Cell-free nucleic acids for the analysis of the human microbiome and components thereof
Methods, devices, compositions and kits are provided for analysis of the microbiome or individual components thereof in an individual. The methods find use in a determination of infection, in analysis of the microbiome structure, in determining the immunocompetence of an individual, and the like. In some embodiments of the invention, the individual is treated with a therapeutic regimen, e.g. drugs, diet, radiation therapy, and the like.
1. A method of detecting at least one bacterium, fungus, or parasite from cell-free microbial nucleic acids derived from a human host, the method comprising:
(i) providing a sample comprising cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite, wherein said cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite are obtained from plasma, serum or cerebrospinal fluid of said human host using a method that recovers sufficient cell-free microbial nucleic acids to enable identification of said at least one bacterium, fungus, or parasite at a strain or species level;
(ii) performing high-throughput sequencing of said cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite to provide cell-free microbial sequence reads to enable identification of said at least one bacterium, fungus, or parasite at a strain or species level; and
(iii) using said cell-free microbial sequence reads from said at least one bacterium, fungus, or parasite in said sample to identify said at least one bacterium, fungus, or parasite at a strain or species level.
2. The method of claim 1 , wherein a presence and prevalence of a plurality of microbes is determined.
3. The method of claim 1 , wherein said high throughput sequencing is performed on a nucleic acid sample that has been amplified by a non-biased method.
4. The method of claim 1 , wherein at least 10 6 sequence reads are performed.
5. The method of claim 1 , wherein (ii) comprises comparing coverage of sequences mapping to a microbial reference sequence to coverage of a host reference sequence.
6. The method of claim 1 , wherein (ii) comprises 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 , wherein (ii) comprises 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 (i) is performed at two or more time points.
10. The method of claim 1 , wherein an amount of said cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite is indicative of infection status of said human host.
11. The method of claim 1 , wherein an amount of said cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite above a predetermined threshold value is indicative of an infection status of said human host.
12. The method of claim 1 , wherein said cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite are derived from said plasma.
13. The method of claim 1 , wherein said cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite are selected from the group consisting of DNA/RNA hybrids, single-stranded RNA, double-stranded RNA and RNA hairpins.
14. The method of claim 1 , wherein said cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite are selected from the group consisting of double-stranded DNA, single-stranded DNA hairpins, single-stranded DNA and cDNA.
15. 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.
16. The method of claim 15 , wherein said assessment of said microbiome components provides a determination of response to therapy of said human host.
17. The method of claim 15 , wherein said assessment of said microbiome components provides a measurement of human physiology.
18. The method of claim 15 , wherein said assessment of said microbiome components is used to calculate a pathogenicity score for microorganisms present in said human host.
19. The method of claim 1 , comprising identifying said at least one bacterium at a strain or species level.
20. The method of claim 1 , comprising identifying said at least one fungus at a strain or species level.
21. The method of claim 1 , comprising identifying said at least one parasite at a strain or species level.
22. The method of claim 1 , further comprising attaching adapters to said cell-free microbial nucleic acids prior to (ii) to produce adapted cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite.
23. The method of claim 22 , wherein (ii) comprises sequencing said adapted cell-free microbial nucleic acids from said at least one bacterium, fungus, or parasite.
24. The method of claim 1 , further comprising quantifying an amount of said at least one bacterium at a strain or species level.
25. The method of claim 1 , further comprising quantifying an amount of said at least one fungus at a strain or species level.
26. The method of claim 1 , further comprising quantifying an amount of said at least one parasite at a strain or species level.
27. The method of claim 1 , wherein a sample is collected from said human host at two or more time points and analyzed.
28. The method of claim 1 , further comprising administering a treatment to said human host.
29. The method of claim 28 , wherein said treatment comprises an anti-microbial treatment.
30. The method of claim 28 , wherein said treatment comprises an antibiotic agent.