# US 2025/0084403 A1 · Compositions and methods for making controls for sequence-based genetic testing

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2025/0084403 A1
- Document ID: US20250084403A1
- Status: Published application
- Kind: A1
- Application number: 18/960,386
- Filed: 2024-11-26
- Published: 2025-03-13
- Earliest claimed: 2018-04-02
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: David Comstock
- Web page: https://patentgrep.com/patent/US20250084403A1

## CPC

- C12N 15/1072: {Differential gene expression library synthesis, e.g. subtracted libraries, differential screening}
- 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 2600/166: Oligonucleotides used as internal standards, controls or normalisation probes
- 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

Provided herein are compositions that include nucleic acid fragments produced from double-stranded template nucleic acids, such as cell free DNA. The compositions can be used as positive or negative controls for quality of library preparation methods, calibration of an instrument such as a sequencing instrument, and/or a validation for a nucleic acid sequencing test. Also provided are methods for making the nucleic acid fragments.

## Claims

### Claim at position 1

1-20. (canceled)

### Claim 21

A method of using a control in a nucleic acid detection test, the method comprising:
providing a plurality of double-stranded template nucleic acids originating from a sample obtained from a subject, wherein the double-stranded template nucleic acids comprise a population of sizes and sequences similar to or identical to the sizes and sequences obtained from the subject;
ligating a universal adapter to both ends and both strands of the template nucleic acids to form a plurality of adapter-template-adapter molecules comprising template nucleic acids flanked by the universal adapter, wherein the universal adapter comprises a region of double stranded nucleic acid and a restriction endonuclease recognition site; and
amplifying the plurality of adapter-template-adapter molecules with a first universal primer and a second universal primer to result in amplified adapter-template-adapter molecules;
removing at least a portion of the universal adapter from both ends of the amplified adapter-template-adapter molecules to result in removed universal adapters and a plurality of regenerated template nucleic acids (reDNAs), wherein the reDNAs comprise a population of template molecules of sizes and sequences similar to or identical to the sizes and sequences obtained from the subject;
performing a nucleic acid detection test on a test sample and on the reDNAs; and
analyzing results from the nucleic acid detection test using the reDNAs as a synthetic control.

### Claim 22 (depends on 21)

The method of claim 21 wherein the double-stranded template nucleic acids comprise cell free DNA (cfDNA).

### Claim 23 (depends on 21)

The method of claim 21 wherein the subject is a pregnant human, and wherein the double-stranded template nucleic acids comprise a mixture of fetal and maternal nucleic acids.

### Claim 24 (depends on 23)

The method of claim 23 wherein the sample comprises cfDNA.

### Claim 25 (depends on 23)

The method of claim 23 wherein the fetus comprises a genetic condition.

### Claim 26 (depends on 25)

The method of claim 25 wherein the genetic condition is an aneuploidy.

### Claim 27 (depends on 21)

The method of claim 21 wherein the subject is suspected of having a neoplasm.

### Claim 28 (depends on 27)

The method of claim 27 wherein the sample comprises circulating tumor DNA and cell free normal DNA.

### Claim 29 (depends on 21)

The method of claim 21, wherein the amplifying comprises an exponential amplification reaction and the exponential amplification reaction is a polymerase chain reaction (PCR).

### Claim 30 (depends on 21)

The method of claim 21, wherein the test sample is from a different subject.

### Claim 31 (depends on 21)

The method of claim 21, wherein the method further comprises analyzing results from the nucleic acid detection test using at least a portion of the reDNAs as a synthetic control.

### Claim 32 (depends on 21)

The method of claim 21, wherein the method further comprising:
ligating a universal adapter to both ends of the reDNAs to form a plurality of adapter-reDNA-adapter molecules comprising an reDNA flanked by the universal adapter, wherein the universal adapter comprises (i) a region of double stranded nucleic acid, and (ii) a region of single-stranded non-complementary nucleic acid strands comprising at least one universal primer binding site, thereby producing a sequencing library for determining the sequence of at least a portion of templates, the sequencing library comprising the plurality of adapter-reDNA-adapter molecules; and
performing a nucleic acid detection test on a test sample and on the sequencing library.

### Claim 33 (depends on 32)

The method of claim 32, wherein the method further comprises analyzing results from the nucleic acid detection test using the at least a portion of the sequencing library as a synthetic control.

### Claim 34 (depends on 21)

The method of claim 21, the method further comprising: further comprising:
providing a surface comprising a plurality of amplification sites,
wherein the amplification sites comprise at least two populations of attached single stranded nuclei acids having a free 3′ end, and
contacting the surface comprising amplification sites with the plurality of adapter-template-adapter molecules under conditions suitable to produce a plurality of amplification sites that each comprise a clonal population of amplicons from an individual adapter-template-adapter molecule.

### Claim 35

A method of using a control in a nucleic acid detection test, the method comprising:
providing a plurality of double-stranded template nucleic acids originating from a sample obtained from a subject, wherein the subject is a pregnant human, wherein the double-stranded template nucleic acids comprise a mixture of fetal and maternal nucleic acids, and wherein the double-stranded template nucleic acids comprise a population of sizes and sequences similar to or identical to the sizes and sequences obtained from the subject;
ligating a universal adaptor to both ends of the template nucleic acids to form a plurality of adaptor-template-adaptor molecules comprising a template nucleic acid flanked by the universal adaptor, wherein the universal adaptor comprises a region of double stranded nucleic acid and a restriction endonuclease recognition site; and
amplifying, such as by an exponential amplification reaction, the plurality of adaptor-template-adaptor molecules with a first universal primer and a second universal primer to result in amplified adaptor-template-adaptor molecules;
removing at least a portion of the universal adaptor from both ends of the amplified adaptor-template-adaptor molecules to result in removed universal adaptors and a plurality of regenerated template nucleic acids (reDNAs), wherein the reDNAs comprise a population of template molecules of sizes and sequences similar to or identical to the sizes and sequences obtained from the subject;
performing a nucleic acid detection test on a test sample and on the reDNAs; and
analyzing results from the nucleic acid detection test using the reDNAs as a synthetic control.

### Claim 36 (depends on 35)

The method of claim 35, wherein the sample comprises cfDNA.

### Claim 37 (depends on 35)

The method of claim 35, wherein the fetus comprises a genetic condition.

### Claim 38 (depends on 37)

The method of claim 37, wherein the genetic condition is an aneuploidy.

### Claim 39 (depends on 35)

The method of claim 35, wherein the subject is suspected of having a neoplasm.

### Claim 40 (depends on 35)

The method of claim 35, wherein the test sample is from a different subject.