Cell-free nucleic acids for the analysis of the human microbiome and components thereof

Abstract

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 detecting a change in immunocompetence of a human patient or transplant rejection in a transplant recipient, comprising:
 (a) receiving a first sample from said human patient or said transplant recipient, wherein said first sample is a body fluid sample comprising first cell-free nucleic acids obtained from said patient at a first time point;   (b) performing a sequencing assay on said first sample, thereby obtaining sequence reads for said first sample; wherein said sequencing assay comprises at least one of: performing PCR on said cell-free nucleic acids using universal primers or ligating adapter nucleic acid molecules to said first cell-free nucleic acids;   (c) comparing said sequence reads to a database containing sequence information of at least one member of an anelloviradae family to obtain a first detected sequence information;   (d) using said first detected sequence information to quantify anelloviridae nucleic acids, thereby obtaining a relative quantitative value for said anelloviridae nucleic acids in said first sample;   (e) receiving a second sample from said human patient or said transplant recipient, wherein said second sample is a body fluid sample comprising second cell-free nucleic acids obtained from said human patient at a second time point;   (f) performing a sequencing assay on said second sample, thereby obtaining sequence reads for said second sample; wherein said sequencing assay comprises at least one of: performing PCR using universal primers or ligating adapter nucleic acid molecules to said second cell-free nucleic acids;   (g) comparing said sequence reads from said second sample to a database containing sequence information of at least one member of the anelloviradae, to obtain a second detected sequence information;   (h) using said second detected sequence information to quantify anelloviridae nucleic acids, thereby obtaining a relative quantitative value for said anelloviridae nucleic acids in said second sample;   (i) comparing said relative quantitative value for said anelloviridae nucleic acids in said first sample to said relative quantitative value for said anelloviridae nucleic acids in said second sample; and   (j) using a computer system to automatically provide a report of said comparing in (i) to said human patient's caregiver or other individual.   
     
     
         2 . The method of  claim 1 , further comprising administering an immunosuppressive agent to said human patient based on said report. 
     
     
         3 . The method of  claim 1 , wherein said second time point occurs after said first time point. 
     
     
         4 . The method of  claim 3 , wherein an increase in said immunocompetence is detected when said relative quantitative value for said anelloviridae nucleic acids in said first sample is higher than said relative quantitative value for said anelloviridae nucleic acids in said second sample. 
     
     
         5 . The method of  claim 3 , wherein a reduction of said immunocompetence is detected when said relative quantitative value for said anelloviridae nucleic acids in said first sample is lower than said relative quantitative value for said anelloviridae nucleic acids in said second sample. 
     
     
         6 . The method of  claim 2 , wherein said immunosuppressive agent comprises at least one immunosuppressive agent selected from the group consisting of: steroid, corticosteroid, calcineurin inhibitor, antibody, monoclonal antibody, and polyclonal antibody. 
     
     
         7 . The method of  claim 2 , wherein said immunosuppressive agent comprises at least one immunosuppressive agent selected from the group consisting of: tacrolimus, prednisone, calcineurin inhibitor, azathioprine, cyclosporine, cyclosporine A, mycophenolate mofetil, sirolimus, muromonab-CD3, anti-CD25 antibodies, basiliximab, daclizumab, and anti-thymocyte globulin, rapamycin, cyclosporin A, and anti-CD40L monoclonal antibody. 
     
     
         8 . The method of  claim 1 , wherein said first sample and said second sample are body fluid samples comprising cell-free nucleic acids and wherein said body fluid is selected from the group consisting of: broncheoalveolar lavage (BAL), serum, plasma, urine, blood, sweat, saliva, semen, vaginal flow, brain fluid, sputum, lymph, tears, ear flow, cerebrospinal fluid, ascites, synovial fluid and respiratory tract secretions. 
     
     
         9 . The method of  claim 8 , wherein said body fluid samples are plasma samples. 
     
     
         10 . The method of  claim 1 , wherein said first and second cell-free nucleic acids are amplified by a non-biased method. 
     
     
         11 . The method of  claim 1 , wherein 10 5  sequence reads are generated. 
     
     
         12 . The method of  claim 1 , wherein said sequencing assay comprises a high-throughput sequencing assay performed without sequence-specific amplification of said anelloviridae nucleic acids. 
     
     
         13 . The method of  claim 1 , wherein said first and second cell-free nucleic acids comprise DNA. 
     
     
         14 . The method of  claim 1 , wherein said first and second cell-free nucleic acids comprise RNA. 
     
     
         15 . The method of  claim 1 , further comprising detecting co-incidence of five or more different microbial species in said first and second samples. 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 1 , wherein said human patient is a transplant recipient, wherein said method further comprises detecting transplant rejection in said transplant recipient and wherein said method further comprises treating said transplant rejection by administering an immunosuppressive agent to said transplant recipient. 
     
     
         18 . The method of  claim 1 , wherein said transplant rejection or said immunocompetence is detected when said anelloviridae nucleic acids are below a threshold value. 
     
     
         19 . The method of  claim 1 , wherein (e) further comprises performing a biopsy on said transplant recipient. 
     
     
         20 - 21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein said transplant recipient received at least one organ selected from the group consisting of: lung, intestine, skin, prostate, pancreas, heart, liver, kidney, pancreas-kidney, and thoracic. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 1 , wherein said transplant rejection is chronic rejection, acute rejection, or allograft failure. 
     
     
         26 . The method of  claim 1 , wherein said transplant rejection is associated with a biopsy grade of ≤2R/3A. 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 1 , wherein said anelloviridae nucleic acids nucleic acids selected from the group consisting of: anellovirus, Torque teno virus or Alphatorquevirus (TTV), Torque teno-like mini virus or Betatorquevirus (TTMV), Torque teno-like midi virus or Gammatorquevirus (TTMDV), TTV1, TTV3, TTV4, TTV6, TTV7, TTV8, TTV10, TTV 12, TTV 14, TTV15, TTV16, TTV19, TTV27, TTV28, TTMV1, TTMV6, TTMDV1, or TTMV8. 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 18 , wherein said threshold value refers to a level of said relative quantitative value for said anelloviridae nucleic acids in said first sample, wherein said first sample is obtained at a time point earlier than said second sample. 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . The method of  claim 1 , wherein said transplant rejection is detected with a sensitivity of at least 50%. 
     
     
         41 . The method of  claim 1 , wherein the method further comprises identifying co-incidence of five or more different microbial species.