Granted Patent B2

US 12,195,814 · App. 16/554,286 · Granted Jan 14, 2025

Inventors: Iwijn de Vlaminck (Stanford, CA); Michael Kertesz (Menlo Park, CA); Kiran Kaur Khush (Stanford, CA); Mark Alec Kowarsky (Victoria, AU); Lance Martin (Half Moon Bay, CA); Stephen R. Quake (Stanford, CA); Hannah Valantine (Stanford, CA)

Assignee: The Board of Trustees of the Leland Stanford Junior University

C12Q1/705C12Q1/6883C12Q1/689C12Q1/6895C12Q1/701G16B20/00G16B20/20G16B30/00G16B30/10

Abstract

Claims (42)

1. A method of processing a sample comprising cell-free nucleic acids obtained from a human host, the method comprising:

(i) providing a synovial fluid sample obtained from said human host, wherein said synovial fluid sample obtained from said human host comprises cell-free nucleic acids that are fragments of nucleic acids from at least one bacterium;

(ii) attaching adapters to said cell-free nucleic acids and producing a sequencing library comprising said cell-free nucleic acids that are fragments of nucleic acids from said at least one bacterium;

(iii) performing high-throughput sequencing of said cell-free nucleic acids to provide sequence reads comprising sequences from said cell-free nucleic acids that are fragments of nucleic acids from said at least one bacterium; and

(iv) using said sequence reads comprising sequences from said cell-free nucleic acids that are fragments of nucleic acids from said at least one bacterium to identify said at least one bacterium.

2. The method of claim 1 , further comprising determining a presence of a plurality of different microbes, wherein said plurality of different microbes comprises the at least one bacterium and at least one fungus.

3. The method of claim 1 , wherein said sequencing library is produced by amplifying said cell-free nucleic acids using a non-biased method.

4. The method of claim 1 , wherein at least 106 sequence reads are generated.

5. The method of claim 1 , further comprising comparing coverage of sequence reads mapping to a microbial reference sequence to coverage of a host reference sequence.

6. The method of claim 1 , further comprising identifying a reference host sequence and masking microbial sequences or microbial mimicking sequences present in a reference host genome.

7. The method of claim 1 , further comprising identifying a reference microbial sequence, and masking host sequences or host mimicking sequences present in a reference microbial genome.

8. The method of claim 1 , wherein a presence of one or more pathogenic microbes is identified.

9. The method of claim 1 , wherein an amount of said cell-free nucleic acids from said at least one bacterium is indicative of infection status of said human host.

10. The method of claim 1 , wherein said cell-free nucleic acids from said at least one bacterium comprise RNA.

11. The method of claim 1 , wherein said cell-free nucleic acids from said at least one bacterium comprise DNA.

12. The method of claim 1 , further comprising providing an assessment of microbiome components to said human host or to a medical provider of said human host.

13. The method of claim 12 , wherein said assessment of said microbiome components provides a determination of a response to a therapy of said human host.

14. The method of claim 12 , wherein said assessment of said microbiome components is used to calculate a pathogenicity score for microorganisms present in said human host.

15. The method of claim 1 , further comprising quantifying an amount of said at least one bacterium at a strain or species level.

16. The method of claim 1 , wherein a sample is collected from said human host at two or more time points and analyzed.

17. The method of claim 1 , further comprising administering an antimicrobial treatment to said human host to treat an infection caused by said at least one bacterium.

18. The method of claim 17 , wherein said antimicrobial treatment comprises an antibiotic agent.

19. The method of claim 1 , further comprising extracting cell-free nucleic acids from said synovial fluid sample obtained from said human host using a method that extracts cell-free nucleic acids.

20. A method of processing a sample comprising cell-free nucleic acids obtained from a human host, the method comprising:

(i) providing a synovial fluid obtained from said human host, wherein said synovial fluid sample obtained from said human host comprises cell-free nucleic acids that are fragments of nucleic acids from at least one fungus;

(ii) attaching adapters to said cell-free nucleic acids and producing a sequencing library comprising said cell-free nucleic acids that are fragments of nucleic acids from at least one fungus;

(iii) performing high-throughput sequencing of said cell-free nucleic acids to provide cell-free sequence reads that comprise sequence reads comprising sequences from said cell-free nucleic acids that are fragments of nucleic acids from at least one fungus; and

(iv) using said cell-free sequence reads that comprise sequence reads comprising sequences from said cell-free nucleic acids that are fragments of nucleic acids from said at least one fungus to identify said at least one fungus.

21. The method of claim 20 , further comprising quantifying an amount of said at least one fungus at a strain or species level.

22. A method of processing a sample comprising cell-free nucleic acids obtained from a human host, the method comprising:

(i) providing a synovial fluid obtained from said human host, wherein said synovial fluid sample obtained from said human host comprises cell-free nucleic acids that are fragments of nucleic acids from at least one parasite;

(ii) attaching adapters to said cell-free nucleic acids and producing a sequencing library comprising said cell-free nucleic acids that are fragments of nucleic acids from said at least one parasite;

(iii) performing high-throughput sequencing of said cell-free nucleic acids to provide sequence reads comprising sequences from said cell-free nucleic acids that are fragments of nucleic acids from said at least one parasite; and

(iv) using said sequence reads comprising sequences from said cell-free nucleic acids that are fragments of nucleic acids from said at least one parasite to identify said at least one parasite.

23. The method of claim 22 , further comprising quantifying an amount of said at least one parasite at a strain or species level.

24. A method of processing a sample comprising cell-free nucleic acids obtained from a human host, the method comprising:

(i) providing a synovial fluid obtained from said human host, wherein said synovial fluid sample obtained from said human host comprises cell-free nucleic acids that are fragments of nucleic acids from at least one virus;

(ii) attaching adapters to said cell-free nucleic acids and producing a sequencing library comprising said cell-free nucleic acids that are fragments of nucleic acids from said at least one virus;

(iii) performing high-throughput sequencing of said cell-free nucleic acids to provide sequence reads comprising sequences from said cell-free nucleic acids that are fragments of nucleic acids from said at least one virus; and

(iv) using said sequence reads comprising sequences from said cell-free nucleic acids that are fragments of nucleic acids from said at least one virus to identify said at least one virus.

25. The method of claim 24 , further comprising quantifying an amount of said at least one virus at a strain or species level.

26. The method of claim 24 , further comprising administering an antiviral agent to said human host to treat an infection caused by said at least one virus.

Assignments (6)

Continuity (4)

References Cited (130)