# US 2020/0399691 A1 · Sample preparation methods, systems and compositions

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- Number: US 2020/0399691 A1
- Document ID: US20200399691A1
- Status: Published application
- Kind: A1
- Application number: 15/930,488
- Filed: 2020-05-13
- Published: 2020-12-24
- Earliest claimed: 2017-04-12
- Assignees: Karius, Inc.
- Applicants: Karius, Inc.
- Inventors: Timothy A. BLAUWKAMP; Rene SIT; Igor D. VILFAN
- Web page: https://patentgrep.com/patent/US20200399691A1

## CPC

- C12Q 1/6869: Methods for sequencing
- C12N 9/1247: {DNA-directed RNA polymerase (2.7.7.6)}
- C12N 9/93: {Ligases (6)}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6855: Ligating adaptors
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 2521/107: RNA dependent DNA polymerase,(i.e. reverse transcriptase)
- C12Q 2533/101: Primer extension
- C12Y 207/07006: DNA-directed RNA polymerase (2.7.7.6)
- C12Y 207/07049: RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
- C12Y 605/01001: DNA ligase (ATP) (6.5.1.1)
- C12Y 605/01003: RNA ligase (ATP) (6.5.1.3)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.

## Claims

### Claim at position 1

1-114. (canceled)

### Claim 115

A method of performing an amplification reaction on a first RNA and a first DNA, comprising:
a) providing a sample comprising a mixture of said first DNA and said first RNA, wherein said first DNA does not comprise a sequence complementary to said first RNA;
b) tagging said first DNA with a first tag without using a transposase;
c) tagging said first RNA with a second tag;
d) performing an amplification or primer extension reaction on said first DNA with a polymerase that is selective for DNA templates; and
e) synthesizing a complementary cDNA strand from said first RNA with a reverse transcriptase.

### Claim 116 (depends on 115)

The method of claim 115, wherein said first DNA is selected from the group comprising single-stranded DNA, double-stranded DNA, triple-stranded DNA, or a Holliday junction.

### Claim 117 (depends on 115)

The method of claim 115, wherein said first RNA is selected from the group comprising single-stranded RNA, double-stranded RNA, or a ribozyme.

### Claim 118 (depends on 115)

The method of claim 115, wherein said first DNA is cell-free DNA.

### Claim 119 (depends on 115)

The method of claim 115, wherein said sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchio-alveolar lavage, urine, stool, saliva, nasal swab, and any combination thereof.

### Claim 120 (depends on 115)

The method of claim 115, comprising performing said amplification to generate amplified products.

### Claim 121 (depends on 120)

The method of claim 120, further comprising sequencing said amplified products.

### Claim 122

A method of sequencing nucleic acids comprising:
a) providing a sample comprising double-stranded nucleic acids and single-stranded nucleic acids; and
b) attaching a first adapter to an end of said double-stranded nucleic acids;
c) denaturing said double-stranded nucleic acids into single-stranded nucleic acids;
d) sequencing nucleic acids ligated to said first adapter and identifying sequences
e) associated with said first adapter as being double-stranded.

### Claim 123

A method for concurrent processing of different nucleic acid forms in a sample comprising:
a) denaturing said nucleic acid forms in a sample;
b) attaching a first adapter to one end of a first nucleic acid form and attaching a second adapter to one end of a second nucleic acid form;
c) primer extending said first and said second attached nucleic acid forms;
d) attaching a third adapter comprising a priming element; and
e) amplifying said first and second nucleic forms.

### Claim 124

A reaction mixture composition comprising:
a) an adapter;
b) a first ligase that has a preference for a first nucleic acid form;
c) a second ligase that has a preference for a second nucleic acid form; and
d) a buffer.

### Claim 125

A reaction mixture comprising:
a) a ligase;
b) a DNA-dependent polymerase that has non-templated activity, wherein said non-templated base is N1; and
c) a RT polymerase that has non-templated activity, wherein said non-templated base is N2, wherein N1 and N2 are different nucleic acid bases.

### Claim 126 (depends on 124)

A kit for preparing the reaction mixture composition of claim 124 comprising:
a) an adapter;
b) a first ligase that has a preference for a first nucleic acid form;
c) a second ligase that has a preference for a second nucleic acid form; and
d) a buffer.

### Claim 127 (depends on 125)

A kit for preparing the reaction mixture composition of claim 125 comprising:
a) a ligase;
b) a DNA-dependent polymerase that has non-templated activity, wherein the non-templated base is N1; and
c) a RT polymerase that has non-templated activity, wherein the non-templated base is N2, wherein N1 and N2 are different nucleic acid bases.

### Claim 128 (depends on 123)

The method of claim 123, wherein said first and said second adapter comprise different identifying sequences; and further comprising sequencing said ligated nucleic acids, thereby identifying said different nucleic acid forms in said sample.

### Claim 129

A method for processing different nucleic acid forms in a sample comprising:
a) denaturing said different nucleic acid forms in a sample, wherein said different nucleic acid forms comprise a first nucleic acid form and a second nucleic acid form;
b) attaching a first adapter to said first nucleic acid form and a second adapter to said second nucleic acid form;
c) amplifying said first nucleic acid form using a DNA-dependent polymerase that has non-templated activity, wherein said non-templated activity comprises adding at least one N1 nucleotide or a first sequence to amplified products of said amplification of said first nucleic acid form; and
d) amplifying said second nucleic acid form using a reverse transcriptase polymerase that has non-templated activity, wherein said non-templated activity comprises adding at least one N2 nucleotide or a second sequence to amplified products of said amplification of said second nucleic acid form, wherein said N1 nucleotide and said N2 nucleotide are different nucleotides or said first sequence is different from said second sequence.

### Claim 130 (depends on 123)

The method of claim 123, wherein attaching said first adapter to one end of a first nucleic acid form comprises using a ligase that has a preference for said first nucleic acid form and wherein attaching said second adapter to one end of a second nucleic acid form comprises using a ligase that has a preference for said second nucleic acid form.