# US 12,601,089 B2 · Direct-to-library methods, systems, and compositions

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- Number: US 12,601,089 B2
- Document ID: US12601089B2
- Status: Granted
- Kind: B2
- Application number: 19/188,895
- Filed: 2025-04-24
- Published: 2025-12-11
- Granted: 2026-04-14
- Earliest claimed: 2018-11-21
- Assignees: Karius, Inc.
- Applicants: Karius, Inc.
- Inventors: Sivan Bercovici; Lily Blair; Timothy A. Blauwkamp; Peter J. Eugster; David K. Hong; Trupti Kawli; Michael J. Rosen; Damek Spacek; Igor D. Vilfan
- Web page: https://patentgrep.com/patent/US12601089B2

## CPC

- C12Q 1/6888: for detection or identification of organisms
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- 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: 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
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are direct-to-library methods, systems, and compositions.

## Claims

### Claim 1

A method of preparing a nucleic acid library from an initial sample, comprising:
(a) adding one or more process control molecules to the initial sample to provide a spiked initial sample, wherein the one or more process control molecules are synthetic nucleic acids; and
(b) generating a nucleic acid library from the spiked initial sample, wherein generating the nucleic acid library comprises attaching one or more adapters to nucleic acids in the spiked initial sample, wherein the one or more adapters comprise an overhang region and a double-stranded region;
wherein nucleic acids used to generate the nucleic acid library are not extracted from the initial sample nor the spiked initial sample before the attaching the one or more adapters.

### Claim 2 (depends on 1)

The method of claim 1, further comprising isolating the nucleic acids after the attaching the one or more adapters.

### Claim 3 (depends on 1)

The method of claim 1, wherein the nucleic acids are single-stranded nucleic acids.

### Claim 4 (depends on 1)

The method of claim 1, wherein said initial sample is selected from the group consisting of plasma, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage, saliva, and a nasal sample.

### Claim 5 (depends on 1)

The method of claim 1, wherein said initial sample is plasma.

### Claim 6 (depends on 1)

The method of claim 1, wherein said initial sample is bronchoalveolar lavage.

### Claim 7 (depends on 1)

The method of claim 1, wherein said initial sample is from a human subject.

### Claim 8 (depends on 1)

The method of claim 1, wherein said initial sample is from a human subject that has undergone an organ transplant.

### Claim 9 (depends on 1)

The method of claim 1, wherein the one or more process control molecules comprises one or more of an ID Spike(s), Spanks, and/or Sparks or GC Spike-in Panel.

### Claim 10 (depends on 1)

The method of claim 1, wherein generating the nucleic acid library from the initial sample comprises:
(a) denaturing the nucleic acids to produce denatured nucleic acids;
(b) attaching an adapter to the 3′-end of the denatured nucleic acids to produce adapted nucleic acids;
(c) attaching an adapter to the 5′-end of the denatured nucleic acids; and
(d) amplifying the complementary strands.

### Claim 11 (depends on 10)

The method of claim 10, further comprising annealing a primer to the adapted nucleic acids and extending the primer with a polymerase to generate complementary strands.

### Claim 12 (depends on 10)

The method of claim 10, wherein attaching the one or more adapters to the nucleic acids comprises ligating with a ligase selected from the group consisting of: T4 DNA ligase, CircLigase II, CircLigase ssDNA Ligase, CircLigase RNA Ligase, Thermostable App-DNA/RNA ligase, T4 RNA ligase 1, T4 RNA Ligase 2, T4 RNA Ligase 2 truncated, and Splint-R ligase.

### Claim 13 (depends on 12)

The method of claim 12, wherein attaching the one or more adapters to the nucleic acids comprises ligating with a T4 DNA ligase.

### Claim 14 (depends on 10)

The method of claim 10, further comprising incubating with a protease prior to denaturing the nucleic acids.

### Claim 15 (depends on 14)

The method of claim 14, wherein the protease comprises proteinase K.

### Claim 16 (depends on 1)

The method of claim 1, wherein the one or more adapters comprise a splint oligonucleotide.

### Claim 17 (depends on 16)

The method of claim 16, further comprising introducing a moiety to the 5′-end of the splint oligonucleotide.

### Claim 18 (depends on 17)

The method of claim 17, wherein the moiety comprises a bulky moiety.

### Claim 19 (depends on 1)

The method of claim 1, wherein the nucleic acids comprise cell-free nucleic acids.

### Claim 20 (depends on 19)

The method of claim 19, wherein the cell-free nucleic acids comprise microbial cell-free nucleic acids.

### Claim 21 (depends on 19)

The method of claim 19, wherein the nucleic acids comprise cell-free DNA (cfDNA), cell-free RNA (cfRNA), or a combination thereof.

### Claim 22 (depends on 21)

The method of claim 21, wherein the nucleic acids comprise a combination of cfDNA and cfRNA.

### Claim 23 (depends on 22)

The method of claim 22, further comprising reverse transcribing the cfRNA into CDNA.

### Claim 24 (depends on 1)

The method of claim 1, wherein said initial sample is selected from the group consisting of whole blood, urine, stool, abdominal fluid, ascites fluid, peritoneal lavage, gastric fluid, interstitial fluid, lymph fluid, bile, abscess fluid, amniotic fluid, meconium, sinus aspirate, cheek swab, skin swab, urethral swab, cervical swab, nasopharyngeal swab, nasopharyngeal aspirate, vaginal swab, semen, vaginal discharge, intercellular fluid, pericardial fluid, rectal swab, and tears.

### Claim 25 (depends on 1)

The method of claim 1, wherein said initial sample is from an animal.

### Claim 26 (depends on 16)

The method of claim 16, wherein the attaching one or more adapters comprises attaching the splint oligonucleotide using a T4 DNA ligase.

### Claim 27 (depends on 1)

The method of claim 1, further comprising heating the nucleic acids in the initial sample to denature the nucleic acids.

### Claim 28 (depends on 1)

The method of claim 1, further comprising size selecting adapted nucleic acids after attaching one or more adapters to nucleic acids in the spiked initial sample.

### Claim 29 (depends on 28)

The method of claim 28, wherein size selecting comprises size selecting through electrophoresis.

### Claim 30 (depends on 28)

The method of claim 28, wherein size selecting comprises immobilizing the adapted nucleic acids on magnetic beads.

### Claim 31 (depends on 20)

The method of claim 20, wherein the microbial cell-free nucleic acids comprise nucleic acids derived from a bacterium, a virus, a fungus, a protozoan parasite, or any combination thereof.