherein the oligonucleotides of the oligonucleotide pool comprise a portion other than the complementarity region and the complementarity region identification polynucleotide, wherein:
(i) the portion other than the complementarity region and the complementarity region identification polynucleotide comprises a nucleic acid binding domain;
(ii) the portion other than the complementarity region and the complementarity region identification polynucleotide comprises all or a portion of a sequencing adapter; or
(iii) a combination of (i) and (ii).

### Claim 17 (depends on 1)

The method of claim 1, wherein oligonucleotides of the oligonucleotide pool are phosphorylated.

### Claim 18 (depends on 1)

The method of claim 1, comprising, prior to combining the nucleic acid composition with the oligonucleotide pool, contacting the target nucleic acids in the composition with an agent comprising a phosphoryl transfer activity under conditions in which a 5′ phosphate is added to a 5′ end of target nucleic acids.

### Claim 19 (depends on 1)

The method of claim 1, comprising contacting the hybridization products with an agent comprising a ligase activity under conditions in which an end of a target nucleic acid is covalently linked to an end of the oligonucleotide to which the target nucleic acid is hybridized.

### Claim 20 (depends on 1)

A method for sequencing target nucleic acids, comprising:
performing a method of claim 1, thereby generating a nucleic acid library;
exposing the nucleic acid library to a sequencing process, wherein the sequencing process generates sequencing reads; and
(a) determining the sequence of one or more overhangs for the target nucleic acids based on the sequencing reads,
(b) determining the sequence of one or more complementarity region identification polynucleotides based on the sequencing reads,
(c) determining lengths of one or more overhangs for the target nucleic acids, or
(d) determining a combination of two or three of (a), (b), and (c).

### Claim 21 (depends on 20)

A method for analyzing one or more particular overhangs in a nucleic acid composition, comprising:
sequencing nucleic acids according to the method of claim 20; and
quantifying the amount of one or more particular overhangs in target nucleic acids, thereby generating one or more overhang quantifications, wherein each overhang quantification is for an overhang characterized as (i) a 5′ overhang, (ii) a 3′ overhang, (iii) a particular sequence, (iv) a particular length, or (v) a combination of two, three or four of (i), (ii), (iii), and (iv).

### Claim 22 (depends on 21)

The method of claim 21, further comprising classifying the sequencing reads by source of origin based on overhang content.

### Claim 23 (depends on 1)

The method of claim 1, wherein each complementarity region identification polynucleotide comprises a nucleic acid sequence that is unique for each oligonucleotide comprising a complementarity region of a particular length.

### Claim 24 (depends on 1)

The method of claim 1, wherein each complementarity region identification polynucleotide comprises a nucleic acid sequence that is unique for each oligonucleotide comprising a complementarity region of a particular length and a particular 5′ or 3′ directionality.