# US 12,215,314 B2 · Integrative DNA and RNA library preparations and uses thereof

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- Number: US 12,215,314 B2
- Document ID: US12215314B2
- Status: Granted
- Kind: B2
- Application number: 17/041,724
- Filed: 2019-03-26
- Published: 2021-01-28
- Granted: 2025-02-04
- Earliest claimed: 2018-03-26
- Assignees: Qiagen Sciences, LLC
- Applicants: Yexun Wang; Quan Peng; Daniel Kim; QIAGEN Sciences, LLC
- Inventors: Yexun Wang; Quan Peng; Daniel Kim
- Web page: https://patentgrep.com/patent/US12215314B2

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- 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

The invention relates to integrated DNA and RNA library preparations and methods of making and uses thereof, wherein the DNA molecules are labelled with a tag identifying the molecule as being a DNA molecule and the RNA molecules are labelled with a tag identifying the molecule as being a RNA molecule. The methods do not require physical separation of DNA and RNA. The methods output two separate libraries from DNA and RNA, respectively, which helps flexible manipulation on downstream sequencing platforms. The application also claims the DNA library and the cDNA library produced by the method and the DNA tag and the RNA tag used in the method.

## Claims

### Claim 1

A method for preparing DNA and cDNA libraries in a same sample comprising DNA and RNA molecules, comprising:
ligating a DNA tag comprising a single-stranded portion to a 3′ end of a double-stranded DNA molecule in the same sample to form a tagged DNA, wherein the DNA tag comprises a unique molecular identifier (UMI) and a DNA identifier;
performing reverse transcription of a RNA molecule in the same sample in the presence of a RNA tag to form a tagged cDNA, wherein the RNA tag comprises a RNA identifier, a UMI, and a poly(T); and
generating DNA and cDNA libraries in the same sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the reverse transcription is performed in the presence of a second RNA tag, wherein the second RNA tag comprises a RNA identifier, a UMI, and a template switching oligonucleotide (TSO).

### Claim 3 (depends on 1)

The method of claim 1, further comprising amplifying the tagged DNA and the tagged cDNA for enrichment with a set of gene specific primers.

### Claim 4 (depends on 3)

The method of claim 3, further comprising separating the same sample into first and second samples.

### Claim 5 (depends on 1)

The method of claim 1, wherein the same sample is a biological sample.

### Claim 6 (depends on 1)

The method of claim 1, wherein the DNA and RNA molecules are fragmented DNA and RNA.

### Claim 7 (depends on 1)

The method of claim 1, wherein the double-stranded DNA molecule contains polished ends for ligation.

### Claim 8 (depends on 1)

The method of claim 1, wherein the RNA molecule is polyadenylated.

### Claim 9 (depends on 1)

The method of claim 1, wherein the method does not require ribosomal depletion.

### Claim 10 (depends on 4)

The method of claim 4, further comprising amplifying the first sample with primers specific for the DNA tag.

### Claim 11 (depends on 10)

The method of claim 10, wherein the amplification generates the DNA library corresponding to the tagged DNA in the first sample.

### Claim 12 (depends on 4)

The method of claim 4, further comprising amplifying the second sample with primers specific for the RNA tag.

### Claim 13 (depends on 12)

The method of claim 12, wherein the amplification generates the cDNA library corresponding to the tagged RNA in the second sample.

### Claim 14 (depends on 1)

The method of claim 1, further comprising sequencing the DNA and/or cDNA libraries.

### Claim 15 (depends on 1)

The method of claim 1, wherein the DNA molecules are genomic DNA.

### Claim 16

A method for preparing targeted DNA and cDNA libraries in a same sample, comprising:
(a) obtaining purified DNA and RNA from a same biological sample;
(b) fragmenting the DNA and RNA;
(c) polishing the ends of double-stranded DNA fragments for ligation;
(d) polishing RNA fragments by polyadenylation;
(e) ligating a DNA tag comprising a single-stranded portion to a 3′ end of the polished double-stranded DNA fragments in the same sample to form a tagged DNA, wherein the DNA tag comprises in a 5′ to 3′ direction a unique molecular identifier (UMI) and a DNA identifier;
(f) performing reverse transcription of the polished RNA fragments in the same sample in the presence of a first RNA tag to form a tagged cDNA, wherein the first RNA tag comprises in a 5′ to 3′ direction a RNA identifier, a UMI, and a poly(T), and a second RNA tag, wherein the second RNA tag comprises in a 5′ to 3′ direction a RNA identifier, a UMI, and a template switching oligonucleotide (TSO);
(g) amplifying the tagged DNA and tagged cDNA for enrichment with a set of gene specific primers to form an amplified sample;
(h)
further amplifying the first amplified sample with primers specific for the DNA tag;
(i) further amplifying the amplified sample with primers specific for the RNA tag; and
(j) generating the targeted DNA and cDNA libraries in the same sample.