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 an therapeutic regimen, e.g. drugs, diet, radiation therapy, and the like.
1. A method of processing a sample comprising microbial DNA, the method comprising:
(a) providing a cell-free sample comprising cell-free DNA from a human subject, wherein the human subject has, is suspected of having, or at risk of developing a microbial infection, and the cell-free DNA comprises microbial cell-free DNA;
(b) performing an initial amplification of the cell-free DNA in a non-biased manner by attaching adapters to the cell-free DNA and amplifying with primers specific for the adapters to produce amplified cell-free DNA attached to the adapters;
(c) performing high-throughput sequencing of the amplified cell-free DNA attached to the adapters to generate sequence reads that comprise sequence reads from at least one microbe;
(d) performing bioinformatics analysis on the sequence reads that comprise sequence reads from at least one microbe to detect the sequence reads from the at least one microbe; and
(e) identifying the at least one microbe at a genus level based on the sequence reads from the at least one microbe, thereby detecting the microbial infection in the human subject.
2. The method of claim 1 , wherein the human subject is an immunocompromised human subject.
3. The method of claim 1 , further comprising selectively extracting cell-free DNA from the cell-free sample to obtain extracted cell-free DNA, wherein the high-throughput sequencing in (c) is performed on the extracted cell-free DNA.
4. The method of claim 1 , wherein the cell-free sample is a body fluid sample.
5. The method of claim 1 , wherein the cell-free sample is selected from the group consisting of plasma, cerebrospinal fluid and synovial fluid.
6. The method of claim 1 , wherein the cell-free sample is a plasma sample.
7. The method of claim 1 , wherein the at least one microbe comprises at least one fungus.
8. The method of claim 1 , wherein the at least one microbe comprises at least one virus.
9. The method of claim 1 , wherein the at least one microbe comprises:
(i) at least one virus selected from the group consisting of: adenovirus, adeno-associated virus, anellovirus, human cytomegalovirus, human respiratory syncytial virus, human rhinovirus, human influenza virus, human coronavirus, human SARS coronavirus, hepatitis virus, human herpesvirus 1, human herpesvirus 2, human herpesvirus 6, BK polyomavirus, JC polyomavirus, cytomegalovirus, parainfluenza virus, human papillomavirus, Epstein-Barr virus, and human T-lymphotropic virus;
(ii) at least one bacterium selected from the group consisting of: Brucella, Treponema, Mycobacterium, Listeria, Helicobacter, Legionella, Streptococcus, Neisseria, Clostridium, Staphylococcus, Pseudomonas, Micrococcus , Multi-drug resistant Staphylococcus Aureus (MRSA), Klebsiella pneumonia , gram positive bacteria, gram negative bacteria, and Bacillus;
(iii) at least one parasite selected from the group consisting of: Trichomonas, Toxoplasma, Giardia, Cryptosporidium, Plasmodium, Leishmania, Trypanosoma, Entamoeba, Schistosoma , Filariae, Ascaria, and Fasciola;
(iv) at least one fungus selected from the group consisting of: Candida, Aspergillus, Cryptococcus, Pneumocystis and yeast; or
(v) any combination thereof.
10. The method of claim 1 , further comprising determining a normalized quantity of the at least one microbe based on the sequence reads from at least one microbe.
11. The method of claim 1 , further comprising detecting antimicrobial resistance in the at least one microbe based on the sequence reads from the at least one microbe.
12. The method of claim 1 , wherein (c) generates at least 100,000,000 sequence reads.
13. The method of claim 1 , wherein the at least one microbe is detected with an AUC of greater than 0.9.
14. The method of claim 1 , further comprising predicting infection in the immunocompromised human subject based at least in part on the identifying the at least one microbe at a genus level based on the sequence reads from the at least one microbe.
15. The method of claim 1 , wherein the high-throughput sequencing comprises sequencing-by-synthesis.
16. The method of claim 1 , further comprising administering an antimicrobial treatment to the human subject to treat the microbial infection after the detecting the microbial infection in the human subject.
17. The method of claim 16 , wherein the antimicrobial treatment comprises an antibiotic.
18. The method of claim 16 , wherein the antimicrobial treatment comprises an antiviral.
19. The method of claim 1 , wherein the at least one microbe comprises at least one bacterium.
20. The method of claim 1 , wherein the at least one microbe comprises at least one parasite.
21. A method of processing a sample comprising microbial RNA, the method comprising:
(a) providing a cell-free sample from a human subject, wherein the human subject has, is suspected of having, or is at risk of developing a microbial infection and the cell-free sample comprises cell-free RNA comprising microbial cell-free RNA;
(b) performing reverse transcription on the cell-free RNA to transform the RNA into cDNA;
(c) performing an initial amplification of the cDNA in a non-biased manner by attaching adapters to the cDNA and amplifying with primers specific for the adapters to produce amplified cDNA attached to the adapters;
(d) performing high-throughput sequencing of the amplified cDNA attached to the adapters to generate sequence reads that comprise sequence reads from at least one microbe;
(e) performing bioinformatics analysis on the sequence reads that comprise sequence reads from at least one microbe to detect the sequence reads from the at least one microbe; and
(f) identifying the at least one microbe at a genus level based on the sequence reads from the at least one microbe, thereby detecting the microbial infection in the human subject.
22. The method of claim 21 , further comprising selectively extracting the cell-free RNA from the cell-free sample to obtain extracted cell-free RNA.
23. The method of claim 21 , wherein the human subject is immunocompromised.
24. The method of claim 21 , wherein the cell-free sample is a body fluid sample.
25. The method of claim 21 , wherein the cell-free sample is selected from the group consisting of plasma, cerebrospinal fluid and synovial fluid.
26. The method of claim 21 , wherein the cell-free sample is a plasma sample.
27. The method of claim 21 , wherein the at least one microbe comprises a microbe selected from the group consisting of at least one bacterium, at least one virus, at least one parasite, and a combination thereof.
28. The method of claim 21 , further comprising administering an antimicrobial treatment to the human subject to treat the microbial infection after the detecting the microbial infection in the human subject.
29. The method of claim 28 , wherein the antimicrobial treatment comprises an antibiotic.
30. The method of claim 28 , wherein the antimicrobial treatment comprises an antiviral.