Granted Patent B2

US 12,595,509 · App. 18/313,716 · Granted Apr 7, 2026

Inventors: Fred C. Christians (Los Altos Hills, CA); Igor D. Vilfan (East Palo Alto, CA); Michael Kertesz (Menlo Park, CA); Timothy A. Blauwkamp (Palo Alto, CA); Shivkumar Venkatasubrahmanyam (San Jose, CA); Michael Rosen (Palo Alto, CA); Rene Sit (Sunnyvale, CA)

Assignee: Karius, Inc.

C12Q1/6874C12N15/1065C12Q1/6869C12Q1/689G16B30/00G16B30/10C12Q2600/166

Abstract

Claims (46)

1 . A method for identifying carry-over between samples using a sample identifier spike-in, the method comprising:

(a) providing a first sample comprising a first microbial nucleic acid from a first subject;

(b) providing a second sample comprising a second microbial nucleic acid from a second subject;

(c) providing a second-sample identifier spike-in, wherein

the second-sample identifier spike-in comprises a synthetic nucleic acid and

does not comprise sequences capable of hybridizing to the first microbial nucleic acid or the second microbial nucleic acid;

(d) adding the second-sample identifier spike-in to the second sample, wherein a number of molecules of the synthetic nucleic acid of the second-sample identifier spike-in is equal to or greater than a number of molecules of the second microbial nucleic acid in the second sample;

(e) assaying the first sample to detect a presence, an absence, or an amount of the second-sample identifier spike-in and the first microbial nucleic acid; and

(f) determining that the first microbial nucleic acid was obtained from the first subject based at least upon, in (e), detection of:

i. the first microbial nucleic acid; and

ii. the absence of the second-sample identifier spike-in or

the amount of the second-sample identifier spike-in below a threshold level.

2 . The method of claim 1 , wherein the second-sample identifier spike-in is less than 150 nucleotides or base pairs in length.

3 . The method of claim 1 , wherein the second-sample identifier spike-in comprises a modified nucleic acid.

4 . The method of claim 1 , wherein the second-sample identifier spike-in comprises DNA.

5 . The method of claim 1 , wherein the first sample is a cell-free bodily fluid sample.

6 . The method of claim 1 , further comprising adding a first reagent synthetic nucleic acid spike-in to a first reagent lot, wherein the first reagent lot is used in sample processing of the first sample.

7 . The method of claim 6 , further comprising detecting the first reagent synthetic nucleic acid spike-in in the assaying of the first sample.

8 . A method for determining whether a positive control nucleic acid has carried-over to a sample, the method comprising:

(a) obtaining the sample comprising a target nucleic acid;

(b) obtaining a control sample comprising the positive control nucleic acid;

(c) obtaining a control sample identifier spike-in, wherein:

(i) the control sample identifier spike-in comprises a control synthetic nucleic acid; and

(ii) the control sample identifier spike-in does not comprise sequences capable of hybridizing to the positive control nucleic acid or the target nucleic acid;

(d) adding the control sample identifier spike-in to the control sample, wherein a number of molecules of the synthetic nucleic acid of the control sample identifier spike-in is equal to or greater than a number of molecules of the positive control nucleic acid in the control sample;

(e) assaying the sample to detect a presence, an absence, or an amount of the control sample identifier spike-in; and

(f) determining whether the control sample identifier spike-in is present in the sample based on the presence, the absence, or the amount below a threshold of the control sample identifier spike-in.

9 . The method of claim 1 , wherein a carry-over result is reported to a patient, caregiver, other person, or any combination thereof.

10 . The method of claim 1 , wherein the first sample comprises cell-free nucleic acids.

11 . The method of claim 1 , wherein the first sample, the second sample, or both are plasma samples.

12 . The method of claim 1 , wherein the second-sample identifier spike-in is a single-stranded DNA, a double-stranded DNA with blunt ends, or a double-stranded DNA with recessed ends.

13 . The method of claim 1 , wherein the first microbial nucleic acid is derived from viruses, bacteria, fungi, parasites, or any combination thereof.

14 . The method of claim 1 , wherein the first microbial nucleic acid are microbial cell-free nucleic acid.

15 . The method of claim 8 , wherein the positive control nucleic acid comprises sheared genomic DNA.

16 . The method of claim 8 , wherein the target nucleic acid are cell-free nucleic acid.

17 . The method of claim 8 , wherein the assaying comprises performing a sequencing assay, and wherein the sequencing assay comprises high-throughput sequencing, massively-parallel sequencing, Next Generation sequencing (NGS), or any combination thereof.

18 . The method of claim 8 , wherein the control sample identifier spike-in is less than 150 nucleotides in length.

19 . The method of claim 8 , wherein the positive control nucleic acid comprises a positive control microbial nucleic acid.

20 . The method of claim 19 , wherein the positive control microbial nucleic acid is derived from viruses, bacteria, fungi, parasites, or any combination thereof.

21 . The method of claim 1 , wherein a ratio of the synthetic nucleic acid of the second-sample identifier spike-in to the second microbial nucleic acid is from 1:1 to 1000:1.

22 . The method of claim 1 , wherein the assaying comprises performing a sequencing assay, and wherein the sequencing assay generates sequence results.

23 . The method of claim 1 , further comprising, prior to (e), adding a first-sample identifier spike-in to the first sample.

24 . The method of claim 23 , wherein (e) further comprises detecting a presence, an absence, or an amount of the first-sample identifier spike-in.

25 . The method of claim 22 , wherein the sequencing assay further comprises adding adapters to the first sample and preparing a sequencing library of the first sample.

26 . The method of claim 22 , wherein the sequence results comprise sequence reads from the first microbial nucleic acid.

27 . The method of claim 22 , wherein the sequencing assay comprises high-throughput sequencing, massively-parallel sequencing, Next Generation sequencing (NGS), or any combination thereof.

Assignments (2)

Continuity (7)

References Cited (182)