WO2020198664A1

WO2020198664A1 - Methods and systems for sequencing cell-free microbiome dna

Info

Publication number: WO2020198664A1
Application number: PCT/US2020/025425
Authority: WO
Inventors: Xianghong Jasmine ZHOU; Xiaohui Ni; Yonggang Zhou; Mary Louisa SAME; Weihua Zhang
Original assignee: The Regents Of The University Of California; Earlydiagnostics, Inc.
Priority date: 2019-03-28
Filing date: 2020-03-27
Publication date: 2020-10-01

Abstract

The present disclosure provides methods, systems, kits, and compositions for enriching cell-free DNA of a microbiome from a collection of cell-free DNA that includes host cell-free DNA. Such methods and systems may provide for more efficient and economical approaches for analyzing microbiome cell-free DNA by excluding at least some portion of host cell-free DNA from a common collection of cell-free DNA. The microbiome cell-free DNA may be enriched by size selection, by a reduction in the amount of linear DNA, and/or by targeting protein(s) to which host cell-free DNA may be associated.

Description

DESCRIPTION

METHODS AND SYSTEMS FOR SEQUENCING CELL-FREE MICROBIOME DNA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62/825,658, filed March 28, 2019, and entitled“METHODS FOR THE EFFECTIVE

SEQUENCING OF CELL-FREE MICROBIOME DNA,” which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

[0002] Embodiments of the disclosure include at least the fields of nucleic acid preparation and analysis, sequencing, molecular biology, cell biology, and medicine.

BACKGROUND

[0003] The microbiome may refer to the collection of microorganisms such as bacteria, fungi, and viruses within a community. Characterization of microbiomes can provide

information about infectious diseases and other health status. The human immune system may be constantly battling against microbiomes and generating microbiome debris. Deoxyribonucleic acid (DNA) from microbiome debris or dead microbiome can be released into the bloodstream, where they may become part of the circulating cell-free DNA (cfDNA) in plasma or other body fluids. Next- generation sequencing-based approaches have the potential of simultaneous diagnosis or characterization of nearly all of the microbiome by analyzing cell-free microbiome DNA (cfmDNA) in the circulation system, however, this type of analysis may be confounded by the abundancy of human DNA flowing in the circulation system. Sequencing data generated from cell-free DNA sequencing may be predominantly originated from unwanted human-derived cfDNA. Therefore, it may be challenging and costly to characterize efficiently the microbiome using sequencing-based approaches.

[0004] The present disclosure provides effective and efficient methods and systems for sequencing cell-free microbiome DNA from a host individual or a subject, such as a human. Utilizing different features between cell-free host and microbiome DNA, the disclosed methods and systems can selectively reduce, deplete, and/or digest contaminating human DNA from admixtures of cell-free human and microbiome DNA. Therefore, sequencing-based microbiome analysis methods and systems may be more efficient and may be more cost-effective.

[0005] The present disclosure provides effective solutions to challenges facing methods and systems for efficient analysis of microbiome cell-free DNA.

SUMMARY

[0006] The present disclosure provides methods, systems, kits, and compositions for enriching microbiome cell-free DNA in a collection ( e.g ., biological sample) comprising nucleic acid. In some embodiments, methods, systems, kits, and compositions comprise the enrichment of microbiome cell-free DNA (cfDNA) in a collection of nucleic acid, including cell-free DNA that comprises cell-free DNA from a host, such as embodiments where the host cell-free DNA is selectively reduced, depleted, or removed, or in embodiments where the non-host cfDNA is selectively increased, isolated, or enriched. Such need may arise from a desire to reduce cost, time, complexity, and/or labor, such that manipulation and/or analysis of microbiome cell-free DNA is more efficient (e.g., performed at higher throughput).

[0007] In some embodiments, a collection of nucleic acid that comprises cell-free DNA is manipulated to reduce, deplete, or remove the amount of a particular type of cell-free DNA, such as cell-free DNA from a host. In some embodiments, a collection of nucleic acid that comprises cell-free DNA is manipulated to reduce, deplete, or remove the amount of cell-free DNA from a host for the purpose of enriching microbiome cell-free DNA in the collection, including increasing, isolating, or enriching the amount of microbiome cell-free DNA in proportion to host cell-free DNA in the collection. Following the practice of methods, systems, kits, and compositions of the present disclosure, the amount of host cell-free DNA that is reduced in the collection may quantitatively be reduced partially or entirely, including to undetectable levels. When reduced partially, the amount of reduction may be of any kind that is detectable by any suitable approach.

[0008] In some embodiments of the methods, systems, kits, and compositions for enriching microbiome cell-free DNA in a common collection with host cell-free DNA leverages one or more structural differences between microbiome cell-free DNA and host cell-free DNA, and any approach to selectively increase, isolate, or enrich in this manner may be utilized alone or in combination with others. In certain aspects, given that much of microbiome cell-free DNA may be circular and not linear as with most if not all host cell-free DNA, and particularly with respect to circular bacterial cell-free DNA, the overall structure differences may be a target for removal of linear host cell-free DNA. The linear nature of at least the majority of host cell-free DNA in comparison to microbiome cell-free DNA may be taken advantage of by focusing on the ends of the linear DNA either by labeling for a targetable label and/or by providing an end for nuclease digestion to reduce, remove, or destroy the linear cell-free DNA from the ends. In addition, or alternatively, the size of host cell-free DNA may be exploited by removing certain ranges of sizes of cell-free DNA, thereby excluding much, if not all, of the microbiome cell-free DNA. In addition, or alternatively, the in vivo configuration of host cell-free DNA may be an approach for removing host cell-free DNA by targeting one or more components to which the host cell-free DNA is bound (directly or indirectly) and that which the microbiome cell-free DNA would not be bound (directly or indirectly). For example, one or more proteins bound by host cell-free DNA that is not bound by microbiome cell-free DNA may be targeted, such as one or more chromatin proteins, including at least histones.

[0009] The present disclosure provides methods, systems, kits, and compositions for selectively reducing, depleting, or removing human DNA from microbiome DNA for any reason, including, for subsequent modification, analysis, quantification, amplification, a combination thereof, and so on. The reduction or removal of human cell-free DNA may be used to facilitate sequencing-based microbiome analysis. Such methods may exploit one or more differences between the microbiome cell-free DNA and human cell-free DNA, including structural differences, including one or more of the following: 1) certain proportion of human cfDNA being associated with one or more proteins as part of chromatin, such as DNA being wrapped around histones; 2) the fragment sizes of human cfDNA, but not microbiome cfDNA, being centered around a particular size range; 3) human cfDNA comprising single-strand linear DNA or double-strand linear DNA, while at least some populations of microbiome cell-free DNA, especially bacterial cfDNA, mainly comprise circular DNA.

[0010] In one aspect, the present disclosure provides a method for preparing cell-free microbiome DNA, comprising the step of subjecting a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA to one or more of the following steps:

subjecting the cell-free DNA in the collection to size selection; subjecting the cell-free DNA in the collection to one or more agents that bind one or more chromatin proteins; and reducing the amount of linear cell-free DNA in the collection, wherein the one or more steps enriches microbiome cell-free DNA in the collection to produce enriched microbiome cell-free DNA. In at least some cases, the method may further comprise the step of assaying for the presence of microbiome cell-free DNA in the collection.

[0011] In an aspect, the present disclosure provides a method for preparing cell-free microbiome deoxyribonucleic acid (DNA), comprising: obtaining a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA; subjecting the collection of cell- free DNA to size selection; subjecting the collection of cell-free DNA to one or more agents that bind one or more chromatin proteins; and reducing the amount of linear cell-free DNA in the collection of cell-free DNA, to enrich microbiome cell-free DNA in the collection, thereby producing an enriched microbiome cell-free DNA. In some embodiments, the method further comprises assaying the collection of cell-free DNA to determine the presence or absence of microbiome cell-free DNA

[0012] In some embodiments, subjecting the cell-free DNA to size selection may comprise reducing the amount of cell-free DNA in the collection that having a size in a certain range, such as the range between 130 base pairs (bp) and 185bp, 130bp and 170bp, 140bp and 170bp, 150bp and 170bp, 160bp and 170bp, 130bp and 160bp, 140bp and 160bp, 150bp and 160bp, 140bp and 150bp, or 150bp and 160bp. In any size selection, subjecting the cell-free DNA to size selection may comprise subjecting the collection of cell-free DNA to separation, enrichment, depletion, or removal by beads or by electrophoresis.

[0013] In some embodiments, the one or more agents that bind histones comprise one or more antibodies, one or more aptamers, one or more fusion proteins, or a mixture thereof. In some embodiments wherein the cell-free DNA is subjected to one or more agents that bind one or more chromatin proteins, the one or more agents that bind histones may comprise one or more antibodies, one or more aptamers, one or more fusion proteins, or mixture thereof.

[0014] In some embodiments, reducing the amount of linear cell-free DNA comprises subjecting the collection of cell-free DNA to one or more modifications (e.g., labels that attach to linear ends of cell-free DNA) to produce modified (e.g., labeled) cell-free linear DNA; and removing the modified (e.g., labeled) cell-free linear DNA from a remainder of the collection of cell-free DNA with an agent that binds the modified cell-free linear DNA (e.g., at the label). In some embodiments, the ends of the linear cell-free DNA are tagged using biotin-dNTP labeling, and the labeled cell-free linear DNA are removed from the collection of cell-free DNA using an agent that binds to biotin. In some embodiments, the one or more modifications comprise subjecting a 3' end of the linear cell-free DNA to conditions sufficient to modify the 3' end with a biotin- deoxynucleotide (dNTP), a dideoxynucleotide (ddNTP) moiety, or a functional analog thereof, or subjecting a 5' end of the linear cell-free DNA to conditions sufficient to

dephosphorylate the 5' end.

[0015] In some embodiments, reducing the amount of linear cell-free DNA comprises subjecting cell-free DNA in the collection to one or more enzymes that digest linear single stranded DNA and/or linear double- stranded DNA, such as exonuclease I, exonuclease VII, DNase, exonuclease V(RecBCD), or a combination thereof.

[0016] The microbiome cell-free DNA may be of any kind, but in some embodiments it comprises DNA from one or more bacteria, one or more fungi, one or more viruses, one or more protozoa, or a combination thereof. Hosts of the microbiome(s) may be a mammal, such as a human, horse, dog, cat, cow, and so forth..

[0017] In some embodiments, the collection of cell-free DNA to be processed and/or analyzed is obtained or derived from a biological sample, such as from a subject or an individual in need of treatment for a medical condition related to the microbiome or suspected of being in need of treatment for a medical condition related to the microbiome, from an individual in need of evaluation of a response to one or more different diet conditions, or from an

immunosuppressed transplant recipient. The biological sample may be obtained or derived from the skin, mouth, nose, gastrointestinal tract, and/or genitourinary tract of the host, and the biological sample may comprise tissue, blood, plasma, urine, fecal matter, saliva, mucus, nipple aspirate, amniotic fluid, cystic fluid, spinal or brain fluid, sweat, tears, or a combination thereof. In some embodiments, methods, systems, kits, and compositions of the disclosure comprise obtaining the biological sample from the host or a repository, for example.

[0018] In some embodiments, the microbiome cell-free DNA obtained from methods of preparation of the disclosure is further processed and/or analyzed. In some embodiments, the enriched microbiome cell-free DNA is further subjected to amplification, modification, analysis, or a combination thereof, which may or may not include sequencing library preparation, for example. In some embodiments, the linear molecules of microbiome cell-free DNA are subjected to polymerase chain reaction (PCR) or nucleic array analysis. [0019] In post-preparation operations, an enriched microbiome cell-free DNA may be digested to produce linear molecules of microbiome cell-free DNA, and the linear molecules of microbiome cell-free DNA may or may not be modified. As an example, the linear molecules of microbiome cell-free DNA are adapter-ligated, and in some embodiments the adapter-ligated linear molecules of microbiome cell-free DNA are subjected to sequencing, such as next- generation sequencing. In some embodiments, sequencing the adapter-ligated linear molecules of microbiome cell-free DNA provides information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; or (5) a combination thereof. In some embodiments, the enriched microbiome cell-free DNA is analyzed for methylation profiling. In some embodiments, at least one therapy or prophylactic for the host is provided upon analysis of the enriched microbiome cell-free DNA. The method may further comprise performing genomic or epigenomic profiling of the enriched microbiome cell-free DNA.

[0020] In another aspect, the present disclosure provides a method of preparing cell-free deoxyribonucleic acid (DNA), comprising: obtaining a collection of cell-free DNA comprising host cell-free DNA; subjecting the collection of cell-free DNA to size selection; subjecting the collection of cell-free DNA to one or more agents that bind histones; and reducing an amount of linear cell-free DNA in the collection of cell-free DNA, to enrich microbiome cell-free DNA in the collection, thereby producing enriched microbiome cell-free DNA. In some embodiments, the method further comprises assaying the collection of cell-free DNA to determine whether or not microbiome cell-free DNA is present in the collection. In some embodiments, the assaying is performed before and/or after the subjecting. The collection of cell-free DNA may be determined to have microbiome cell-free DNA, or the collection may be determined not to have microbiome cell-free DNA.

[0021] In another aspect , the present disclosure provides a method of preparing cell-free DNA, comprising the step of subjecting a collection of cell-free DNA comprising host cell-free DNA to one or more of the following steps: subjecting the cell-free DNA in the collection to size selection; subjecting the cell-free DNA in the collection to one or more agents that bind histones; and reducing an amount of linear cell-free DNA in the collection to enrich microbiome cell-free DNA in the collection when present in the collection to produce enriched microbiome cell-free DNA. In specific embodiments, the method further comprises the step of assaying whether or not microbiome cell-free DNA is present in the collection. In some embodiments, the assaying is performed before and/or after the subjecting step or steps. The collection of cell-free DNA may be determined to have microbiome cell-free DNA, or the collection may be determined not to have microbiome cell-free DNA.

[0022] In another aspect, is the present disclosure provides a method of preparing cell- free microbiome DNA, comprising an optional step of obtaining a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA, subjecting a collection of cell- free DNA comprising microbiome cell-free DNA and host cell-free DNA to size selection, to enrich microbiome cell-free DNA in the collection of cell-free DNA, thereby producing enriched microbiome cell-free DNA. In some embodiments, subjecting the collection to size selection comprises reducing the amount of cell-free DNA in the collection that are sized in a range between about 100 base pairs (bp) and about 200bp, about l lObp and 190bp, about 120bp and 180bp, about 130bp and about 170bp, about 140bp and about 170bp, about 150bp and about 170bp, about 160bp and about 170bp, about 130bp and 160bp, about 140bp and 160bp, about 150bp and 160bp about 140bp and 150bp, or about 150bp and 160bp. In some embodiments, and subjecting the cell-free DNA to size selection comprises subjecting the collection of cell-free DNA to separation by beads or by electrophoresis.

[0023] In another aspect, the present disclosure provides a method of preparing cell-free microbiome DNA, comprising the step of optionally obtaining a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA; subjecting the collection of cell- free DNA comprising microbiome cell-free DNA and host cell-free DNA to one or more agents that bind one or more chromatin proteins, thereby producing enriched microbiome cell-free DNA. In some embodiments, the chromatin protein is one or more histone. In some

embodiments, the one or more agents that bind chromatin protein comprise one or more antibodies, one or more aptamers, one or more fusion proteins, or mixture thereof. In these embodiments, binding refers to specific binding or specifically binding of one or more compounds or agents.

[0024] In another aspect, is the present disclosure provides a method of preparing cell- free microbiome DNA, comprising optionally obtaining a collection of cell-free DNA

comprising microbiome cell-free DNA and host cell-free DNA;, and reducing the amount of linear cell-free DNA in the collection of cell-free DNA, to enrich microbiome cell-free DNA in the collection, thereby producing enriched microbiome cell-free DNA. In some embodiments, reducing the amount of linear cell-free DNA comprises: subjecting cell-free DNA in the collection of cell-free DNA to one or more modifications ( e.g ., labels that attach to linear ends of cell-free DNA) to produce modified (e.g., labeled) cell-free DNA; and removing the modified e.g., labeled) cell-free DNA from the collection with an agent that binds the modified (e.g., labeled) cell-free DNA. In some embodiments, reducing the amount of linear cell-free DNA comprises subjecting cell-free DNA in the collection to one or more enzymes that digest linear single stranded DNA and/or that digest linear double stranded DNA.

[0025] In another aspect, is the present disclosure provides a method for processing and/or analyzing a plurality of cell-free deoxyribonucleic (cfDNA) molecules from a subject, comprising: (a) subjecting a plurality of cfDNA molecules comprising both subject-derived cfDNA and microbiome cell-free DNA (cfmDNA) to one or more enrichment methods to permit subject-derived cfDNA molecules to be (i) separated from a remainder of said cfmDNA molecules or (ii) digested (for example, with one or more nucleases) in the plurality of cfDNA molecules; (b) coupling adapters to ends of said plurality of DNA molecules to provide a plurality of tagged DNA molecules; (c) subjecting the plurality of tagged DNA molecules or tagged derivatives thereof to nucleic acid sequencing to yield a plurality of sequence reads; and (d) processing the plurality of sequence reads to identify sequences from the microbiome. In some embodiments, the enrichment comprises removing cfDNA molecules of specific sizes to enrich the cfmDNA in the plurality of cfDNA molecules (for example, lengths from 145 to 185 nucleic acid bases or lengths greater than 100 nucleic acid bases). In some embodiments, , the removal of molecules of specific sizes is performed with beads (e.g., Ampure® or Solid Phase Reversible Immobilization (SPRI) beads) and/or may be performed with gel electrophoresis.

[0026] In some embodiments, the enrichment comprises: (a) using protein binding reagents (for example, anti-histone antibodies, peptides, aptamers, fusion proteins, or a combination thereof) to bind to histone proteins to allow a plurality of histone-associated cell- free DNA molecules to be separated from the remainder of the plurality of cfDNA molecules; and (b) removing the histone binding reagents to deplete histone-associated cell-free DNA molecules from the plurality of cfDNA molecules.

[0027] In some embodiments, the enrichment comprises: (a) modifying one or both ends of each of at least a portion of the plurality of cf DNA molecules to provide a plurality of modified cfDNA molecules having ends that are (i) configured for separation from a remainder of the plurality of cfDNA or (ii) incapable of coupling with adapters; and (b) removing the modified DNA molecules from a remainder of the plurality of cfDNA.

[0028] In some embodiments, the protein binding reagents may be biotinylated; for example, they may be conjugated to magnetic beads. In some embodiments, streptavidin beads or protein A or protein G or secondary antibody conjugated magnetic beads are used for removing the histone binding reagents. In some embodiments, histone protein binding and removal is performed before the extraction of the plurality of cfDNA molecules.

[0029] In some embodiments, wherethe enrichment comprises modifying one or both ends of each of at least a portion of plurality of cell-free DNA molecules, the modifying comprises subjecting a 3' end of each of at least a portion of plurality of cfDNA molecules to conditions sufficient to modify 3' ends with a deoxynucleotide (dNTP) moiety, a

dideoxynucleotide (ddNTP) moiety, or a functional analog thereof. In some embodiments, modifying comprises subjecting a 5' end of each of at least the portion of the plurality of cfDNA molecules to conditions sufficient to dephosphorylate the 5' end. In some embodiments, the modified cfDNA molecules are coupled to magnetic beads. For example, the modified cfDNA molecules may be separated using magnetic separation. In some embodiments, ends of modified cfDNA molecules are incapable of undergoing ligation and/or primer extension.

[0030] In a certain embodiment, there is a method for processing and/or analyzing a plurality of cell-free deoxyribonucleic (cfDNA) molecules from a subject, comprising (a) subjecting a plurality of cfDNA molecules comprising both subject-derived cfDNA and microbiome cell-free DNA (cfmDNA) to one or more enrichment methods to permit subject- derived cfDNA molecules to be (i) separated from a remainder of said cfmDNA molecules or (ii) digested in the plurality of cfDNA molecules; (b) coupling adapters to ends of said plurality of DNA molecules to provide a plurality of tagged DNA molecules; (c) subjecting said plurality of tagged DNA molecules or tagged derivatives thereof to nucleic acid sequencing to yield a plurality of sequence reads; and (d) processing said plurality of sequence reads to identify sequences from said microbiome. In specific cases, the enrichment method comprises removing cfDNA molecules of specific sizes to enrich the cfmDNA in the plurality of cfDNA molecules. The enrichment method may comprise (a) using protein binding reagents to bind to histone proteins to allow a plurality of histone-associated cell-free DNA molecules to be separated from the remainder of said plurality of cfDNA molecules; and (b) removing the histone binding reagents to deplete histone-associated cell-free DNA molecules from the plurality of cfDNA molecules. In certain cases, the enrichment method comprises: (a) modifying one or both ends of each of at least a portion of said plurality of cf DNA molecules to provide a plurality of modified cfDNA molecules having ends that are (i) configured for separation from a remainder of said plurality of cfDNA or (ii) incapable of coupling with adapters; and (b) removing the modified DNA molecules from a remainder of said plurality of cfDNA. The enrichment method may comprise digesting the plurality of linear cfDNA molecules using one or more nucleases.

[0031] In cases wherein certain sizes of cfDNA is removed, the specific sizes may have lengths from 100 base pairs (bp) and 200bp, l lObp and 190bp, 120bp and 180bp, 130bp and 170bp, 140bp and 170bp, 150bp and 170bp, 160bp and 170bp, 130bp and 160bp, 140bp and 160bp, 150bp and 160bp about 140bp and 150bp, or 150bp and 160bp. The specific sizes may have lengths larger than lOObp, l lObp, 120bp, 130bp, 140bp, 150bp, or 160bp. The removal of molecules of specific sizes may be performed with Ampure® or Solid Phase Reversible Immobilization (SPRI) beads and/or with gel electrophoresis.

[0032] In cases wherein protein binding reagents are used to bind to histone proteins to allow a plurality of histone-associated cell-free DNA molecules to be separated from the remainder of said plurality of cfDNA molecules, the protein binding reagents may comprise anti histone antibodies, peptides, aptamers, fusion proteins, or a combination thereof. The protein binding reagents may be biotinylated, and the protein binding reagents may be conjugated to magnetic beads. Streptavidin beads or protein A or protein G or secondary antibody conjugated magnetic beads may be used for removing the histone binding reagents. The histone protein binding and removal may be performed before the extraction of the plurality of cfDNA molecules, in certain cases.

[0033] In particular embodiments, modifying comprises subjecting a 3' end of each of said at least said portion of said plurality of cfDNA molecules to conditions sufficient to modify said 3' end with a dideoxynucleotide (ddNTP) moiety, or a functional analog thereof. The modifying may comprise subjecting a 5' end of each of said at least said portion of said plurality of cfDNA molecules to conditions sufficient to dephosphorylate said 5' end. In specific cases, modified cfDNA molecules are coupled to magnetic beads, and wherein said modified cfDNA molecules are separated using magnetic separation. Ends of modified cfDNA molecules may be incapable of undergoing ligation and primer extension. [0034] In specific embodiments, sequences from the microbiome are identified in the method. The identifying of sequences from the microbiome may be to determine the presence and/or prevalence of microbial sequences. Determining the presence and/or prevalence of microbial sequences may be to provide information about one or more of the following: (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof. As used herein, prevalence refers to frequency or abundance of one or more particular microbial sequences. In specific cases, a user of the method may be interested in the abundance of one or more specific microbes, such as one or more specific bacteria. Thus, in specific embodiments, methods of the disclosure provide quantitative measurements of one or more particular microbes.

[0035] In some embodiments, the nuclease comprises exonuclease I, exonuclease VII, adenosine triphosphate (ATP) -dependent Dnase, exonuclease V(RecBCD) or a functional analog thereof or a combination thereof. In some embodiments, identifying sequences from said microbiome comprises determining the presence and prevalence of microbial sequences. In some embodiments, determining the presence and/or prevalence of microbial sequences comprises providing information about one or more of the following: (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof.

[0036] In another aspect, the present disclosure provides a computer system for preparing cell-free microbiome deoxyribonucleic acid (DNA), comprising: a controller configured to obtain a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA; a controller configured to subject the collection of cell-free DNA to size selection;

subjecting the collection of cell-free DNA to one or more agents that bind one or more chromatin proteins; and a controller configured to reduce the amount of linear cell-free DNA in the collection of cell-free DNA, to enrich microbiome cell-free DNA in the collection, thereby producing an enriched microbiome cell-free DNA. In some embodiments, the computer system further comprises an electronic display operatively coupled to the one or more computer processors, wherein the electronic display comprises a graphical user interface configured to allow a user to interface with the computer system. [0037] In another aspect, the present disclosure provides a computer system for preparing cell-free deoxyribonucleic acid (DNA), comprising: a controller configured to obtain a collection of cell-free DNA comprising host cell-free DNA; a controller configured to subject the collection of cell-free DNA in the collection to size selection; and a controller configured to reduce an amount of linear cell-free DNA in the collection of cell-free DNA, to enrich microbiome cell- free DNA in the collection, thereby producing enriched microbiome cell-free DNA. In some embodiments, the computer system further comprises an electronic display operatively coupled to the one or more computer processors, wherein the electronic display comprises a graphical user interface configured to allow a user to interface with the computer system.

[0038] In another aspect, the present disclosure provides a computer system for preparing cell-free microbiome deoxyribonucleic acid (DNA), comprising: a controller configured to obtain a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA; a controller configured to subject the collection of cell-free DNA to size selection, to enrich microbiome cell-free DNA in the collection of cell-free DNA, thereby producing enriched microbiome cell-free DNA. In some embodiments, the computer system further comprises an electronic display operatively coupled to the one or more computer processors, wherein the electronic display comprises a graphical user interface configured to allow a user to interface with the computer system.

[0039] In another aspect, the present disclosure provides a computer system for preparing cell-free microbiome deoxyribonucleic acid (DNA), comprising: a controller configured to obtain a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA; and a controller configured to subject the collection of cell-free DNA to one or more agents that bind one or more chromatin proteins, thereby producing enriched microbiome cell- free DNA. In some embodiments, the computer system further comprises an electronic display operatively coupled to the one or more computer processors, wherein the electronic display comprises a graphical user interface configured to allow a user to interface with the computer system.

[0040] In another aspect, the present disclosure provides a computer system for preparing cell-free microbiome deoxyribonucleic acid (DNA), comprising: a controller configured to obtain a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA; and a controller configured to reduce the amount of linear cell-free DNA in the collection of cell-free DNA, to enrich microbiome cell-free DNA in the collection of cell-free DNA, thereby producing enriched microbiome cell-free DNA. In some embodiments, the computer system further comprises an electronic display operatively coupled to the one or more computer processors, wherein the electronic display comprises a graphical user interface configured to allow a user to interface with the computer system.

[0041] In another aspect, the present disclosure provides a computer system for processing and/or analyzing a plurality of cell-free deoxyribonucleic (cfDNA) molecules from a subject, comprising: a controller configured to subject a plurality of cfDNA molecules comprising both subject-derived cfDNA and microbiome cell-free DNA (cfmDNA) to one or more enrichment methods to permit subject-derived cfDNA molecules to be (i) separated from a remainder of said cfmDNA molecules or (ii) digested (for example, with one or more nucleases) in the plurality of cfDNA molecules; a controller configured to couple adapters to ends of said plurality of DNA molecules to provide a plurality of tagged DNA molecules; a controller configured to subject the plurality of tagged DNA molecules or tagged derivatives thereof to nucleic acid sequencing to yield a plurality of sequence reads; and a controller configured to process the plurality of sequence reads to identify sequences from the microbiome. In some embodiments, the computer system further comprises an electronic display operatively coupled to the one or more computer processors, wherein the electronic display comprises a graphical user interface configured to allow a user to interface with the computer system.

[0042] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0043] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein

[0044] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.

[0045] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better

understood. Additional features and advantages will be described hereinafter which form the subject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present designs. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features which are believed to be characteristic of the designs disclosed herein, both as to the organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure. Additional objects, features, aspects and advantages of the present invention will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by practice of the invention. Various embodiments of the disclosure will be described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that changes may be made without departing from the scope of the invention. The following detailed description is, therefore, not be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also“Figure” and“FIG.” herein), of which: [0047] FIG. 1 provides a flowchart of performing genomic and epigenomic microbiome profiling of cell-free DNA (cfDNA).

[0048] FIG. 2 illustrates an example of performing microbiome profiling of cfDNA with size selection.

[0049] FIG. 3 illustrates an example of performing microbiome profiling of cfDNA with antibody binding followed by removal by streptavidin magnetic beads.

[0050] FIG. 4 illustrates an example of performing microbiome profiling of cfDNA with end-labeling followed by removal by streptavidin magnetic beads.

[0051] FIG. 5 illustrates an example of performing microbiome profiling of cfDNA with digestion of linear DNA by nucleases.

[0052] FIG. 6 illustrates an example of a computer system 601 that is programmed or otherwise configured to implement methods of the disclosure.

[0053] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

DETAILED DESCRIPTION

[0054] The present disclosure generally relates to preparation of nucleic acids, including improved preparation methods, systems, kits, and compositions. The nucleic acids may be obtained or derived from biological materials of any kind, including biological samples in need of analysis. In some embodiments, the nucleic acids comprise cell-free DNA that is present in a collection of different types of cell-free DNA and optionally other types of nucleic acids. The present disclosure provides for methods, systems, kits, and compositions of removing, reducing, or depleting at least some of certain type(s) of cell-free DNA such that another type(s) of cell- free DNA is thereby enriched, increased or isolated. I. [0055] Examples of Definitions

[0056] In keeping with long-standing patent law convention, the words“a” and“an” when used in the present specification in concert with the word comprising, including the claims, denote“one or more.” As used in the specification and claims, the singular form“a”,“an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term“a nucleic acid” includes a plurality of nucleic acids, including mixtures thereof. Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and/or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0057] Throughout this specification, unless the context requires otherwise, the words “comprise”,“comprises” and“comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By“consisting of’ is meant including, and limited to, whatever follows the phrase“consisting of.” Thus, the phrase“consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By“consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase“consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0058] As used herein, the terms“or” and“and/or” are utilized to describe multiple components in combination or exclusive of one another. For example,“x, y, and/or z” can refer to“x” alone,“y” alone,“z” alone,“x, y, and z,”“(x and y) or z,”“x or (y and z),” or“x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an

embodiment.

[0059] Throughout this application, the term“about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. [0060] Reference throughout this specification to“one embodiment,”“an embodiment,” “a particular embodiment,”“a related embodiment,”“a certain embodiment,”“an additional embodiment,” or“a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0061] The term“host” as used herein generally refers to an organism in which one or more microbiomes reside. The host may be an animal or plant and may be living or deceased. The host may be of any age and any condition of health. A single host may comprise one or more microbiomes, and the one or more microbiomes may be located in vivo in different regions of the body.

[0062] The terms“medical condition associated with a microbiome” or“medical condition related with a microbiome” or“microbiome-related medical condition”, or similar terms, as used herein, generally refer to medical condition(s) of an individual that are directly or indirectly caused by the presence or absence of one or more particular microbes in a host’s microbiome. In some embodiments, the individual may not have the medical condition were it not for the presence or absence of one or more particular microbes in the microbiome or were it not for the presence or absence of a certain level (including threshold level) of one or more particular microbes in the microbiome. An example of a medical condition that is directly associated with the presence of a microbe in the microbiome is a vims that causes a viral infection in the host. An example of a medical condition that is indirectly caused by the presence of a microbe in the microbiome is antibiotic-associated diarrhea, such as with Clostridia difficile treatment for a host.

[0063] The term“microbiome,” as used herein, generally refers to collection of microorganisms (that also may be referred to as microbes), such as bacteria, fungi, viruses, and/or protozoa, within a community in a host, including within a particular location and/or tissue and/or organ of a host.

[0064] The term“profile,” as used herein, generally refers to the biological constitution of different microbes in a microbiome, including the identity and, in some embodiments, levels, of one or more different microbes, including with respect to one or more other microbes in the microbiome in some embodiments.

[0065] The term“sample,” as used herein, generally refers to a biological sample. The sample may be taken from tissue or cells or from the environment of tissue or cells. In some examples, the sample may comprise, or be derived from, a tissue biopsy, blood ( e.g ., whole blood), blood plasma, extracellular fluid, dried blood spots, cultured cells, culture media, discarded tissue, bacterial and/or viral samples, fungal tissue, archaea, and/or protozoans. The sample may have been isolated from the source prior to collection. Non-limiting examples include blood, cerebral spinal fluid, pleural fluid, amniotic fluid, lymph fluid, saliva, urine, stool, tears, sweat, or mucosal excretions, and other bodily fluids isolated from the primary source prior to collection. In some examples, the sample is isolated from its primary source (cells, tissue, bodily fluids such as blood, environmental samples, etc.) during sample preparation. The sample may be obtained from a living individual or a deceased individual. The sample may be derived from an extinct species, such as samples derived from fossils. The sample may or may not be purified or otherwise enriched from its primary source. In some embodiments, the primary source is homogenized prior to further processing. The sample may be filtered or centrifuged to remove buffy coat, lipids, or particulate matter. The sample may also be purified or enriched for nucleic acids, or may be treated with RNases. The sample may contain tissues or cells that are intact, fragmented, or partially degraded.

[0066] The term“subject,” as used herein, generally refers to an individual having a biological sample that is undergoing processing or analysis and, in some embodiments, has one or more microbiomes associated therewith. A subject can be an animal or plant. The subject can be any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The subject can be a patient, e.g., have or be suspected of having a disease (that may be referred to as a medical condition), such as one or more infectious diseases, one or more genetic disorders, one or more cancers, or any combination thereof. The disease may be pathogenic. The subject may be undergoing or having undergone antibiotic treatment. The subject may be asymptomatic. The subject may be healthy individuals. The term “individual” may be used interchangeably, in at least some embodiments. The“subject” or "individual", as used herein, may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may be receiving one or more medical compositions via the internet. An individual may comprise any age of a human or non-human animal and therefore includes both adult and juveniles ( e.g .., children) and infants and includes in utero individuals. A subject may or may not have a need for medical treatment; an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies.

[0067] As used herein, the term“nucleic acid” generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs), or analogs thereof. Nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of nucleic acids include deoxyribonucleic (DNA), ribonucleic acid (RNA), coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be made before or after assembly of the nucleic acid. The sequence of nucleotides of a nucleic acid may be interrupted by non-nucleotide components. A nucleic acid may be further modified after polymerization, such as by conjugation or binding with a reporter agent.

[0068] As used herein, the term“target nucleic acid” generally refers to a nucleic acid molecule in a starting population of nucleic acid molecules having a nucleotide sequence whose presence, amount, and/or sequence, or changes in one or more of these, are desired to be determined. A target nucleic acid may be any type of nucleic acid, including DNA, RNA, and analogs thereof. As used herein, a“target ribonucleic acid (RNA)” generally refers to a target nucleic acid that is RNA. As used herein, a“target deoxyribonucleic acid (DNA)” generally refers to a target nucleic acid that is DNA.

[0069] As used herein, the terms“amplifying” and“amplification” generally refer to increasing the size or quantity of a nucleic acid molecule. The nucleic acid molecule may be single-stranded or double-stranded. Amplification may include generating one or more copies or “amplified product” of the nucleic acid molecule. Amplification may be performed, for example, by extension (e.g., primer extension) or ligation. Amplification may include performing a primer extension reaction to generate a strand complementary to a single- stranded nucleic acid molecule, and in some cases generate one or more copies of the strand and/or the single-stranded nucleic acid molecule. The term“DNA amplification” generally refers to generating one or more copies of a DNA molecule or“amplified DNA product.” The term “reverse transcription amplification” generally refers to the generation of deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template via the action of a reverse transcriptase.

II. [0070] Methods

[0071] the present disclosure provides methods, systems, media, kits, and compositions for enriching microbiome cell-free DNA from other type(s) of cell-free DNA, including at least cell-free DNA of one or more hosts. In some embodiments, the method enriches for microbiome cell-free DNA by increasing, enriching, or isolating the amount of microbiome cell-free DNA with respect to one or more other type(s) of cell-free DNA, such as host cell-free DNA, for example in comparison to the amount of both types of cell-free DNA that is present without performing the disclosed methods. In some embodiments, the methods enrich for or isolate microbiome cell-free DNA by increasing the proportion of microbiome cell-free DNA compared to host cell-free DNA in the context of a collection that comprises them both.

[0072] The present disclosure provides methods, systems, media, kits, and compositions for removing unwanted human cell-free DNA prior to cell-free microbiome DNA library preparation or other cell-free microbiome assays such as nucleic acid array based on their DNA feature differences. The methods may comprise one or more of the following:

[0073] (1) A size selection approach may be performed to remove, deplete, or decrease host DNA at specific size ranges. The sizes of human (as an example) cfDNA fragments, but not the microbiome DNA fragments, may be peaked around 165 bp. DNA fragments with sizes around 165 bp, for example, sizes between 130 bp and 185 bp, may be removed by bead-based, gel-based, or other size selection strategies. The size selection approach may also be used to remove cfDNA fragments of larger sizes, for examples, sizes larger than 100 bp, to enrich cfmDNA that are at smaller sizes.

[0074] (2) A protein- specific depletion approach may be performed to deplete, decrease, or remove host DNA based on its histone wrapping property. Anti-histone antibodies or any other class of protein- specific binding reagents such as aptamers and fusion proteins, for example, can specifically bind to histones wrapped with host cfDNA; the antibodies then may be depleted, for example with magnetic beads conjugated to them.

[0075] (3) A DNA depletion approach may be performed to remove, deplete, or decrease linear DNA. Human cfDNA may comprise single- and/or double-strand linear DNA. Linear DNA may be end-modified (e.g., 3 '-end-labeled, such as biotin-dNTP-labeled). The modified DNA may be removed using approaches such as magnetic removal (e.g., by streptavidin magnetic beads). Circular cfDNA that originated from at least some populations of the microbiome may remain for further analysis.

[0076] (4) An enzymatic digestion approach may be performed to digest human cfDNA. Enzymatic digestion approach may use one or more nucleases, such as exonuclease I, exonuclease VII, Dnase, and/or exonuclease V(RecBCD), to digest either single-strand DNA or double-strand DNA or both. Thus, circular cfDNA that originated from the microbiome may remain for further analysis.

[0077] The different approaches to reduce host cell-free DNA may be utilized separately or in conjunction. In some embodiments, the method only utilizes operations for size selection to enrich for, increase, or isolate microbiome cell-free DNA in a particular collection of cell-free DNA from a sample. In some embodiments, the method only utilizes protein- specific depletion operations to enrich for microbiome cell-free DNA. In some embodiments, the method only utilizes operations that exploit structural differences between microbiome cell-free DNA and host cell-free DNA (e.g., being linear and/or strandededness). In some embodiments, two or more of the approaches are utilized with the same particular collection of cell-free DNA from a biological sample. In some embodiments, the collection of cell-free DNA is subjected at least to size selection and protein-specific depletion. In some embodiments, the collection of cell-free DNA is subjected at least to size selection and/or operations that exploit structural differences.

In some embodiments, the collection of cell-free DNA is subjected at least to protein- specific depletion and operations that exploit structural differences. In some embodiments, size selection is not utilized to enrich for microbiome cell-free DNA.

[0078] The present disclosure provides methods of removing human cell-free DNA from non-human cell-free DNA in a collection of cell-free DNA by depleting and/or digesting the human cell-free DNA based on one or more physical characteristic differences between the human cell-free DNA and the non-human cell-free DNA. Examples of physical characteristic differences include size distribution, protein association, linearality, and/or strandedness, for example. Embodiments of the disclosure include methods of removing human cell-free DNA from microbial cell-free DNA in a collection of cell-free DNA by depleting and/or digesting the human cell-free DNA based on one or more physical characteristic differences between the human cell-free DNA and the microbial cell-free DNA. Examples of physical characteristic differences include size distribution, protein association, linearality, and/or strandedness, for example.

[0079] The cell-free biological samples may be obtained or derived from a healthy subject, a patient with a disease or disorder (e.g., a cancer), a patient suspected of having a disease or disorder (e.g., a cancer), a pregnant female subject, or a female subject suspected of being pregnant. The cell-free samples may be stored in a variety of storage conditions before processing, such as different temperatures (e.g., at room temperature, under refrigeration or freezer conditions, at 25°C, at 4°C, at -18°C, -20°C, or at -80°C) or different suspensions (e.g., EDTA collection tubes, cell-free RNA collection tubes, or cell-free DNA collection tubes).

[0080] The cell-free biological sample may be obtained from a subject with a disease or disorder (e.g., a cancer), from a subject that is suspected of having a disease or disorder (e.g., a cancer), or from a subject that does not have or is not suspected of having the disease or disorder (e.g., a cancer).

[0081] The cell-free biological sample may be taken before and/or after treatment of a subject with the disease or disorder (e.g., a cancer). Cell-free biological samples may be obtained from a subject during a treatment or a treatment regime. Multiple cell-free biological samples may be obtained from a subject to monitor the effects of the treatment over time. The cell-free biological sample may be taken from a subject known or suspected of having a disease or disorder (e.g., a cancer) for which a definitive positive or negative diagnosis is not available via clinical tests. The sample may be taken from a subject suspected of having a disease or disorder (e.g., a cancer). The cell-free biological sample may be taken from a subject experiencing unexplained symptoms, such as fatigue, nausea, weight loss, aches and pains, weakness, or bleeding. The cell-free biological sample may be taken from a subject having explained symptoms. The cell-free biological sample may be taken from a subject at risk of developing a disease or disorder (e.g., a cancer) due to factors such as familial history, age, hypertension or pre-hypertension, diabetes or pre-diabetes, overweight or obesity, environmental exposure, lifestyle risk factors (e.g., smoking, alcohol consumption, or drug use), or presence of other risk factors.

[0082] In some embodiments, a plurality of nucleic acid molecules is extracted from the cell-free biological sample and subjected to sequencing to generate a plurality of sequencing reads. The nucleic acid molecules may comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The nucleic acid molecules (e.g., RNA or DNA) may be extracted from the cell-free biological sample by a variety of methods, such as a FastDNA Kit protocol from MP

Biomedicals, a QIAamp DNA cell-free biological mini kit from Qiagen, or a cell-free biological DNA isolation kit protocol from Norgen Biotek. The extraction method may extract all RNA or DNA molecules from a sample. Alternatively, the extract method may selectively extract a portion of RNA or DNA molecules from a sample. Extracted RNA molecules from a sample may be converted to DNA molecules by reverse transcription (RT).

[0083] The sequencing may be performed by any suitable sequencing methods, such as massively parallel sequencing (MPS), paired-end sequencing, high-throughput sequencing, next- generation sequencing (NGS), shotgun sequencing, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, pyrosequencing, sequencing-by- synthesis (SBS), sequencing-by-ligation, and sequencing-by-hybridization, RNA-Seq (Illumina).

[0084] The sequencing may comprise nucleic acid amplification (e.g., of RNA or DNA molecules). In some embodiments, the nucleic acid amplification is polymerase chain reaction (PCR). A suitable number of rounds of PCR (e.g., PCR, qPCR, reverse-transcriptase PCR, digital PCR, etc.) may be performed to sufficiently amplify an initial amount of nucleic acid (e.g., RNA or DNA) to a desired input quantity for subsequent sequencing. In some cases, the PCR may be used for global amplification of target nucleic acids. This may comprise using adapter sequences that may be first ligated to different molecules followed by PCR amplification using universal primers. PCR may be performed using any of a number of commercial kits, e.g., provided by Life Technologies, Affymetrix, Promega, Qiagen, etc. In other cases, only certain target nucleic acids within a population of nucleic acids may be amplified. In some embodiments, the plurality of DNA is subjected to enzymatic or chemical reactions to distinguish methylated vs.

unmethylated bases. In some embodiments, the plurality of DNA undergoes bisulfite conversion. Specific primers, possibly in conjunction with adapter ligation, may be used to selectively amplify certain targets for downstream sequencing. The PCR may comprise targeted amplification of one or more genomic loci, such as genomic loci associated with cancer or pregnancy. The sequencing may comprise use of simultaneous reverse transcription (RT) and polymerase chain reaction (PCR), such as a OneStep RT-PCR kit protocol by Qiagen, NEB, Thermo Fisher Scientific, or Bio-Rad.

[0085] RNA or DNA molecules isolated or extracted from a cell-free biological sample may be tagged, e.g., with identifiable tags, to allow for multiplexing of a plurality of samples. Any number of RNA or DNA samples may be multiplexed. For example a multiplexed reaction may contain RNA or DNA from at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 initial cell-free biological samples. For example, a plurality of cell-free biological samples may be tagged with sample barcodes such that each DNA molecule may be traced back to the sample (and the subject) from which the DNA molecule originated. Such tags may be attached to RNA or DNA molecules by ligation or by PCR amplification with primers. The barcodes may uniquely tag the cfDNA molecules in a sample. Alternatively, the barcodes may non-uniquely tag the cfDNA molecules in a sample. The barcode(s) may non-uniquely tag the cfDNA molecules in a sample such that additional information taken from the cfDNA molecule (e.g., at least a portion of the endogenous sequence of the cfDNA molecule), taken in combination with the non-unique tag, may function as a unique identifier for (e.g., to uniquely identify against other molecules) the cfDNA molecule in a sample. For example, cfDNA sequence reads having unique identity (e.g., from a given template molecule) may be detected based on sequence information comprising one or more contiguous -base regions at one or both ends of the sequence read, the length of the sequence read, and the sequence of the attached barcodes at one or both ends of the sequence read. DNA molecules may be uniquely identified without tagging by partitioning a DNA (e.g., cfDNA) sample into many (e.g., at least about 50, at least about 100, at least about 500, at least about 1 thousand, at least about 5 thousand, at least about 10 thousand, at least about 50 thousand, or at least about 100 thousand) different discrete subunits (e.g., partitions, wells, or droplets) prior to amplification, such that amplified DNA molecules can be uniquely resolved and identified as originating from their respective individual input molecules of DNA.

[0086] The plurality of DNA molecule or derivatives may be subject to conditions sufficient to permit distinction between methylated nucleic acid bases and unmethylated nucleic acid bases. In some cases, subjecting the plurality of DNA molecules or derivatives thereof to conditions to distinguish methylated vs. unmethylated bases comprises performing bisulfite conversion on the plurality of DNA molecules. In some cases, subjecting the plurality of DNA molecules or derivatives thereof to conditions to distinguish methylated vs. unmethylated bases comprises enzymatic or chemical reactions to oxidize the methylated cytosine nucleic acid bases and/or hydroxymethylated cytosine nucleic acid bases followed by reduction and/or deamination of oxidation reaction products.

[0087] Biological samples of the present disclosure may be sequenced using various nucleic acid sequencing approaches. Such samples may be processed prior to sequencing, such as by being subjected to purification, isolation, enrichment, nucleic acid amplification (e.g., polymerase chain reaction (PCR)). Sequencing may be performed using, for example, Sanger sequencing, high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), Next generation sequencing (e.g., Illumina, Pacific Biosciences of California, Ion Torrent), Single Molecule Sequencing by Synthesis (SMSS)(Helicos), massively-parallel sequencing, Clonal Single Molecule Array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, primer walking, sequencing using PacBio, SOLiD, Ion Torrent, or Nanopore platforms and any other sequencing methods known in the art. Simultaneous sequencing reactions may be performed using multiplex sequencing. Sequencing may generate sequencing reads (“reads”), which may be processed by a computer. In some examples, reads may be processed against one or more references to identify copy number variants (CNVs).

[0088] In some examples, sequencing can be performed on cell-free polynucleotides that may comprise a variety of different types of nucleic acids. Nucleic acids may be polynucleotides or oligonucleotides. Nucleic acids included, but are not limited to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), single-stranded or double- stranded DNA, complementary DNA (cDNA), or a RNA/cDNA pair.

[0089] In some embodiments, a sample (e.g., biological sample) comprising a collection of cell-free DNA is subjected to any methods encompassed by the disclosure. In some embodiments, it is unknown whether or not the cell-free DNA comprises microbiome cell-free DNA. The sample may be suspected of comprising microbiome cell-free DNA that is of an unhealthy constitution, such as having reduced or increased levels of one or more pathogens. In embodiments wherein the sample is suspected of comprising microbiome cell-free DNA, the sample may come from an individual having a medical condition, suspected of having a medical condition, at risk of having a medical condition, or the individual may be healthy and the method is performed because of routine medical care, for example. The individual may or may not have one or more symptoms of a medical condition. In some embodiments, the medical condition is the result of the presence of the microbiome, such as one or more pathogens therein.

[0090] In some embodiments, methods are performed when it is unknown if the initial collection of cell-free DNA comprises microbiome cell-free DNA, or sufficient levels of microbiome cell-free DNA to be detected; the collection of cell-free DNA may be suspected of having microbiome cell-free DNA. In such embodiments, performance of the operations of the method may not enrich for microbiome cell-free DNA that is not present, but the method may nevertheless be performed, for example in the absence of predetermined confirmation of a sample having microbiome cell-free DNA. In other embodiments, the method is performed to enrich for certain cell-free DNA, for example, by enriching for cell-free DNA of any kind that is (1) of a certain size; (2) not protein-bound (of at least certain protein(s)); and/or (3) circular.

[0091] In some embodiments, the method operations of the disclosure are dictated by analysis needed for an expected or suspected microbe. For example, one may utilize certain enrichment approaches dependent upon one or more microbes suspected or known to be the cause of a medical condition or its symptoms. As an example, an individual having a medical condition or at risk of or susceptible to the medical condition associated with a bacteria is subjected to enrichment approaches that target the difference between bacterial circular cell-free DNA versus host linear cell-free DNA.

[0092] The present disclosure provides methods, systems, kits, and compositions for enriching one type of cell-free DNA with respect to another type of cell-free DNA. In some embodiments, the methods are performed for enriching microbiome cell-free DNA in a collection with respect to host cell-free DNA. In some embodiments, the enrichment comprises increasing in a collection of cell-free DNA the amount of microbiome cell-free DNA; in some aspects the increasing is with respect to the amount of host cell-free DNA in the same collection. In some embodiments, after performing methods of the disclosure, the proportion of microbiome cell-free DNA to host cell-free DNA increases in a common collection of cell-free DNA. Any increase in amount of microbiome cell-free DNA in a collection may occur, but in some embodiments the increase is 2-fold, 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1000-fold, 10,000-fold, 10⁵-fold, 10⁶-fold, 10⁷-fold, and so forth. In specific embodiments, the amount of host cell-free DNA reduces by a particular amount in the collection, such as 2-fold, 10-fold, 50- fold, 100-fold, 250-fold, 500-fold, 1000-fold, 10,000-fold, 10⁵-fold, 10⁶-fold, 10⁷-fold, etc. Following the practice of methods of the disclosure, the amount of host cell-free DNA that is reduced in the collection may quantitatively be reduced partially or entirely, such asto undetectable levels. When reduced partially, the amount of reduction may be of any kind that is detectable by any approach. In some embodiments, the amount of host cell-free DNA is reduced in the collection by a particular quantity following practice of the method, such as by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the amount of microbiome cell-free DNA is increased in the collection by a particular quantity after perfoming the method, such as by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0093] The methods of the disclosure may comprise assaying for the presence of microbiome cell-free DNA in the collection, for example prior to the onset of the method. In some embodiments, the sample is determined not to have any detectable microbiome cell-free DNA, and the enrichment method may not be needed, although in other embodiment the method still applies to enrichment of certain types of cell-free DNA over others. When the presence of microbiome cell-free DNA is detected, the method may be performed at that time or at a later time. The presence of microbiome cell-free DNA may be confirmed subsequent to completion of the enrichment method, and in some embodiments the quantity of the enriched microbiome cell-free DNA is determined.

[0094] Methods of the disclosure may be performed on cell-free DNA from any microbiome of any organism. In some embodiments, the microbiome comprises one or more pathogens. The microbiome may comprise one or more pathogenic microorganisms and/or one or more non-pathogenic microorganisms. The microbiome may comprise one or more bacteria, one or more viruses, one or more fungi, and/or one or more protozoans. The microbiome may or may not be associated with a medical condition, including directly or indirectly associated with a medical condition. The microbiome may or may not be part of a symbiotic relationship with the host and in some embodiments the host is in need of determining the profile of the microbiome for therapeutic or prophylactic purposes. The individual may be in need of ascertaining one or more causes of one or more symptoms of a medical condition, in some embodiments. In specific embodiments, the host is in need of determining the profile of a microbiome to be aware of the risk or susceptibility to a medical condition. Any medical condition may be of concern, including at least one of the skin, mouth, nose, gastrointestinal tract (including at least the stomach and the intestines), and/or genitourinary tract (including kidneys, bladder, fallopian tubes, penis, prostate, uterus, and vagina as examples) of the host, for example.

[0095] The individual may be in need of determining the health of the microbiome, such as in need of determining the presence or absence of a particular profile of the microbiome. In some embodiments, the microbiome is in need of analysis for the presence or absence of one or more specific microorganisms. The one or more specific microorganisms may be associated with a medical condition or associated with a beneficial presence for the host.

[0096] In an example, an individual having a symptom of a medical condition associated with the gastrointestinal tract is in need of determining the cause of the symptom. In a particular case the individual has diarrhea, stomach cramps, blood in the gastrointestinal tract, and/or nausea, for example, and it is desired to determine the profile of the microbiome in the gastrointestinal tract. Determination of the microbiome profile utilizing one or more methods of the disclosure may provide information for treatment of the individual. In an example, an individual having a personal or family history of a medical condition associated with the gastrointestinal tract is in need of determining the profile of the microbiome in the

gastrointestinal tract to determine if prophylactic operations are needed.

[0097] In another example, an individual with one or more symptoms of a medical condition associated with the reproductive tract (including the vagina) may have a biological sample subjected to methods of the disclosure. The individual may have a clinical symptom, such as an itching, a change in the consistency and/or color of discharge, swelling or soreness of the reproductive tract, etc. Such an individual may be in need of knowing the microbiome profile of the reproductive tract to treat the symptoms.

[0098] The profile of the microbiome may depend on the in vivo location of the microbiome from which the sample comprising the collection of cell-free DNA is obtained. The profile may be characteristic of a certain location in vivo because of the presence of one or more microorganisms and/or on the absence of one or more microorganisms. The makeup of the microbiome profile may originate from what is acquired at birth, from the genetics of the host with respect to a physiological interaction with microorganisms, such as over time, and/or from what may be acquired from the environment over time or from one or more specific events, for example.

[0099] In some embodiments, the microbiome of any location in vivo may comprise one or more particular bacteria, including one or more particular genera, one or more particular bacteria species, one or more particular strains, and/or one or more particular clades. The bacteria may be Gram positive or Gram negative. The bacteria may be spherical, rod-shaped, or other shaped. The bacteria may be anaerobic or aerobic. In some embodiments, the bacteria comprise Firmicutes, Bacteroidetes, Actinobacteria, and/or Proteobacteria. In some

embodiments, the bacteria belong to the genera Bacteroides, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Escherichia, Lactobacillus, and/or Bifidobacterium.

[0100] Examples of fungal genera that may be present in the microbiome include

Candida, Saccharomyces, Aspergillus, Penicillium, Rhodotomla, Trametes, Pleospora,

Sclerotinia, Bullera, Candida, and Galactomyces, among others.

[0101] Examples of viruses that may be present in the microbiome include Adenoviruses, Coronavimses, Orthomyxoviruses, Paramyxoviruses, Herpesviruses, Papillomaviruses,

Polyomaviruses, Astroviruses, Caliciviruses, Picomaviruses, Reoviruses, Retroviruses, etc. In some embodiments, the profile of a microbiome with respect to viruses may be characterized by the location of the microbiome in vivo. For example, the viral profile of an oral microbiome may comprise Herpesviruses and/or Papillomaviruses, but there may or may not be others, and in some embodiments one or both of these are not present. In a respiratory microbiome, for example, there may be one or more of Adenoviruses, Coronavimses, Orthomyxoviruses, and Paramyxoviruses and there may or may not be others and in some embodiments one or more of these viruses are not present. For a dermal microbiome, for example, there may be one or more of Herpesviruses, Papillomaviruses, and Polyomaviruses, yet there may or may not be others and in some embodiments one or more of these viruses are not present. In a gastrointestinal microbiome, as an example, there may be one or more of Adenoviruses, Astroviruses,

Caliciviruses, Picomaviruses, Reoviruses, and Retroviruses, yet there may or may not be others and in some cases one or more of these viruses are not present. In a genitourinary tract, there may be one or more of Adenoviruses, Herpesviruses, Papillomaviruses, and Retroviruses, yet there may or may not be others and in some embodiments one or more of these viruses are not present.

[0102] In some embodiments, a microbiome is analyzed for one or more protozoa, some of which that are pathogenic and in some embodiments, some of which that are non-pathogenic. The protozoa may be Intestinal protozoa, e.g. Entamoeba histolytica, Giardia lamblia and Cryptosporidium parvum, or they may be Urogenital protozoa, e.g. Trichomonas vaginalis, or they may be Blood and Tissue protozoa, e.g. Toxoplasma gondii and Plasmodium falciparum.

[0103] In some embodiments, methods, systems, kits, and compositions of the present disclosure are used to prepare cell-free microbiome DNA from a particular microbiome to determine whether or not there is a standard or otherwise particular profile that indicates that the individual is healthy or free from risk of a medical condition related to the microbiome. In other embodiments, the absence of the standard or otherwise particular profile indicates the individual is not healthy or is at risk for a medical condition, including one related to the microbiome. In such methods, in some embodiments, the analysis of the microbiome is a directed analysis for one or more particular microbes, whereas in other embodiments the analysis is not directed toward any one or more particular microbes.

[0104] In some embodiments, methods of the disclosure comprise providing particular information about one or more microbiomes from one or more hosts. Examples of information that may be obtained with or as a result of methods of the disclosure include the richness (the number of distinct members ("species") in the community); diversity (a measure of the richness and evenness characteristics of a community, often calculated as a specific "diversity index") and/or dysbiosis (a term used to refer to a microbiota community associated with a diseased state that can be differentiated from the microbiota community associated with a healthy control state) (as described by Shreiner et al. Curr Opin Gastroenterol. 2015 Jan; 31(1): 69-75, incorporated by reference).

[0105] Such analysis for one or more microbiomes from one or more hosts may exploit any differential between a host cell-free DNA and its microbiome cell-free DNA. Examples of approaches to enrich microbiome cell-free DNA from a collection of cell-free DNA are provided herein. A. Size Selection

[0106] In some embodiments, cell-free microbiome DNA is sorted to remove host cell- free microbiome DNA based upon size of certain subpopulations of cell-free DNA in a collection of cell-free DNA. In some embodiments, cell-free DNA that comprises host cell-free DNA and that is suspected of having cell-free microbiome DNA (or known to have cell-free microbiome DNA) is subjected to size selection to remove certain sizes of cell-free DNA. The size selection may be performed by any suitable method, and in specific embodiments is performed using beads or gels ( e.g ., electrophoresis).

[0107] In some embodiments, a range of cell-free DNA for selection is between about 100 base pairs (bp) and about 200 bp. In specific examples, the DNA is in a range of 100-200, 100-180, 100-175, 100-160, 100-150, 100-140, 100-130, 100-125, 100-120, 100-110, 125-200, 125-175, 125-150, 130-200, 130-185, 130-175, 130-165, 130-155, 130-145, 130-135, 140-200, 140-185, 140-175, 140-165, 140-155, 140-150, 150-200, 150-180, 150-175, 150-165, 150-160, 150-155, 160-200, 160-180, 160-175, 160-170, 160-165, 170-180, 175-200, 175-180, 180-200, or 190-200 bp or bases.

[0108] In some embodiments, the size selection is utilized in methods to remove cfDNA fragments of DNA sizes larger than 100 bp, thereby enriches cell-free microbiome DNA that are at smaller sizes than lOObp. The cell-free microbiome DNA to be enriched may be in the range of 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 25-100, 25-90, 25-80, 25-70, 25-60, 25-50, 25-40, 25-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100 bp or bases, as examples.

[0109] Once the collection of cell-free DNA has been subjected to size selection of a desired size associated with host cell-free DNA to remove the undesired host cell-free DNA, the remaining cell-free may be enriched for cell-free microbiome DNA as a result. Examples of size selection approaches are provided herein:

1. Bead-Based Selection

[0110] In some embodiments, beads are employed to size select certain cell-free DNA sizes. As an example, bead-based size selection of nucleic acids may or may not be performed such as with commercially available kits. As an example, the kits may utilize solid phase reversible immobilization (SPRI) beads, which reversibly bind DNA in the presence of a crowding agent (e.g., polyethylene glycol) and salt. In such embodiments, the concentration of the crowding agent determines the size of the fragments selected by the beads, and this can be manipulated to select for a desired range of sized cell-free DNA. In commercial embodiments, size selection may vary in a range of 100 bp to 500 bp. An example of SPRI beads is AMPure XP beads. In this approach, one may adjust the type and composition of the bead dependent upon the desired sizes of cell-free DNA to be bound.

2. Gel-Based Selection

[0111] In some embodiments, in conjunction with or alternatively to bead-based size selection, one may perform gel-based size selection of the cell-free nucleic acids. Gel-based size selection may be performed, for example, in a variety of precast gels through electrophoresis, such as agarose gel electrophoresis for nucleic acids. Such exposure to gels may provide a high resolution of DNA molecules, for examle in the range of 10 to 20,000 bp, or greater. A type of gel used may be agarose gel employing various buffers, such as Tris-acetate-EDTA (TAE) buffer or Tris-boric acid-EDTA (TBE) buffer. The type of different buffers and amount used may impact the fragment separation in electrophoresis. Some examples of commercial products for gel-based size selection include Novex ® TBE Gel, Novex ® DNA Retardation Gel, and E-Gel SizeSelect II Gel. One may adjust the type and composition of the gel dependent upon the desired sizes of cell-free DNA to be separated.

B. Protein-Specific Depletion

[0112] In some embodiments, a collection of cell-free DNA is subjected to depletion, reduction, or removal of one or more types of certain proteins to enrich for cell-free DNA that is not associated with the proteins. In some embodiments, a collection of cell-free DNA is subjected to depletion, reduction, or removal of one or more types of chromatin proteins to enrich for cell-free DNA that is not associated with the chromatin proteins. In some examples, the chromatin protein(s) comprise one or more histones. The histones may be of any kind, including core histones or linker histones. In some embodiments, the histones comprise H1/H5, H2A, H2B, H3, or H4. Cell-free DNA associated with chromatin proteins may be separated based on molecular weight from cell-free DNA that is not associated with the protein by centrifugal filter. For example, an Amiocon Ultra-2 Centrifugal Filter Unit with Ultracel-50 membrane may enable the concentration and depletion of cell-free-DNA-associated chromatin protein (above 50kDa) with a membrane. However, the abundance of other proteins in the plasma may make molecular weight based protein depletion difficult. [0113] Thus, in some embodiments, a collection of cell-free DNA that is suspected of having microbiome cell-free DNA or known to have it is subjected to one or more agents that binds one or more chromatin proteins. The agents may be one or a combination of chromatin protein-binding agents, in particular. The agents may target the protein and be extracted by a feature of the agent, thereby extracting with it anything that the agent/protein complex is bound to, including cell-free DNA.

[0114] In some embodiments, the one or more agents that bind one or more chromatin proteins are antibodies, and they may be antibodies of any type. In some embodiments, the antibodies encompass any immunologic binding agent, such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM may be utilized because they are the most common antibodies in the physiological situation and because they are easily made in a laboratory setting. The term "antibody," as used herein refers to any antibody-like molecule that has an antigen binding region, and may include antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. Various techniques for preparing and using various antibody-based constructs and fragments may be used. Various methods for preparing and characterizing antibodies may be used (See, e.g., Antibodies: A Faboratory Manual, Cold Spring Harbor Faboratory, 1988; which is incorporated herein by reference).

[0115] There are many commercially available anti-histone antibodies, as an example. Specific, non-limiting examples that work with human DNA (as opposed to mouse, rabbit, sheep, goat, rat or chicken, for example) include at least the following: IGX4228H anti-histone H2B antibody; IGX4696H anti-histone H4 antibody; EPR21201 anti-histone H2A.X antibody; AE-4 anti-histone HI antibody; HH1/957 anti-histone HI antibody; r 1415- 1 anti-histone HI antibody; and HH1/1784R anti-histone HI antibody.

[0116] In other embodiments, agents that are not antibodies are employed as chromatin binding agents. As an example, aptamers may be employed. Aptamers may be synthetic oligonucleotide or peptide molecules that target and bind with antigens. Aptamers may be commercially available but also may be synthetically generated. As examples, aptamer 4.33 histone H4 n-terminal tail or H4K16Ac aptamer may be utilized. Other examples of commercial products include aptamer 4.13 and aptamer 4.15, which are both anti-histone H4 aptamers. Custom aptamers can also be made (e.g., by IBA Fife Sciences). [0117] In some embodiments, instead of antibodies and/or aptamers, one may utilize one or more fusion proteins as chromatin protein-binding agents. An example of a fusion protein is the H2B-GFP fusion protein, which targets the H2B histone (as described by Kanda et al., 1998; Curr Biol. 1998 Mar 26;8(7):377-85, which is incorporated by reference).

[0118] In some embodiments, the chromatin protein-binding agent is marked such that it (and anything to which it is bound) may be removed, such as cell-free DNA that is bound to the chromatin, including a protein of the chromatin. For example, magnetized beads may be utilized; streptavidin/biotin, FITC/anti-FITC, a secondary antibody in embodiments wherein the chromatin protein-binding agent is an antibody, and so forth.

C. Removing Linear Cell-free DNA

[0119] In some embodiments, one can reduce the amount of linear cell-free DNA in a collection of cell-free DNA to reduce host cell-free DNA that is often linear in nature. In some embodiments, this allows enrichment of cell-free DNA that is not linear, such as that is circular. In some embodiments, the circular cell-free DNA is from the microbiome, such as from bacteria and some viruses and from natural plasmids of filamentous fungi or extrachromosomal DNA as in prokaryotes, for example.

[0120] In some embodiments, reducing the amount of linear cell-free DNA comprises subjecting cell-free DNA in the collection to one or more end-modifications. The end modified cell-free DNA may be (i) configured for separation from the remaining cell-free DNA or (ii) incapable of coupling with adapters so that they are not sequenced in next-generation sequencing based analysis. In some embodiments, the end modification configured for separation comprises labeling the 3' end of cell-free DNA with a biotin-deoxynucleotide (dNTP) moiety, followed by streptavidin beads binding and magnetic separation. In some embodiments, the end modification to block the adapter ligation with cell-free DNA comprises labeling 3' end of cell-free DNA with a dideoxynucleotide (ddNTP) moiety or dephosphorylating the 5' end.

[0121] In some embodiments, reducing the amount of linear cell-free DNA comprises subjecting cell-free DNA in the collection to one or more enzymes that digest linear single stranded DNA and/or that digest linear double stranded DNA. In some embodiments, the digestion occurs from the ends to effect successive removal of base pairs from the open ends, as opposed to restriction enzymes. In some embodiments, the one or more enzymes comprise exonuclease I, exonuclease VII, DNAase, exonuclease V(RecBCD), or a combination thereof. D. Further Modification and/or Analyzing or Processing

[0122] Following enrichment of the microbiome cell-free DNA with respect to the host cell-free DNA, the resulting enriched microbiome cell-free DNA may be further manipulated or processed or analyzed in any manner. For example, the enriched microbiome cell-free DNA may be further modified, it may be analyzed, it may be stored for future analysis, it may be amplified, it may be sequenced, or a combination thereof. Further modifications may include modifying the enriched microbiome cell-free DNA to allow the molecules to be subjected to a high-throughput analysis of any kind, such as tagging the enriched microbiome cell-free DNA with a label or adaptor to be used then or later for the high-throughput analysis.

[0123] The present disclosure generally relates to methods of enriching cell-free microbiome deoxyribonucleic (DNA) molecules from cell-free DNA molecules by the depletion or digestion of host-derived cell free DNA molecules. In an aspect, the present disclosure provides methods for processing or analyzing a plurality of DNA molecules of a subject, comprising: (a) subjecting the plurality of cell-free DNA to enrichment to permit the host- derived cell-free DNA molecules to be (i) separated from a remainder of the microbiome cell- free DNA molecules and/or (ii) digested in the plurality of cell-free DNA molecules containing both human-derived and cell-free microbiome DNA; coupling the adapters to ends of the plurality of DNA molecules to provide a plurality of tagged DNA molecules; (b) subjecting the plurality of tagged DNA molecules or derivatives thereof to nucleic acid sequencing to yield a plurality of sequence reads; and (c) processing the plurality of sequence reads to obtain a microbiome composition, a diagnosis, to inform the risk of infectious diseases or other health conditions, or to determine or monitor therapeutic treatments.

[0124] In some embodiments, one may sequence all or part of the enriched microbiome cell-free DNA, and the sequencing may of any kind, including High-Throughput Sequencing (HTS) techniques or next-generation Sequencing (NGS). Part or all of the resulting sequences may provide information of the profile of part or all of the microbiome from which the cell-free DNA was obtained.

[0125] The present disclosure provides cost-effective methods for sequencing cell-free DNA to obtain the microbiome profiles. The methods may utilize feature differences between microbiome cell-free DNA and human-derived cell-free DNA. For example, human-derived cell-free DNA can be either depleted or digested from cell-free DNA based on their distinct features such as a characteristic size distribution, histone wrapping property, and linear single- or double-stranded fragments with opened ends. Thus, sequencing and profiling of remaining microbiome-derived cell-free DNA in cell-free DNA may be achieved efficiently and with reduced cost.

[0126] In some embodiments, cell-free microbiome DNA-enriched cell-free DNA with the methods provided herein or other enrichment methods can be directly used for next- generation sequencing, or may be further enriched with targeted probes before next-generation sequencing, or as a template for polymerase chain reaction (PCR)-based analysis, as examples. However, the analysis of the trace amounts of cell-free microbiome DNA (typically less than one nanogram per milliliter of plasma) may be challengingln light of these challenges, the present disclosure provides methods for performing analysis of trace amounts of cfmDNA that are enriched with the methods provided herein. In some embodiments, the method comprises: a) [0127] amplifying cell-free microbiome DNA-enriched cell-free DNA with one or more whole-genome amplification(WGA) methods to produce one or more WGA products; and b) [0128] subjecting the WGA product to next-generation sequencing analysis.

[0129] In another aspect, a method of the present disclosure further comprises processing the information of the microbiome profile to obtain or determine microbiome therapeutic composition and/or to determine a diagnosis and/or to inform the risk of infectious diseases or other health conditions, including to determine or monitor therapeutic treatments, for example.

[0130] In some embodiments, the present disclosure provides methods for treating an individual for a medical condition associated with a microbiome of the individual. The medical condition may be a microbial infection, including bacterial infection, viral infection, fungal infection, and/or protozoan infection; diarrhea, including antibiotic-associated diarrhea;

vaginosis; acne; asthma/allergies; autism; one or more autoimmune diseases; cancer; dental cavities; depression and anxiety; diabetes; eczema; gastric ulcers; hardening of the arteries; inflammatory bowel diseases; malnutrition; and obesity. Treatment of the medical condition may occur directly as a result of the analysis of the microbiome using methods of the present disclosure. [0131] In some embodiments, the method produces a collection of enriched microbiome cell-free DNA for analysis of methylation status and/or profiles, for example in the process of diagnosis of cancer. In some embodiments, the methods utilize the produced cell-free DNA to allow for focused enrichment of fragments having two or more enzyme digestion sites and containing at least one CpG site, following which methylation analysis may occur by any suitable method.

[0132] In an aspect, once analysis of the enriched microbial cell-free DNA is performed, or as part of the analysis, the method comprises comparing the level of one or more microbes from the microbiome from a normal subject to a level of one or more microbes from the microbiome of a potential patient. Here, the method may be used to determine the level of the microbe(s) using cell-free DNA samples from both the normal subject and a potential patient.

[0133] In an aspect, once analysis of the enriched microbial cell-free DNA is performed, or as part of the analysis, one may compare the level of one or more microbes from the microbiome from a patient known to have a medical condition related to the microbiome to that of the same one or more microbes from the microbiome from a potential patient. In another aspect, the present disclosure provides methods for comparing the level of one or more microbes in the microbiome from a patient with a known disease or condition to that of the same biological composition from a potential patient Here, the method may be used to determine the level of the one or more microbes from the microbiome using cfDNA samples from both the normal subject and a potential patient.

[0134] In some embodiments, the present disclosure provides methods of diagnosing a patient based on determining whether the patient has a methylation profile indicative of a medical condition. In some embodiments, the method comprises generating a methylation profile that indicates whether the patient has a pathogenic infection, cancer, or another disease or condition, and if so, from what organ. In some embodiments, this is performed using a biological sample from the patient that comprises cell free DNA.

[0135] Some methods may further involve performing one or more additional operations to obtain information about a medical condition associated with a microbiome. Some examples of operations include biopsy, CAT or CT scan, ultrasound, mammogram, culture of microbe(s), or otherwise evaluating tissue and/or fluids from an individual suspected of having or known to have a medical condition associated with a microbiome. In some embodiments, one or more microbes in a microbiome are identified as confirmation of outcomes of methods encompassed provided herein.

[0136] In some embodiments, the sample is obtained or derived from an individual suspected of having a microbiome-related medical condition or known to have one or at risk for having one. The sample may comprise diseased tissue; cancer tissue; tissue from a specific organ or tissue, such as gastrointestinal tract, reproductive tract, skin, mouth, rectum, liver tissue, lung tissue, kidney tissue, colon tissue, T-cells, B-cells, neutrophils, small intestines tissue, pancreas tissue, adrenal glands tissue, esophagus tissue, adipose tissue, heart tissue, brain tissue, placenta tissue, and combinations thereof. Methods, systems, kits, and compositions provided herein can be applied to any information obtained from methods of the disclosure, including information related to a microbiome-related medical condition, and including a condition in which a difference exists in the cell-free microbiome DNA from affected versus unaffected individuals or individuals at a different stage of the disease or condition or having a different prognosis. For example, one can identify the abnormal profiles of cell-free DNAs from any microbiome to diagnose disease and/or to determine a therapy to be used and/or to monitor success of a therapy. Therefore, in some embodiments, the methods, systems, kits, and compositions provided herein comprise obtaining or generating a microbiome profile of cell free DNA from biological samples with the disease related to the microbiome are included. In other embodiments, obtaining or generating a microbiome profile of cell free DNA from biological samples without a disease or considered disease-free are included.

[0137] Turning to the figures, in system 100 of FIG. 1, an example is provided of a flow chart of cell-free DNA preparation followed by examples of subsequent modification and analysis operations. In this example, cell-free DNA is obtained (as in operation 110), and this operation may or may not be performed by the individual that performs one or more subsequent operations. The cell-free DNA may be obtained from a host, such as a human, by standard approaches of extraction, including biopsy, swabbing, scraping, with a needle, by scalpel, by tube, by catheter, by scoopula, by syringe, and so forth. Following obtaining of the cell-free DNA 110, host-derived (in this example, human-derived) cell-free DNA is removed (as in operation 120), such as with methods provided herein. Following this preparation of cell-free DNA, a sequencing library is prepared with the cell-free DNA (as in operation 130). Once the sequence library is prepared as in operation 130, profiling of the cell-free DNA may occur, including using genomic and epigenomic profiling (as in operation 140); in this example, the microbiome is profiled using genomic and epigenomic approaches.

[0138] FIG. 2 illustrates an example of enriching cell-free DNA (cfDNA) for particular cfDNA, such as from a microbiome. In method 200, a collection of cfDNA 201 is used as a material that is known to have or at least suspected of having cfDNA from a micriobiome, and the cfDNA from a microbiome is desired to be enriched in a collection of cfDNA, such as upon reducing the amount of host cfDNA in the collection. The collection of cfDNA 201 comprises different types of DNA, including different sizes of linear DNA, including linear double stranded DNA, and circular DNA. In some embodiments, the circular DNA may be from a microbiome. In some aspects, linear DNA, including linear double stranded DNA, is from a host, although certain sizes may be from a microbiome. The collection of cfDNA 201 is subjected to size selection (as in operation 202), as in methods of the disclosure, and in specific embodiments the size selection occurs using beads or by gel electrophoresis. The size selection operation 202 allows for removal of cfDNA of certain sizes that are generally from host cfDNA. Following the size selection operation 202, a collection of cfDNA enriched for microbiome cfDNA 203 is produced. The collection of cfDNA enriched for microbiome cfDNA 203 is enriched for microbiome cfDNA with respect to host cfDNA compared to the absence of the size selection operation. The collection of cfDNA enriched for microbiome cfDNA 203 is further modified for subsequent analysis. Although the modification may be of any kind to facilitate analysis of the enriched microbiome cfDNA, in specific embodiments, the collection of cfDNA enriched for microbiome cfDNA 203 is subjected to sequencing library preparation (as in operation 204). The sequencing library preparation operation 204 may comprise any type of sequencing library preparation, although in specific embodiments the sequencing library preparation comprises generating linear molecules from circular microbiome cfDNA and modifying the linear ends of the microbiome cfDNA to comprise adapter sequences, thereby producing adapter-ligated microbiome cfDNA 205, for example. Once the adapter-ligated microbiome cfDNA 205 is produced, the adapter-ligated microbiome cfDNA may be subjected to genomic and epigenomic profiling 206.

[0139] FIG. 3 illustrates an example of enrichment for microbiome cfDNA utilizing targeting of nucleic acid bound to chromatin proteins 300. In some embodiments, this approach exploits the in vivo nature of host (for example, human) cfDNA in which the host cfDNA is bound or associated with one or more types of proteins. In an aspect, the proteins are histones. As depicted by example in FIG. 3, a collection of cfDNA 301 includes host cfDNA that is associated with histones, in addition to cfDNA that is circular in nature and linear DNA un associated with histones. In some aspects the circular DNA may be from a microbiome. In some aspects, linear DNA, including linear double stranded DNA, is from a host, although certain sizes may be from a microbiome. To extract protein-bound host cfDNA, one targets the protein with an agent that binds the protein, and the agent may have the ability to be extracted along with the nucleic acid to which it is bound; the agent may or may not be labeled. In some embodiments, operation 302 illustrates one example of antibody binding to histones to which host cfDNA is bound, all among a collection of cfDNA comprising circular and linear cfDNA that is not bound to protein(s) 303. Element 303 illustrates antibodies that comprise a moiety on the antibody that is directly or indirectly able to be utilized for removal of protein-bound cfDNA. In some embodiments, one or more moieties on the antibody are recognizable by magnetic beads that are then removed by magnetic force (as in operation 304). Removal of the antibody-targeted protein-bound host cfDNA in operation 304 produces an enriched collection of microbiome cfDNA 305. Following this, the enriched collection of microbiome cfDNA 305 may be subjected to sequencing library preparation operation 306 by any suitable approach. In a specific embodiment, circular microbiome cfDNA is linearized and the microbiome cfDNA is then subjected to adapter ligation (as in operation 307) to produce adapter-ligated microbiome cfDNA that facilitates subsequent analysis. The adapter-ligated microbiome cfDNA is then subjected to genomic and epigenomic profiling (as in operation 308).

[0140] FIG. 4 illustrates an example in which linear DNA is removed from a collection of cfDNA based on the nature of being the cfDNA being linear. For example, a collection 401 of cfDNA comprising linear DNA, both single stranded and double stranded, and circular DNA is subjected to biotin-dNTP labeling 402 to produce a collection 403 of cfDNA that comprises circular DNA and biotin end labeled double- stranded and single-stranded linear DNA. In operation 404 the biotin end labeled double-stranded and single- stranded linear DNA is removed, thereby leaving a collection of circular microbiome cfDNA 405. Following this, the circular microbiome cfDNA 405 (as in operation 406) is subjected to sequencing library preparation, for example by being linearized and subjected to adapter ligation to produce adapter-ligated microbiome cfDNA 407 that facilitates subsequent analysis. The adapter- ligated microbiome cfDNA 407 is then subjected to genomic and epigenomic profiling (as in operation 408). [0141] FIG. 5 illustrates an example of enriching for microbiome cfDNA by exploiting the linear nature of host cfDNA. In method 500, a collection 501 of cfDNA comprising linear DNA, both single-stranded and double-stranded, and circular DNA are subjected to one or more nucleases (as in operation 502) that digest linear double- stranded and/or single-stranded DNA, essentially depleting the collection of linear DNA, thereby leaving a collection of circular microbiome cfDNA 503. The collection of circular microbiome cfDNA 503 (as in operation 504) is subjected to sequencing library preparation, for example by linearizing the circular DNA and adding adapters to the free ends, thereby producing a collection of adapter-ligated microbiome cfDNA 505 that facilitates subsequent analysis. The adapter-ligated microbiome cfDNA 505 is then subject to genomic and epigenomic profiling (as in operation 506).

III. [0142] Kits

[0143] Any of the compositions described herein may be included in a kit. In a non limiting example, cfDNA; one or more apparatuses for collection of cfDNA; enzymes;

antibodies; adapters; primers; adapters; deoxynucleoside triphosphates (dNTPs); one or more end-modification agents; beads; buffers, and other chemicals, including adenosine triphosphate (ATP), dioxythreitol (DTT), and so forth may be included in a kit.

[0144] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container of the kits may generally include at least one vial, test tube, flask, bottle, or other container, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also may generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present disclosure also may include a container for containing component(s) in close confinement for commercial sale. Such containers may include blow -molded plastic containers into which the desired vials are retained.

Kits of the present disclosure may include instructions for performing methods provided herein, such as methods for enrichment of cell-free microbiome DNA and methods for subjecting the enriched cell-free microbiome for further analysis ( e.g ., PCR, nucleic acid array, next- generation sequencing). Such instructions may be in physical form (e.g., printed instructions) or electronic form. Kits of the present disclosure may include a software package or a web link to a server or cloud-computing platform for analyzing the sequencing data generated from sequencing library prepared with the kit. The analysis may provide information about the microbiome composition of the host.

[0145] Kits of the present disclosure may include a report generated by a software package provided with the kit, or by a server or cloud-computing platform. The report may provide information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome. For example, the report may provide the load of viruses of the Anelloviridae family and predict the likelihood of organ transplant rejection.

IV. [0146] Computer systems

[0147] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 6 shows a computer system 601 that is programmed or otherwise configured to, for example, obtain a plurality of sequencing reads; sequence a plurality of cell-free nucleic acids; assay a collection of cell-free DNA to determine the presence or absence of microbiome cell-free DNA; and analyze enriched microbiome cell-free DNA, such as to provide information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof; and perform genomic or epigenomic profiling of enriched microbiome cell-free DNA.

[0148] The computer system 601 can regulate various aspects of analysis, calculation, and generation of the present disclosure, such as, for example, obtaining a plurality of sequencing reads; sequencing a plurality of cell-free nucleic acids; assaying a collection of cell- free DNA to determine the presence or absence of microbiome cell-free DNA; analyzing enriched microbiome cell-free DNA, such as to provide information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof; and performing genomic or epigenomic profiling of enriched microbiome cell-free DNA. The computer system 601 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device. [0149] The computer system 601 includes a central processing unit (CPU, also “processor” and“computer processor” herein) 605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 601 also includes memory or memory location 610 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 615 (e.g., hard disk), communication interface 620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 625, such as cache, other memory, data storage and/or electronic display adapters. The memory 610, storage unit 615, interface 620 and peripheral devices 625 are in communication with the CPU 605 through a communication bus (solid lines), such as a motherboard. The storage unit 615 can be a data storage unit (or data repository) for storing data. The computer system 601 can be operatively coupled to a computer network (“network”) 630 with the aid of the

communication interface 620. The network 630 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.

[0150] The network 630 in some cases is a telecommunication and/or data network. The network 630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. For example, one or more computer servers may enable cloud computing over the network 630 (“the cloud”) to perform various aspects of analysis, calculation, and generation of the present disclosure, such as, for example, obtaining a plurality of sequencing reads; sequencing a plurality of cell-free nucleic acids; assaying a collection of cell-free DNA to determine the presence or absence of microbiome cell-free DNA; analyzing enriched microbiome cell-free DNA, such as to provide information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof; and performing genomic or epigenomic profiling of enriched microbiome cell-free DNA. Such cloud computing may be provided by cloud computing platforms such as, for example, Amazon Web Services (AWS), Microsoft Azure, Google Cloud Platform, and IBM cloud. The network 630, in some cases with the aid of the computer system 601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 601 to behave as a client or a server.

[0151] The CPU 605 may comprise one or more computer processors and/or one or more graphics processing units (GPUs). The CPU 605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 610. The instructions can be directed to the CPU 605, which can subsequently program or otherwise configure the CPU 605 to implement methods of the present disclosure. Examples of operations performed by the CPU 605 can include fetch, decode, execute, and writeback.

[0152] The CPU 605 can be part of a circuit, such as an integrated circuit. One or more other components of the system 601 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0153] The storage unit 615 can store files, such as drivers, libraries and saved programs. The storage unit 615 can store user data, e.g., user preferences and user programs. The computer system 601 in some cases can include one or more additional data storage units that are external to the computer system 601, such as located on a remote server that is in communication with the computer system 601 through an intranet or the Internet.

[0154] The computer system 601 can communicate with one or more remote computer systems through the network 630. For instance, the computer system 601 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 601 via the network 630.

[0155] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 601, such as, for example, on the memory 610 or electronic storage unit 615. The machine-executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 605. In some cases, the code can be retrieved from the storage unit 615 and stored on the memory 610 for ready access by the processor 605.

In some situations, the electronic storage unit 615 can be precluded, and machine-executable instructions are stored on memory 610.

[0156] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre compiled or as-compiled fashion. [0157] Aspects of the systems and methods provided herein, such as the computer system 601, can be embodied in programming. Various aspects of the technology may be thought of as “products” or“articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.

“Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine“readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0158] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0159] The computer system 601 can include or be in communication with an electronic display 635 that comprises a user interface (UI) 640 for providing, for example, a visual display of data indicative of sequencing reads; sequencing data; information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof; and genomic or epigenomic profiles of enriched microbiome cell-free DNA. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0160] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 605. The algorithm can, for example, obtain a plurality of sequencing reads; sequence a plurality of cell-free nucleic acids; assay a collection of cell-free DNA to determine the presence or absence of microbiome cell-free DNA; analyze enriched microbiome cell-free DNA, such as to provide information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof; and perform genomic or epigenomic profiling of enriched microbiome cell- free DNA.

EXAMPLES

[0161] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

EFFICIENT SEQUENCING OF CELL-FREE MICROBIOME DNA

[0162] FIG. 1 illustrates a flowchart of performing genomic and epigenomic microbiome profiling of cell-free DNA (cfDNA). A plurality of cell-free DNA (cfDNA) molecules may be obtained from a subject, as an example a human. Next, human-derived cfDNA may be removed or digested or derived from the cfDNA. Next, libraries may be prepared from the cfDNA enriched for cell-free microbiome DNA (cfmDNA). Next, genomic and epigenomic profiles of cfmDNA-enriched cfDNA may be generatedusing the prepared libraries.

[0163] FIG. 2 illustrates an example of performing microbiome profiling of cfDNA with size selection. The sizes of human cfDNA fragments, but not the microbiome DNA fragments, may be peaked around 165 bp. DNA fragments with sizes around 165 bp, for example, sizes between 145 bp and 185 bp, may be removed by bead-based, gel-based, or other size selection strategies. Next, libraries may be prepared from the cfDNA enriched for cell-free microbiome DNA (cfmDNA). Next, genomic and epigenomic profiles of cfmDNA-enriched cfDNA may be performed using the prepared libraries.

[0164] FIG. 3 illustrates an example of performing microbiome profiling of cfDNA with antibody binding followed by removal by streptavidin magnetic beads. Anti-histone antibodies may bind to histones wrapped with human DNA. Next, the antibodies may be depleted using magnetic removal (e.g., by streptavidin magnetic beads or protein A or protein G conjugated magnetic beads). Next, libraries may be prepared from the cfDNA enriched for cell-free microbiome DNA (cfmDNA). Next, genomic and epigenomic profiles of cfmDNA-enriched cfDNA may be performed using the prepared libraries.

[0165] FIG. 4 illustrates an example of performing microbiome profiling of cfDNA with end-labeling followed by removal by streptavidin magnetic beads. The linear cfDNA may be subjected to modification of one or both ends, for example, labeled with biotinylated dNTP.

Next, the fragments that have been end-modified can be removed using magnetic removal (e.g., by streptavidin magnetic beads). Next, the remaining cfDNA can be subjected to sequencing library preparation. Next, genomic and epigenomic profiles of cfmDNA-enriched cfDNA may be performed using the prepared libraries.

[0166] FIG. 5 illustrates an example of performing microbiome profiling of cfDNA with digestion of linear DNA by nucleases. Nucleases (e.g., exonuclease I, exonuclease VII, ATP- dependent DNase, or exonuclease V(RecBCD)) may digest either single-strand DNA or double strand DNA or both. Next, the remaining cfDNA enriched for circular cfDNA from microbiome cfDNA can be subject to sequencing library preparation. Next, genomic and epigenomic profiles of cfmDNA-enriched cfDNA may be performed using the prepared libraries.

[0167] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims

CLAIMS What is claimed is:

1. A method for preparing cell-free microbiome deoxyribonucleic acid (DNA), comprising: optionally obtaining a collection of cell-free DNA comprising microbiome cell-free

DNA and host cell-free DNA; subjecting the collection of cell-free DNA to size selection; subjecting the collection of cell-free DNA to one or more agents that bind one or more

chromatin proteins; and reducing the amount of linear cell-free DNA in the collection of cell-free DNAto enrich

microbiome cell-free DNA in the collection, thereby producing an enriched microbiome cell-free DNA.

2. The method of claim 1, further comprising assaying the collection of cell-free DNA to determine the presence or absence of microbiome cell-free DNA.

3. The method of claim 1 or 2, wherein subjecting the cell-free DNA to size selection comprises reducing the amount of cell-free DNA that have a size in the range between 100 base pairs (bp) and 200bp, 130bp and 170bp, 140bp and 170bp, 150bp and 170bp, 160bp and 170bp, 130bp and 160bp, 140bp and 160bp, 150bp and 160bp 140bp and 150bp, or 150bp and 160bp, lOObp and 175bp, lOObp and 150bp, lOObp and 125bp, 125bp and 200bp, about 125bp and 175bp, 125bp and 150bp, 150bp and 200bp, or 175bp and 200bp.

4. The method of any one of claims 1- 3, wherein subjecting the cell-free DNA to size selection comprises subjecting the collection of cell-free DNA to separation by beads or by electrophoresis.

5. The method of any one of claims 1-4, wherein the one or more agents that bind histones comprise one or more antibodies, one or more aptamers, one or more fusion proteins, or mixture thereof.

6. The method of any one of claims 1-5, wherein reducing the amount of linear cell-free DNA comprises: subjecting the collection of cell-free DNA to one or more modifications to linear ends of the cell-free DNA to produce modified cell-free linear DNA that are (i) configured for separation from a remainder of the cell-free DNA or (ii) incapable of coupling with adapters; and removing the modified cell-free linear DNA that are configured for separation from a

remainder of the collection of cell-free DNA with an agent that binds the modified cell-free linear DNA.

7. The method of claim 6, wherein the one or more modifications comprise subjecting a 3' end of the linear cell-free DNA to conditions sufficient to modify the 3' end with a biotin- deoxynucleotide (dNTP), a dideoxynucleotide (ddNTP) moiety, or a functional analog thereof, or subjecting a 5' end of the linear cell-free DNA to conditions sufficient to dephosphorylate the 5' end.

8. The method of any one of claims 1-7, wherein reducing the amount of linear cell-free DNA comprises subjecting the collection of cell-free DNA to one or more enzymes that digest linear single stranded DNA and/or linear double-stranded DNA.

9. The method of claim 8, wherein the one or more enzymes comprise exonuclease I, exonuclease VII, DNAase, exonuclease V(RecBCD), or a combination thereof.

10. The method of any one of claims 1-9, wherein the microbiome cell-free DNA comprises DNA from one or more bacteria, one or more fungi, one or more viruses, one or more protozoa, or a combination thereof.

11. The method of any one of claims 1-10, wherein the host is a mammal.

12. The method of claim 11, wherein the mammal is a human.

13. The method of any one of claims 1-12, wherein the collection of cell-free DNA is obtained or derived from a biological sample.

14. The method of claim 13, wherein the biological sample is from an individual in need of treatment for a medical condition related to the microbiome or suspected of being in need of treatment for a medical condition related to the microbiome, from an individual in need of evaluation of a response to one or more different diet conditions, or from an immunosuppressed transplant recipient.

15. The method of claim 13 or 14, wherein the biological sample is from the skin, mouth, nose, gastrointestinal tract, and/or genitourinary tract of the host.

16. The method of any one of claims 13-15, wherein the biological sample comprises tissue, blood, plasma, urine, fecal matter, saliva, mucus, nipple aspirate, amniotic fluid, cystic fluid, spinal or brain fluid, sweat, tears, or a combination thereof.

17. The method of any one of claims 13-16, further comprising obtaining the biological sample from the host.

18. The method of any one of claims 1-17, wherein the enriched microbiome cell-free DNA is digested to produce linear molecules of microbiome cell-free DNA.

19. The method of any one of claims 1-18, wherein the enriched microbiome cell-free DNA is further subjected to amplification, modification, analysis, or a combination thereof.

20. The method of claim 19, wherein the amplification, modification, analysis, or a combination thereof comprises sequencing library preparation.

21. The method of any one of claims 1-19, wherein the linear molecules of microbiome cell- free DNA are subjected to polymerase chain reaction (PCR) or nucleic array analysis.

22. The method of any one of claims 1-19, wherein the linear molecules of microbiome cell- free DNA are adapter-ligated.

23. The method of claim 22, wherein adapter-ligated linear molecules of the microbiome cell- free DNA are subjected to sequencing.

24. The method of claim 23, wherein the sequencing comprises next generation sequencing.

25. The method of claim 23 or 24, wherein sequencing the adapter- ligated linear molecules of microbiome cell-free DNA provides information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof.

26. The method of any one of claims 1-25, wherein the enriched microbiome cell-free DNA is analyzed for methylation profiling.

27. The method of any one of claims 18-26, wherein at least one therapy or prophylactic for the host is provided upon analysis of the enriched microbiome cell-free DNA.

28. The method of any one of claims 18-27, further comprising performing genomic or epigenomic profiling of the enriched microbiome cell-free DNA.

29. A method for preparing cell-free DNA, comprising: optionally obtaining a collection of cell-free DNA comprising host cell-free DNA; subjecting the collection of cell-free DNA to size selection; subjecting the collection of cell-free DNA to one or more agents that bind histones; and reducing an amount of linear cell-free DNA in the collection of cell-free DNA to enrich

microbiome cell-free DNA in the collection when present in the collection, thereby producing an enriched microbiome cell-free DNA.

30. The method of claim 29, further comprising assaying the collection of cell-free DNA to determine whether or not microbiome cell-free DNA is present in the collection.

31. The method of claim 30, wherein the assaying is performed before and/or after the subjecting.

32. The method of claim 31, wherein the collection of cell-free DNA is determined to have microbiome cell-free DNA.

33. The method of claim 31 , wherein the collection of cell-free DNA is determined not to have microbiome cell-free DNA.

34. The method of any one of claims 29-33, wherein the microbiome cell-free DNA is from one or more bacteria, one or more fungi, one or more viruses, one or more protozoa, or a combination thereof.

35. The method of any one of claims 29-34, wherein the host is a mammal.

36. The method of claim 35, wherein the mammal is a human.

37. The method of any one of claims 29-36, wherein the collection of cell-free DNA is obtained or derived from a biological sample.

38. The method of claim 37, wherein the biological sample is from an individual in need of treatment for a medical condition related to the microbiome or suspected of being in need of treatment for a medical condition related to the microbiome, from an individual in need of evaluation of the response to one or more different diet conditions, or from an immunosuppressed transplant recipient.

39. The method of claim 37 or 38, wherein the biological sample is from the skin, mouth, nose, gastrointestinal tract, and/or genitourinary tract of the host.

40. The method of any one of claims 37-39, wherein the sample comprises tissue, blood, plasma, urine, fecal matter, saliva, mucus, nipple aspirate, amniotic fluid, cystic fluid, spinal or brain fluid, sweat, tears, or a combination thereof.

41. The method of any one of claims 37-40, further comprising obtaining the biological sample from the host.

42. The method of claim 37-41, wherein the enriched microbiome cell-free DNA is digested to produce linear molecules of microbiome cell-free DNA.

43. The method of any one of claims 29-42, wherein the enriched microbiome cell-free DNA is further subjected to amplification, modification, analysis or a combination thereof.

44. The method of claim 43, wherein the amplification, modification, analysis or a combination thereof comprises sequencing library preparation.

45. The method of any one of claims 29-44, wherein the linear molecules of microbiome cell- free DNA are subjected to PCR or nucleic array analysis.

46. The method of any one of claims 29-44, wherein the linear molecules of microbiome cell- free DNA are adapter-ligated.

47. The method of claim 46, wherein adapter- ligated linear molecules of microbiome cell-free DNA are subjected to sequencing.

48. The method of claim 47, wherein the sequencing is next generation sequencing.

49. The method of claim 47 or 48, wherein sequencing of the adapter-ligated linear molecules of microbiome cell-free DNA comprises providing information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof.

50. The method of any one of claims 42-49, wherein the enriched microbiome cell-free DNA is analyzed for methylation profiling.

51. The method of any one of claims 29-50, wherein at least one therapy or prophylactic for the host is provided upon analysis of the enriched microbiome cell-free DNA.

52. The method of any one of claims 29-51, further comprising performing genomic or epigenomic profiling of the enriched microbiome cell-free DNA.

53. A method for preparing cell-free microbiome DNA, comprising optionally obtaining a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA, and subjecting the collection of cell-free DNA comprising microbiome cell-free DNA and host cell- free DNA to size selection to enrich microbiome cell-free DNA in the collection of cell-free DNA to produce enriched microbiome cell-free DNA.

54. The method of claim 53, wherein subjecting the collection to size selection comprises reducing the amount of cell-free DNA in the collection that are sized in a range between 100 base pairs (bp) and 200bp, l lObp and 190bp, 120bp and 180bp, 130bp and 170bp, 140bp and 170bp, 150bp and 170bp, 160bp and 170bp, 130bp and 160bp, 140bp and 160bp, 150bp and 160bp 140bp and 150bp, or 150bp and 160bp.

55. The method of claim 53 or 54, wherein subjecting the cell-free DNA to size selection comprises subjecting the collection of cell-free DNA to separation by beads or by electrophoresis.

56. The method of any one of claims 53-55, wherein the microbiome cell-free DNA in the collection of cell-free DNA is from one or more bacteria, one or more fungi, one or more viruses, one or more protozoa, or a combination thereof.

57. The method of any one of claims 53-56, wherein the host is a mammal.

58. The method of claim 57, wherein the mammal is a human.

59. The method of any one of claims 53-58, wherein the collection of cell-free DNA is from a biological sample.

60. The method of claim 59, wherein the biological sample is from an individual in need of treatment for a medical condition related to the microbiome or suspected of being in need of treatment for a medical condition related to the microbiome, from an individual in need of evaluation of the response to different diet conditions, or from an immunosuppressed transplant recipient.

61. The method of claim 59 or 60, wherein the biological sample is from the skin, mouth, nose, gastrointestinal tract, and/or genitourinary tract of the host.

62. The method of any one of claims 59-61, wherein the biological sample comprises tissue, blood, plasma, urine, fecal matter, saliva, mucus, nipple aspirate, amniotic fluid, cystic fluid, spinal or brain fluid, sweat, tears, or a combination thereof.

63. The method of any one of claims 59-62, further comprising obtaining the biological sample from the host.

64. The method of any one of claims 59-63, wherein the enriched microbiome cell-free DNA is digested to produce linear molecules of microbiome cell-free DNA.

65. The method of any one of claims 53-64, wherein the enriched microbiome cell-free DNA is further subjected to amplification, modification, analysis or a combination thereof.

66. The method of claim 65, wherein the amplification, modification, analysis, or a combination thereof comprises sequencing library preparation.

67. The method of any one of claims 53-66, wherein the linear molecules of microbiome cell- free DNA are subjected to PCR or nucleic array analysis.

68. The method of any one of claims 53-66, wherein the linear molecules of microbiome cell- free DNA are adapter-ligated.

69. The method of claim 68, wherein adapter-ligated linear molecules of microbiome cell-free DNA are subjected to sequencing.

70. The method of claim 69, wherein the sequencing is next generation sequencing.

71. The method of claim 69 or 70, wherein sequencing of the adapter- ligated linear molecules of microbiome cell-free DNA comprises providing information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof.

72. The method of any one of claims 53-71, wherein the enriched microbiome cell-free DNA is analyzed for methylation profiling.

73. The method of any one of claims 53-72, wherein at least one therapy or prophylactic for the host is provided upon analysis of the enriched microbiome cell-free DNA.

74. The method of any one of claims 53-73, wherein the enriched microbiome cell-free DNA is subjected to genomic or epigenomic profiling.

75. A method of preparing cell-free microbiome DNA, comprising subjecting a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA to one or more agents that bind one or more chromatin proteins, thereby enriching microbiome cell-free DNA in the collection of cell-free DNA.

76. The method of claim 75, wherein the chromatin protein is one or more histones.

77. The method of claim 75 or 76, wherein the one or more agents that bind chromatin protein comprise an antibody, aptamer, fusion protein, or mixture thereof.

78. The method of any one of claims 75-77, wherein enriching the microbiome cell-free DNA comprises using beads to remove antibody-binding protein.

79. The method of any one of claims 75-78, wherein the microbiome cell-free DNA is from one or more bacteria, one or more fungi, one or more viruses, one or more protozoa, or a combination thereof.

80. The method of any one of claims 75-79, wherein the host is a mammal.

81. The method of claim 80, wherein the mammal is a human.

82. The method of any one of claims 75-81, wherein the collection of cell-free DNA is from a biological sample.

83. The method of claim 82, wherein the biological sample is from an individual in need of treatment for a medical condition related to the microbiome or suspected of being in need of treatment for a medical condition related to the microbiome, from an individual in need of evaluation of the response to different diet conditions, or from an immunosuppressed transplant recipient.

84. The method of claim 82 or 83, wherein the biological sample is from the skin, mouth, nose, gastrointestinal tract, and/or genitourinary tract of the host.

85. The method of any one of claims 82-84, wherein the biological sample comprises tissue, blood, plasma, urine, fecal matter, saliva, mucus, nipple aspirate, amniotic fluid, cystic fluid, spinal or brain fluid, sweat, tears, or a combination thereof.

86. The method of any one of claims 82-85, further comprising obtaining the biological sample from the host.

87. The method of any one of claims 82-86, wherein the enriched microbiome cell-free DNA is digested to produce linear molecules of microbiome cell-free DNA.

88. The method of any one of claims 75-87, wherein the enriched microbiome cell-free DNA is further subjected to amplification, modification, analysis, or a combination thereof.

89. The method of claim 88, wherein the amplification, modification, analysis or a combination thereof comprises sequencing library preparation.

90. The method of any one of claims 75-89, wherein the linear molecules of microbiome cell- free DNA are subjected to PCR or nucleic array analysis.

91. The method of any one of claims 75-89, wherein the linear molecules of microbiome cell- free DNA are adapter-ligated.

92. The method of claim 91, wherein adapter- ligated linear molecules of microbiome cell-free DNA are subjected to sequencing.

93. The method of claim 92, wherein the sequencing is next- generation sequencing.

94. The method of claim 91 or 92, wherein sequencing of the adapter- ligated linear molecules of microbiome cell-free DNA provides information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof.

95. The method of any one of claims 75-94, wherein the enriched microbiome cell-free DNA is analyzed for methylation profiling.

96. The method of any one of claims 75-95, wherein at least one therapy or prophylactic for the host is provided upon analysis of the enriched microbiome cell-free DNA.

97. The method of any one of claims 75-96, wherein the enriched microbiome cell-free DNA is subject to genomic or epigenomic profiling.

98. A method of preparing cell-free microbiome DNA, comprising: obtaining a collection of cell-free DNA comprising microbiome cell-free DNA and host cell-free DNA, and reducing the amount of linear cell-free DNA in the collection of cell-free DNA, to enrich microbiome cell-free DNA in the collection of cell-free DNA, thereby producing enriched microbiome cell-free DNA.

99. The method of claim 98, wherein reducing the amount of linear cell-free DNA comprises: subjecting the collection of cell-free DNA to one or more modifications to linear ends

of the cell-free DNA to produce modified cell-free linear DNA that are (i) configured for separation from a remainder of cell-free DNA or (ii) incapable of coupling with adapters; and removing the labeled cell-free linear DNA configured for separation from the collection

with an agent that binds the modified cell-free linear DNA ends.

100. The method of claim 98 or 99, wherein reducing the amount of linear cell-free DNA comprises subjecting the collection of cell-free DNA to one or more enzymes that digest linear single-stranded DNA aor linear double-stranded DNA.

101. The method of claim 100, wherein the one or more enzymes comprise exonuclease I, exonuclease VII, DNAase, exonuclease V(RecBCD), or a combination thereof.

102. The method of any one of claims 98-101, wherein the microbiome cell-free DNA is from one or more bacteria, one or more fungi, one or more viruses, one or more protozoa, or a combination thereof.

103. The method of any one of claims 98-102, wherein the host is a mammal.

104. The method of claim 103, wherein the mammal is a human.

105. The method of any one of claims 98-103, wherein the collection of cell-free DNA is from a biological sample.

106. The method of claim 105, wherein the biological sample is from an individual in need of treatment for a medical condition related to the microbiome or suspected of being in need of treatment for a medical condition related to the microbiome, from an individual in need of evaluation of the response to different diet conditions, or from an immunosuppressed transplant recipient.

107. The method of claim 105 or 106, wherein the biological sample is from the skin, mouth, nose, gastrointestinal tract, and/or genitourinary tract of the host.

108. The method of any one of claims 105-107, wherein the biological sample comprises tissue, blood, plasma, urine, fecal matter, saliva, mucus, nipple aspirate, amniotic fluid, cystic fluid, spinal or brain fluid, sweat, tears, or a combination thereof.

109. The method of any one of claims 105-108, further comprising obtaining the biological sample from the host.

110. The method of claim any one of claims 105-109, wherein the enriched microbiome cell- free DNA is digested to produce linear molecules of microbiome cell-free DNA.

111. The method of any one of claims 105-110, wherein the enriched microbiome cell-free DNA from the collection is further subjected to amplification, modification, analysis or a combination thereof.

112. The method of claim 111, wherein the amplification, modification, analysis or a combination thereof comprises sequencing library preparation.

113. The method of claim 112, wherein the sequencing is next-generation sequencing.

114. The method of any one of claims 105-113, wherein the linear molecules of microbiome cell-free DNA are subjected to PCR or nucleic acid array analysis.

115. The method of any one of claims 105-113, wherein the linear molecules of microbiome cell-free DNA are adapter-ligated.

116. The method of claim 115, wherein adapter-ligated linear fragments of microbiome cell- free DNA are subject to sequencing.

117. The method of claim 116, wherein the sequencing is next-generation sequencing.

118. The method of claim 115 or 116, wherein sequencing of the adapter- ligated linear molecules of microbiome cell-free DNA provides information for (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof.

119. The method of any one of claims 98-118, wherein the enriched microbiome cell-free DNA is analyzed for methylation profiling.

120. The method of any one of claims 98-119, wherein at least one therapy or prophylactic for the host is provided upon analysis of the enriched microbiome cell-free DNA.

121. The method of any one of claims 98-120, wherein the enriched microbiome cell-free DNA is subjected to genomic or epigenomic profiling.

122. A method for processing or analyzing a plurality of cell-free deoxyribonucleic (cfDNA) molecules from a subject, comprising:

(a) subjecting a plurality of cfDNA molecules comprising both subject-derived cfDNA

and microbiome cell-free DNA (cfmDNA) to enrichment to permit subject-derived cfDNA molecules to be (i) separated from a remainder of said cfmDNA molecules or (ii) digested in the plurality of cfDNA molecules;

(b) coupling adapters to ends of the plurality of DNA molecules to provide a plurality

of tagged DNA molecules; (c) subjecting the plurality of tagged DNA molecules or tagged derivatives thereof to nucleic acid sequencing to yield a plurality of sequence reads; and

(d) processing the plurality of sequence reads to identify sequences from the

microbiome.

123. The method of claim 122, wherein the enrichment comprises removing cfDNA molecules of specific sizes to enrich the cfmDNA in the plurality of cfDNA molecules.

124. The method of claim 122 or 123, wherein the enrichment comprises:

(a) using protein binding reagents to bind to histone proteins to allow a plurality of

histone-associated cell-free DNA molecules to be separated from the remainder of the plurality of cfDNA molecules;

(b) removing the histone binding reagents to deplete histone-associated cell-free DNA

molecules from the plurality of cfDNA molecules.

125. The method of any one of claims 122-124, wherein the enrichment comprises:

(a) modifying one or both ends of each of at least a portion of the plurality of cf DNA

molecules to provide a plurality of modified cfDNA molecules having ends that are (i) configured for separation from a remainder of the plurality of cfDNA or (ii) incapable of coupling with adapters; and

(b) removing the modified DNA molecules from a remainder of the plurality of cfDNA

molecules.

126. The method of any one of claims 122-125, wherein the enrichment comprises digesting the plurality of linear cfDNA molecules using one or more nucleases.

127. The method of claim 123, wherein the specific sizes have lengths from 100 base pairs (bp) and 200bp, l lObp and 190bp, 120bp and 180bp, 130bp and 170bp, 140bp and 170bp, 150bp and 170bp, 160bp and 170bp, 130bp and 160bp, 140bp and 160bp, 150bp and 160bp about 140bp and 150bp, or 150bp and 160bp.

128. The method of claim 123, wherein the specific sizes have lengths larger than lOObp, 1 lObp, 120bp, 130bp, 140bp, 150bp, or 160bp.

129. The method of claim 123, wherein the removal of molecules of specific sizes is performed with Ampure® or Solid Phase Reversible Immobilization (SPRI) beads.

130. The method of claim 123, wherein the removal of molecules of specific sizes is performed with gel electrophoresis.

131. The method of claim 123, wherein the protein binding reagents comprise anti-histone antibodies, peptides, aptamers, fusion proteins, or a combination thereof.

132. The method of claim 124, wherein the protein binding reagents are biotinylated.

133. The method of claim 124, wherein the protein binding reagents are conjugated to magnetic beads.

134. The method of claim 124, wherein streptavidin beads or protein A or protein G or secondary antibody conjugated magnetic beads are used for removing the histone binding reagents.

135. The method of claim 124, wherein the histone protein binding and removal can be performed before the extraction of the plurality of cfDNA molecules.

136. The method of claim 124, wherein the modifying comprises subjecting a 3' end of each of said at least said portion of the plurality of cfDNA molecules to conditions sufficient to modify the 3' end with a dideoxynucleotide (ddNTP) moiety, or a functional analog thereof.

137. The method of claim 125, wherein the modifying comprises subjecting a 5' end of each of the at least the portion of said plurality of cfDNA molecules to conditions sufficient to dephosphorylate the 5' end.

138. The method of claim 125, wherein the modified cfDNA molecules are coupled to magnetic beads, and optionally wherein the modified cfDNA molecules are separated using magnetic separation.

139. The method of claim 125, wherein in (a), ends of the modified cfDNA molecules are incapable of undergoing ligation and/or primer extension.

140. The method of claim 126, wherein the nuclease comprises exonuclease I, exonuclease VII, ATP-dependent Dnase, exonuclease V(RecBCD) or a functional analog thereof or a combination thereof.

141. The method of claim 122, wherein identifying sequences from said microbiome comprises determining the presence and prevalence of microbial sequences.

142. The method of claim 141, wherein determining the presence and/or prevalence of microbial sequences comprises providing information about one or more of the following: (1) microbiome composition of the host; (2) diagnosis and/or prophylaxis of a medical condition; (3) therapy for a medical condition; (4) therapy monitoring; (5) optimal diet conditions; (6) prediction of transplant outcome; or (7) a combination thereof.