Granted Patent B2

US 12,320,036 · App. 17/323,843 · Granted Jun 3, 2025

Inventors: Sivan Bercovici (Redwood City, CA); Lily Blair (Redwood City, CA); Timothy A. Blauwkamp (Redwood City, CA); Peter J. Eugster (Redwood City, CA); David K. Hong (Redwood City, CA); Trupti Kawli (Redwood City, CA); Michael J. Rosen (Redwood City, CA); Damek Spacek (Redwood City, CA); Igor D. Vilfan (Redwood City, CA)

Assignee: KARIUS, INC.

C40B50/06C12N15/1093C12Q1/6806

Abstract

Claims (28)

1. A method of preparing a nucleic acid library from an initial sample, comprising generating a nucleic acid library from the initial sample,

wherein the initial sample comprises a plasma sample, a synovial fluid sample, a bronchoalveolar lavage sample, a cerebrospinal fluid sample or a urine sample,

wherein nucleic acids used to generate the nucleic acid library are not extracted from the initial sample before preparing the nucleic acid library and wherein generating the nucleic acid library from the initial sample comprises:

(a) dephosphorylating the nucleic acids in the initial sample to produce a group of dephosphorylated nucleic acids;

(b) denaturing the dephosphorylated nucleic acids to produce denatured nucleic acids;

(c) attaching a 3′-end adapter to the denatured nucleic acids to produce adapted nucleic acids;

(d) annealing a primer to the adapted nucleic acids and extending the primer with a polymerase to generate complementary strands;

(e) attaching a 5′-end adapter; and

(f) amplifying the complementary strands.

2. The method of claim 1 , further comprising isolating the nucleic acids after (f).

3. The method of claim 1 , further comprising adding one or more process control molecules to the initial sample to provide a spiked initial sample and generating the nucleic acid library from the spiked initial sample, wherein nucleic acids used to generate the nucleic acid library are not extracted from the spiked initial sample before preparing the nucleic acid library.

4. The method of claim 1 , wherein the initial sample is a plasma sample.

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

6. The method of claim 5 , wherein said human subject has undergone an organ transplant.

7. The method of claim 3 , wherein the one or more process control molecules comprise one or more of an ID Spike, a Spank, a Spark, and a GC Spike-in Panel.

8. The method of claim 1 , further comprising immobilizing the adapted nucleic acids on functionalized beads or columns.

9. The method of claim 8 , wherein the immobilizing occurs on magnetic beads.

10. The method of claim 1 , wherein attaching a 3′-end adapter to the denatured nucleic acids comprises attaching with a splint oligonucleotide.

11. The method of claim 10 , wherein a 5′-end of the splint oligonucleotide comprises a bulky moiety, optionally wherein the bulky moiety comprises digoxigenin, and optionally further comprising contacting the digoxigenin with an anti-digoxigenin antibody.

12. The method of claim 1 , wherein attaching a 3′-end adapter to the denatured nucleic acids and/or attaching an adapter to the 3′-end of the complementary strands comprises ligating with an enzyme comprising a T4 DNA ligase.

13. The method of claim 1 , further comprising using a Klenow fragment in step (d).

14. The method of claim 2 , wherein the nucleic acids used to generate the library comprise DNA or RNA.

15. The method of claim 1 , wherein the step of attaching a 3′-end adapter to the denatured nucleic acids and/or attaching an adapter to the 3′-end of the complementary strands comprises the steps of incubating with a polymerase that has non-templated activity and, subsequently using a template switching reaction to attach a 3′-end adapter.

16. The method of claim 1 , further comprising incubating with a protease prior to denaturing the nucleic acids.

17. The method of claim 16 , wherein the protease comprises a Proteinase K.

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

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

20. The method of claim 1 , wherein the nucleic acids comprise microbial nucleic acids from one or more microbes selected from the group consisting of a bacterium, a virus, a fungus, or a protozoan parasite.

Assignments (3)

Continuity (3)

References Cited (173)

Cited By (1)

US 12,601,089