Split sample processing

Abstract

This disclosure relates to methods of clinical sample processing by simultaneous split sample processing that provides microbial enrichment, identification, and quantification from the same clinical sample.

Description

[0001] SPLIT SAMPLE PROCESSING

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 611,691, filed December 18, 2023, the content of which is herein incorporated by reference in its entirety.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with U.S. Government support under 1R43AI167193-01 awarded by the National Institutes of Health and under Agreement Number 75A50122C00028 awarded by the U.S. Department of Health and Human Services. The U.S. Government has certain rights in the invention.

[0006] BACKGROUND

[0007] Treatment of infectious disease relies on rapidly and accurately detecting, identifying, and characterizing one or more microbial species in a clinical sample. However, clinical samples are typically dominated by human DNA (huDNA). Human nuclei require harsh chemical conditions for lysis and release of genomic DNA (gDNA), a step needed for enzymatic depletion of huDNA. There is also variation in how well different microbial species survive this treatment, as well as in how different microbial species handle mitigation measures for augmenting the capture of as many microbial species as possible.

[0008] SUMMARY

[0009] Clinical samples are complex mixtures of subject (e.g., human) cells, cells from one or more microbial species (e.g., bacteria, fungi, or parasites), and / or viruses. Modern identification techniques (e.g., polymerase chain reaction or sequencing) rely on isolation of pathogen DNA from the human and microbial cells and / or viral particles in a clinical sample as an input. However, different cells often require different lysis conditions to ensure effective release of nucleic acids of interest and to prevent degradation of these nucleic acids of interest (e.g., DNA or RNA). Additionally, treatment of a sample with DNA amplification can bias microbial species abundance outputs. A split sample approach where one subsample is amplified and the other is not can be used to address this.

[0010] Currently available clinical sample processing protocols typically provide a single lysis condition, resulting in differences in lysis efficiency across the many cell types in a sample. These protocols also proceed with a single clinical sample being processed or with multiple clinical samples split from the original clinical sample being processed identically. Universal treatment of clinical samples with a single processing protocol inhibits the accurate detection and quantification of certain cell populations (e.g., microbial cells).

[0011] Described herein, in some aspects, are methods for enriching an array of microbes in a clinical sample (e.g., blood, urine, or bronchoalveolar lavage). Also provided herein are methods for simultaneously identifying and quantifying one or more microbial species (e.g., bacteria, fungi, or parasites) and / or viruses in a clinical sample.

[0012] In some aspects, the present disclosure provides a method for enriching a set of microbes in a sample, comprising: (i) obtaining a clinical sample from a human subject comprising human cells and microbial cells or receiving a clinical sample previously obtained from a human subject and comprising human cells and microbial cells; (ii) dividing the clinical sample into at least a first subsample and a second subsample; (iii) lysing human cells in the first subsample in the presence of a first detergent and human cells in the second subsample in the presence of a second detergent and a surfactant; and (iv) removing human DNA from the lysed cells in the first subsample and the second subsample; wherein the enrichment of the array of microbes in the first subsample and / or the second subsample is increased relative to a control (e.g., human cells, microbial cells, human DNA, microbial DNA, synthetic DNA). Enrichment of a set of microbes is relative to a sample that has not been through steps (i) and (iv), and the enrichment is achieved by removing human cells and human DNA from the sample.

[0013] In some embodiments, the method further comprises (v) amplifying microbial DNA in the first subsample and / or the second subsample. In some embodiments, the method further comprises (vi) sequencing microbial DNA in the first subsample and the second subsample. In some embodiments, the method further comprises (vii) identifying one or more microbial species present in the first subsample and / or the second subsample based on the sequenced microbial DNA. In some embodiments, the method further comprises (viii) quantifying the abundance of the one or more microbial species present in the first subsample and / or the second subsample based on the sequenced microbial DNA.

[0014] In some aspects, the present disclosure provides a method for processing a sample, comprising: (i) obtaining a clinical sample from a human subject comprising human cells and microbial cells or receiving a clinical sample previously obtained from a human subject and comprising human cells and microbial cells; (ii) dividing the clinical sample into at least a first subsample and a second subsample; (iii) lysing human cells in the first subsample in the presence of a first detergent and human cells in the second sub sample in the presence of a second detergent and a surfactant; and (iv) removing human DNA from the lysed cells in the first subsample and the second subsample; wherein the enrichment of the array of microbes in the first subsample and / or the second subsample is increased relative to a control (e.g., human cells, microbial cells, human DNA, microbial DNA, synthetic DNA). Enrichment of a set of microbes is relative to a sample that has not been through steps (i) and (iv), and the enrichment is achieved by removing human cells and human DNA from the sample.

[0015] This disclosure also provides, in some aspects, a method for simultaneously identifying one or more microbial species in a clinical sample, comprising: (i) obtaining a clinical sample from a human subject; (ii) dividing the clinical sample into at least a first subsample and a second subsample; (iii) amplifying microbial DNA in the first subsample to achieve whole genome amplification; (iv) sequencing microbial DNA in the first subsample and the second subsample; (v) identifying one or more microbial species present in the first subsample and the second subsample based on the sequenced microbial DNA.

[0016] In embodiments, the method further comprises: (vi) quantifying the relative clinical abundance of the one or more microbial species in the second subsample.

[0017] This disclosure also provides, in some aspects, a method for simultaneously identifying and quantifying one or more microbial species in a clinical sample, comprising: (i) obtaining a clinical sample from a human subject or receiving a clinical sample previously obtained from a human subject; (ii) dividing the clinical sample into at least a first subsample and a second subsample; (iii) amplifying microbial DNA in the first subsample to achieve whole genome amplification; (iv) sequencing microbial DNA in the first subsample and the second subsample; (v) identifying one or more microbial species present in the first subsample and the second subsample based on the sequenced microbial DNA; and (vi) quantifying the relative clinical abundance of the one or more microbial species in the second sub sample.

[0018] Relative clinical abundance, as provided herein, refers to abundance of a microbial species relative to other microbial species in a clinical sample that has been through steps (i) - (v) above.

[0019] This disclosure also provides, in some aspects, a method for simultaneously identifying and quantifying one or more microbial species in a clinical sample, comprising: (i) obtaining a clinical sample from a human subject or receiving a clinical sample previously obtained from a human subject; (ii) dividing the clinical sample into at least a first subsample and a second subsample; (iii) amplifying microbial DNA in the first subsample; (iv) sequencing microbial DNA in the first subsample and the second subsample; (v) identifying one or more microbial species present in the first subsample and the second subsample based on the sequenced microbial DNA; and (vi) quantifying the relative abundance, in the second subsample, of one or more microbial species identified in step (v).

[0020] This disclosure further provides, in some aspects, a method comprising: (i) obtaining a clinical sample from a human subject or receiving a clinical sample previously obtained from a human subject; (ii) dividing the clinical sample into at least a first subsample and a second subsample; (iii) quantifying the relative abundance of the one or more microbial species in the first subsample; (iv) splitting the second subsample into a third subsample and a fourth subsample; (v) lysing human cells in the third subsample in the presence of a first detergent and human cells in the fourth subsample in the presence of a second detergent and a surfactant; (vi) removing human DNA from the third subsample and the fourth subsample; (vii) lysing microbial cells in the third subsample and the fourth subsample; and (viii) amplifying microbial DNA in the third subsample and the fourth subsample to achieve whole genome amplification. In some embodiments, this method further comprises (ix) identifying one or more microbial species present in the third subsample and / or the fourth subsample based on the sequence microbial DNA.

[0021] Relative clinical abundance, as provided herein, refers to abundance of a microbial species relative to other microbial species in a clinical sample that has been through steps (i) - (ii) above.

[0022] This disclosure further provides, in some aspects, a method for simultaneously identifying and quantifying one or more microbial species in a clinical sample, comprising: (i) obtaining a clinical sample from a human subject or receiving a clinical sample previously obtained from a human subject; (ii) dividing the clinical sample into a least a first subsample and a second subsample; (iii) amplifying microbial DNA in the first subsample to achieve whole genome amplification (WGA); (iv) sequencing microbial DNA in the first subsample and the subsample; (v) identifying one or more microbial species present in the first subsample and the second subsample based on the sequenced microbial DNA; (vi) using the amplified DNA from the first subsample to create complete sequences of each strain found in the sample; and (vii) using complete sequences from the first subsample for genomic drug susceptibility predictions and / or as a mapping reference for microbial abundance quantification in the second subsample. Thus, the method may comprise at step (vii) performing a genomic drug susceptibility prediction using complete sequences from the first subsample and / or quantifying microbial abundance in the second subsample using complete sequences from the first subsample as a mapping reference for microbial abundance quantification. In some embodiments, WGA is achieved by multiple displacement amplification (MDA).

[0023] In some embodiments provided herein, prior to step (ii) dividing the clinical sample into at least a first subsample and a second subsample, the clinical sample is processed to enrich microbial cells relative to human cells using a method provided herein. In some embodiments, the method further comprises (vii) determining drug susceptibility of the one or more microbial species in the first subsample. In some embodiments, the method further comprises (viii) treating the human subject with a drug to which the one or more microbial species are susceptible, or recommending treatment of the human subject (or selecting the human subject for treatment) with a drug to which the one or more microbial species have been determined to be susceptible.

[0024] In some embodiments, the method is accomplished in 6 hours or less. In some embodiments, the method is accomplished in 12 hours or less. In some embodiments, the clinical sample is a urine sample. In some embodiments, the clinical sample is a blood sample. In some embodiments, the clinical sample is a bronchoalveolar lavage sample.

[0025] In some embodiments, processing a clinical sample comprises centrifuging the first subsample and the second subsample to separate human cells from the rest of the sample at the bottom of separate tubes containing the first subsample and the second subsample. In some embodiments, the method further comprises removing supernatant from above the centrifuged first and second subsamples and placing it into separate tubes. In some embodiments, the supernatant is used for further processing steps (e.g., amplifying microbial DNA, sequencing microbial DNA, identifying one or more microbial species, using the amplified DNA, using complete sequences from the first subsample for comprehensive genomic drug susceptibility predictions and / or as a mapping reference).

[0026] In some embodiments, the microbial DNA and human DNA in the first subsample are amplified by polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, digital droplet PCR (ddPCR), primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HAD), transcription mediated amplification (TMA), or recombinase polymerase amplification (RPA).

[0027] In some embodiments, identifying the one or more microbial species comprises classifying microbial DNA according to a reference genome. In some embodiments, identifying the one or more microbial species comprises comparing the sequenced microbial DNA to one or more reference genomes. In some embodiments, the one or more microbial species are bacteria, fungi, or a combination thereof. In some embodiments, determining drug sensitivity comprises identifying patterns in whole genomes of the one or more microbial species that are associated with drug sensitivity.

[0028] These and other aspects and embodiments of the invention are illustrated and described below. Other compositions, methods, and features will be apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional compositions and methods and features are within the scope of the present invention. The combination of the aspects and preferred features described are all explicitly envisaged except where such a combination is clearly impermissible or expressly avoided.

[0029] BRIEF DESCRIPTION OF THE FIGURES

[0030] The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

[0031] FIG. 1 illustrates a split sampling protocol for a urine sample. Step 1 : a clinical urine sample is collected from a subject. Step 2: size-based velocity sedimentation separates smaller microbial cells from larger human cells. Step 3: selective human cell lysis and removal of released human DNA. Step 4: the clinical urine sample is divided into at least 2 subsamples. Step 5A: microbial DNA in subsample 1 is amplified via an isothermal amplification step, resulting in the enrichment of microbial DNA (relative to human DNA) by a factor of 109. Step 5B: microbial DNA in subsample 2 is not subjected to a DNA amplification step. Step 6: Subsamples 1 and 2 are sequenced with whole genome sequencing (WGS) and the sequencing data are analyzed to determine species identification (ID), antibiotic sensitivity (AST), and relative microbial abundance.

[0032] FIG. 2 illustrates microbial enrichment isolation and sequencing depth using a split sample protocol. Bacteria (Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis)) were spiked at IxlO5colony-forming units per milliliter (CFU / mL) into urine samples from three independent donors. After whole genome amplification, all samples were sequenced.

[0033] FIGs. 3A-3B illustrate DNA amplification for microbial species identification with E. coli (triangles) and E. faecalis (circles). FIG. 3A shows the percentage of spiked-in microbial genomes recovered. FIG. 3B shows the breadth of E. coli and E. faecalis genomes recovered. The dotted line indicates the normalized bacterial ID value, “ns” means not significant, means p<0.05, and “****” means p<0.0001. FIGs. 4A-4B illustrate bacterial enrichment in the presence of excess human white blood cells (WBCs). FIG. 4A shows bacterial enrichment of control urine samples containing IxlO5CFU / mL bacteria and no WBCs with 0 (Excluded), 1 (Included IX), or 2 (Included 2X) rounds of velocity sedimentation. FIG. 4B shows bacterial enrichment of urine samples containing IxlO5CFU / mL bacteria and 5xl05white blood cells per milliliter (WBCs / mL) with 0 (Excluded), 1 (Included IX), or 2 (Included 2X) rounds of velocity sedimentation, “ns” means not significant, means p<0.05, “**” means p<0.01, and “Mbs” means megabases.

[0034] FIG. 5 illustrates sequence abundance values over a range of bacterial loads.

[0035] FIGs. 6A-6C illustrate Gram-negative bacteria Acinetobacter baumannii (A. baumannii) survival in buffer containing surfactant (Tween) as measured in CFUs at the end of clinical sample processing. FIG. 6A provides an exemplary split-sample processing protocol, with Arm A having a lower percentage detergent (SDS) in the presence of a surfactant (Tween) and Arm B having a higher percentage detergent (SDS) in the absence of surfactant. FIG. 6B shows total A. baumannii CFUs recovered and FIG. 6C shows A. baumannii CFUs recovered relative to input from samples in Arm A (circles) and Arm B (squares).

[0036] FIGs. 7A-7C illustrate d, baumannii survival in a buffer containing a detergent (SDS) with or without a surfactant (Tween). FIG. 7A shows A. baumannii survival measured in CFUs at the end of clinical sample processing as measured by total megabases (MBs) of amplified A. baumannii DNA. FIG. 7B shows the breadth of A. baumannii whole genome coverage in amplified DNA. FIG. 7C shows the (log) ratio of A. baumannii DNA to human DNA.

[0037] FIGs. 8A-8D illustrate the effect of higher percentage detergent (SDS) in the absence of a surfactant or lower percentage detergent (SDS) in the presence of a surfactant on P. mirabilis and S. pneumoniae detection following sample processing. FIG. 8A shows the total MBs of P. mirabilis and S. pneumoniae post-enrichment. FIG. 8B shows the breadth of P. mirabilis and S. pneumoniae genome coverage in amplified DNA. FIG. 8C shows the percentage of P. mirabilis and S. pneumoniae that have at least IX whole genome coverage. FIG. 8D shows the percentage of P. mirabilis and S. pneumoniae that have at least 5X whole genome coverage. “Both arms A” means that the entire clinical sample was treated with a lower percentage detergent in the presence of surfactant, “Both arms B” means that the entire clinical sample was treated with a higher percentage detergent in the absence of surfactant, and “Split: One arm A, one arm B” means that the clinical sample was split as in FIG. 6A. FIG. 9 illustrates a split sampling protocol for a clinical sample. Step 1 : a clinical sample is collected from a subject. Step 2: the clinical sample is divided into at least 2 subsamples. Step 3: selective human cell lysis and removal of released human DNA. Step 4A: microbial (e.g., bacterial) cells are lysed and microbial DNA in subsample 1 is amplified via an isothermal amplification step, resulting in the enrichment of microbial DNA (relative to human DNA) by a factor of 109. Step 4B: microbial cells are lysed but microbial DNA in subsample 2 is not subjected to a DNA amplification step. Step 5: Subsamples 1 and 2 are sequenced with whole genome sequencing (WGS) and the sequencing data are analyzed to determine species identification (ID), antibiotic sensitivity (AST), and relative microbial (pathogen) abundance.

[0038] FIGs. 10A-10E illustrate pathogen (microbial) identification and abundance in lower respiratory tract infection (LRTI) samples as assessed based on the split sampling protocol shown in FIG. 9. FIG. 10A shows the identification value of A. baumannii, K. pneumoniae, P. aeurognisa, S. aureus, and S. pneumoniae based on a 104CFU / mL input, where a value of 1.0 indicates that the full 104CFU / mL was recovered. FIG. 10B shows the identification value of A. baumannii, K. pneumoniae, P. aeruginosa, S. aureus, and S. pneumoniae based on a 105CFU / mL input, where a value of 1.0 indicates that the full 105CFU / mL was recovered. FIG. 10C shows human DNA recovery in mapped megabases, with the Abundance Module being processed as in subsample 2 in FIG. 9 and the ID module being processed as in subsample 1 in FIG. 9. FIG. 10D shows the genome recovery (IX coverage) of A. baumannii, K. pneumoniae, P. aeruginosa, S. aureus, and S. pneumoniae based on a 104CFU / mL input, where a value of 100% indicates that the full 104CFU / mL was recovered. FIG. 10E shows the genome recovery (IX coverage) of 4. baumannii, K. pneumoniae, P. aeruginosa, S. aureus, and S. pneumoniae based on a 105CFU / mL input, where a value of 100% indicates that the full 105CFU / mL was recovered.

[0039] FIGs. 11A-11B illustrates predicted signal from K. pneumoniae in clinical samples processed as in FIG. 9 based on actual measured signal. FIG. 11A shows the K. pneumoniae signal (dotted line) in LRTI clinical samples based on the concentration of K. pneumoniae (CFU / mL) and the normalized signal of the process produced by carrier DNA in the clinical samples. Data points are plotted from 3 donors. FIG. 11B shows the actual versus predicted burden from K. pneumoniae from the 3 donors in FIG. 11A with bucketed microbial abundance bins.

[0040] FIG. 12 illustrates a possible lower respiratory tract infection (LRTI) split sample processing workflow, where the amplified sample and the unamplified sample provide information to the other sample to power measurement of microbial abundance, identification of microbial species, and determination of antibiotic susceptibility for present microbes in the original LRTI sample.

[0041] DETAILED DESCRIPTION

[0042] Clinical samples (e.g., blood, urine, or bronchoalveolar lavage) are processed to identify microbes present in subjects (e.g., human subjects) from whom the clinical samples are obtained. Rapid, accurate identification of these microbes is critical for detecting and treating microbial infections in subjects. Lysis conditions adapted so that the nuclear contents (e.g., DNA or RNA) of cells in a clinical sample (e.g., blood, urine, or bronchoalveolar lavage) are equally released help to promote accurate species identification, determination of drug susceptibility, and quantification of the microbial abundance of the microbes in the clinical sample. The present disclosure therefore advances the field by providing inventive approaches for split clinical sample processing, delivering a comprehensive clinical result, to drive rapid and appropriate treatment of infections.

[0043] The present disclosure provides, in part, a method for enriching an array of microbes in a clinical sample (e.g., blood or urine). In contrast to existing sample preparation strategies, methods provided herein efficiently and selectively remove human cells, leaving behind microbial cells for DNA isolation. This is accomplished by splitting a clinical sample into parallel subsamples that may be treated with different lysis conditions (e.g., detergents, surfactants, etc.) to allow microbial cells to remain intact while human cells are lysed. Also provided herein are methods for simultaneously identifying and quantifying one or more microbial species in a clinical sample. This is accomplished by splitting a clinical sample into parallel subsamples, one of which can be used for microbial identification and measuring microbial clinical abundance without amplification and the other for microbial identification and drug susceptibility testing with amplification. Methods combining both approaches are also described. The sample splitting approaches provided herein enable the processing conditions for each subsample to be utilized for the recovery of a subset of microbial pathogens, allowing for rapid molecular characterization of microbial infections and the detection of a broad array of microbial pathogens from a single clinical sample. Further, this unbiased approach allows for diagnostic completeness, with at least microbial identification, microbial abundance, and drug identification being provided from a single clinical sample. Clinical Sample Processing

[0044] Clinical samples obtained from subject (e.g., human subjects) contain complex mixtures of subject cells and microbes (e.g. microbial cells and / or viral particles). Identifying and quantifying microbes in these clinical samples so that the subject may receive appropriate treatment for a microbial infection requires accurate identification and quantification of microbes (e.g. microbial cells) in the clinical sample. The present application provides, in some embodiments, methods for clinical sample processing that include splitting a clinical sample for parallel processing of the resulting subsamples. This parallel processing permits rapid: enrichment of microbes (e.g. microbial cells and / or viruses) relative to subject cells, identification of one or more microbial species, quantification of microbial species, or some combination thereof.

[0045] Sample Splitting

[0046] A clinical sample may be split at any point in sample processing, although typically it will be split before any processing occurs. In some embodiments, a clinical sample is split immediately after it is obtained (e.g., from a subject). In some embodiments, a clinical sample is split after some steps of sample processing (e.g., velocity sedimentation (e.g., by centrifugation), lysis, etc.) have occurred. In some embodiments, a clinical sample is split before velocity sedimentation. In some embodiments, a clinical sample is split after velocity sedimentation. In some embodiments, a clinical sample is split before lysis and enrichment. In some embodiments, a clinical sample is split after lysis and enrichment.

[0047] In some embodiments, a clinical sample is split into at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more subsamples. In some embodiments, a clinical sample is split into 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3- 6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10 or more subsamples.

[0048] In some embodiments, a clinical sample is split into at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more subsamples that are equal in weight and / or volume. In some embodiments, a clinical sample is split into at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more subsamples. In some embodiments, a clinical sample is split into 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5- 10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10 subsamples that are equal in weight and / or volume. In some embodiments, a clinical sample is split into at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more subsamples that are unequal in weight and / or volume. In some embodiments, a clinical sample is split into 2, 3, 4, 5, 6, 7, 8, 9, 10, or more subsamples. In some embodiments, a clinical sample is split into 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4- 10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10 subsamples that are unequal in weight and / or volume. In some embodiments, a clinical sample is split into at least a first subsample (e.g., subsample 1) and a second subsample (e.g., subsample 2).

[0049] Subsamples (split from a clinical sample) may be recombined into a single sample after clinical sample processing. In some embodiments, all subsamples (e.g., 1-10 or more subsamples) are recombined into a single sample after clinical sample processing. In some embodiments, some, but not all subsamples, are recombined into a single sample after clinical sample processing. In some embodiments, all subsamples are recombined into multiple (e.g., 1-10 more) samples after clinical sample processing. In some embodiments, a clinical sample is recombined before velocity sedimentation. In some embodiments, a clinical sample is recombined after velocity sedimentation. In some embodiments, a clinical sample is recombined before lysis and enrichment. In some embodiments, a clinical sample is recombined after lysis and enrichment.

[0050] Enriching

[0051] Methods provided herein include enriching an array of microbes in a clinical sample. As used herein, the term “array of microbes” refers to a mixture of microbes that may include different bacteria, fungi, viruses, parasites, or some combination thereof. An array of microbes may include only one type of microbe (e.g., bacteria, fungi, viruses, parasites, etc.), two types of microbes, three types of microbes, four types of microbes, five types of microbes, six types of microbes, seven types of microbes, eight types of microbes, nine types of microbes, or ten or more types of microbes.

[0052] As used herein, the term “enriching” refers to the process of increasing the concentration and / or abundance of one or more microbial species in a clinical sample relative to another type of cell in a clinical sample (e.g., a control) or decreasing the concentration and / or abundance of human cells in a clinical sample, thus enriching any remaining microbial species that remain in the clinical sample relative to human cells, relative to unenriched sample, or relative to human cells and unenriched samples. In some embodiments, enriching may include amplifying microbial DNA (e.g., whole genome microbial DNA) from one or more microbial species in a clinical sample. In some embodiments, microbial DNA is genomic microbial DNA. In some embodiments, amplifying microbial DNA comprise whole genome amplification of microbial DNA.

[0053] In some embodiments, enriching may include lysing cells of one or more microbial species in a clinical sample (e.g., blood, urine, or bronchoalveolar lavage). In some embodiments, lysing cells of one or more microbial species in a clinical sample occurs after human DNA is removed from the clinical sample. In some embodiments, lysing cells of one or more microbial species in a subsample occurs after human DNA is removed from the subsample. In some embodiments, lysing cells of one or more microbial species in a clinical sample occurs simultaneously with lysing human cells in the clinical sample. In some embodiments, lysing cells of one or more microbial species in a subsample occurs simultaneously with lysing human cells in the subsample.

[0054] As used herein, the term “simultaneously” refers to one or more parallel processes that occur in close temporal proximity. In some embodiments, when lysing cells of one or more microbial species in a clinical sample or in a subsample occurs simultaneously with lysing human cells in the clinical sample or subsample, the cells of the one or more microbial species and the human cells may be lysed in the same clinical sample or subsample (e.g., in the same container, vial, or tube). In some embodiments, when lysing cells of one or more microbial species in a clinical sample or in a subsample occurs simultaneously with lysing human cells in the clinical sample or subsample, the cells of the one or more microbial species and the human cells may be lysed in different subsamples. The cells may be lysed in the different subsamples at approximately the same step in the protocol (e.g., in parallel). In other words, the cells may be lysed in the different subsamples prior to the different subsamples being subject to a next step of the protocol. In some embodiments, when cells of one or more microbial species and human cells are lysed in different subsamples, e.g. at approximately the same step in the protocol (e.g., in parallel), the lysis conditions (e.g., buffer composition, time, temperature) may vary in each subsample. Method for lysing cells of one or more microbial species are known to those of skill in the art and are discussed herein.

[0055] In some embodiments, enriching may include amplifying microbial DNA (e.g., whole genome microbial DNA) from one or more microbial species in a clinical sample. In some embodiments, when a clinical sample is divided into at least a first subsample (e.g., subsample 1) and a second subsample (e.g., subsample 2), nucleic acids (e.g., microbial DNA) in the first subsample are amplified and nucleic acids (e.g., microbial DNA) in the second subsample are not amplified. In some embodiments, when a clinical sample is divided into at least a first subsample (e.g., subsample 1) and a second subsample (e.g., subsample 2), nucleic acids (e.g., microbial DNA) in the first and the second subsample are amplified. In some embodiments, enriching may include amplifying subject (e.g., human) DNA.

[0056] Non-limiting examples of methods for amplifying microbial DNA include polymerase chain reaction (PCR), quantitative PCR (qPCR), reverse-transcriptase PCR (RT-PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, strand displacement amplification (SDA), helicase dependent amplification (HAD), transcription mediated amplification (TMA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), whole genome amplification (WGA), multiple displacement amplification (MDA), random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), and rolling circle amplification (RCA). In some embodiments, whole genome microbial DNA from one or more lysed microbial species is amplified using MDA.

[0057] In some embodiments, a method for enriching an array of microbes in a clinical sample comprises: (i) obtaining a clinical sample (e.g., blood, urine, or bronchoalveolar lavage) from a human subject comprising human cells and microbial cells; (ii) dividing the clinical sample (e.g., blood, urine, or bronchoalveolar lavage) into at least a first subsample and a second subsample; (iii) lysing human cells in the first subsample in the presence of a first detergent and human cells in the second subsample in the presence of a second detergent and a surfactant; and (iv) removing human DNA from the lysed cells in the first subsample and the second subsample; wherein the enrichment of the array of microbes in the first subsample and / or the second subsample is increased relative to a control. A “control” may be, in some instances, one or more microbial species present in a clinical sample, human DNA present in a clinical sample, human cells present in a clinical sample, one or more microbial species introduced into a clinical sample, or a synthetic control (e.g., synthetic DNA sequence) introduced into a clinical sample.

[0058] In some embodiments, the amount of one or more microbial species DNA may be enriched by at least 10-fold, at least 100-fold, at least 103-fold, at least 104-fold, at least 105- fold, at least 106-fold, at least 107-fold, at least 108-fold, at least 109-fold, at least 1010-fold, at least 10n-fold, or at least 1012-fold more than the amount of human DNA in a clinical sample. In some embodiment, the amount of one or more microbial species DNA may be enriched by 10-fold to 100-fold, 10-fold to 103-fold, 10-fold to 104-fold, 10-fold to 105-fold, 10-fold to 106-fold, 10-fold to 107-fold, 10-fold to 108-fold, 10-fold to 109-fold, 10-fold to 1010-fold, 10-fold to 10n-fold, 10-fold to 1012-fold, 100-fold to 103-fold, 100-fold to 104-fold, 100-fold to 105-fold, 100-fold to 106-fold, 100-fold to 107-fold, 100-fold to 108-fold, 100-fold to 109-fold, 100-fold to 1010-fold, 100-fold to 10n-fold, 100-fold to 1012-fold, 103-foldto 104- fold, 103-foldto 105-fold, 103-foldto 106-fold, 103-foldto 107-fold, 103-foldto 108-fold, 103- foldto 109-fold, 103-foldto 1010-fold, 103-foldto 10n-fold, 103-foldto 1012-fold, 104-foldto

[0059] 105-fold, 104-foldto 106-fold, 104-foldto 107-fold, 104-foldto 108-fold, 104-foldto 109-fold, 104-foldto 1010-fold, 104-foldto 10n-fold, 104-foldto 1012-fold, 105-foldto 106-fold, 105-fold to 107-fold, 105-foldto 108-fold, 105-foldto 109-fold, 105-foldto 1010-fold, 105-foldto 1011- fold, 105-foldto 1012-fold, 106-foldto 107-fold, 106-foldto 108-fold, 106-foldto 109-fold, 106- foldto 1010-fold, 106-foldto 10n-fold, 106-foldto 1012-fold, 107-foldto 108-fold, 107-foldto 109-fold, 107-foldto 1010-fold, 107-foldto 10n-fold, 107-foldto 1012-fold, 108-foldto 109- fold, 108-foldto 1010-fold, 108-foldto 10n-fold, 108-foldto 1012-fold, 109-foldto 1010-fold, 109-foldto 10n-fold, 109-foldto 1012-fold, 1010-foldto 10n-fold, 1010-foldto 1012-fold, or 10n-fold to 1012-fold more than the amount of human DNA in a clinical sample.

[0060] In some embodiments, the total amount of the one or more microbial species DNA may be enriched by at least 10-fold, at least 100-fold, at least 103-fold, at least 104-fold, at least 105-fold, at least 106-fold, at least 107-fold, at least 108-fold, at least 109-fold, at least 1010-fold, at least 10n-fold, or at least 1012-fold more than the amount of human DNA in a clinical sample. In some embodiment, the total amount of the one or more microbial species DNA may be enriched by 10-fold to 100-fold, 10-fold to 103-fold, 10-fold to 104-fold, 10- fold to 105-fold, 10-fold to 106-fold, 10-fold to 107-fold, 10-fold to 108-fold, 10-fold to 109- fold, 10-fold to 1010-fold, 10-fold to 10n-fold, 10-fold to 1012-fold, 100-fold to 103-fold, 100- fold to 104-fold, 100-fold to 105-fold, 100-fold to 106-fold, 100-fold to 107-fold, 100-fold to

[0061] 108-fold, 100-fold to 109-fold, 100-fold to 1010-fold, 100-fold to 10n-fold, 100-fold to 1012- fold, 103-foldto 104-fold, 103-foldto 105-fold, 103-foldto 106-fold, 103-foldto 107-fold, 103- foldto 108-fold, 103-foldto 109-fold, 103-foldto 1010-fold, 103-foldto 10n-fold, 103-foldto 1012-fold, 104-foldto 105-fold, 104-foldto 106-fold, 104-foldto 107-fold, 104-foldto 108-fold, 104-foldto 109-fold, 104-foldto 1010-fold, 104-foldto 10n-fold, 104-foldto 1012-fold, 105-fold to 106-fold, 105-foldto 107-fold, 105-foldto 108-fold, 105-foldto 109-fold, 105-foldto 1O10- fold, 105-foldto 10n-fold, 105-foldto 1012-fold, 106-foldto 107-fold, 106-foldto 108-fold,

[0062] 106-foldto 109-fold, 106-foldto 1010-fold, 106-foldto 10n-fold, 106-foldto 1012-fold, 107-fold to 108-fold, 107-foldto 109-fold, 107-foldto 1010-fold, 107-foldto 10n-fold, 107-foldto 1012- fold, 108-foldto 109-fold, 108-foldto 1010-fold, 108-foldto 10n-fold, 108-foldto 1012-fold,

[0063] 109-foldto 1010-fold, 109-foldto 10n-fold, 109-foldto 1012-fold, 1010-foldto 10n-fold, 1O10- fold to 1012-fold, or 10n-fold to 1012-fold more than the amount of human DNA in a clinical sample. In some embodiments, amplified DNA from a clinical sample is used to create complete sequences of each microbial strain found in the clinical sample. Complete sequences means complete microbial genome sequences, also referred to herein as whole microbial genome sequences. A complete sequence of a microbial strain may refer to a whole genome sequence for the strain. A complete microbial genome sequence of each microbial strain may be 75% - 100% of the known microbial sequence (e.g., from a public database, including, but not limited to, National Center for Biotechnology Information (NCBI)). In some embodiments, a complete microbial sequence of each microbial strain is 80% - 95% or 85% - 90% of the known microbial sequence. In some embodiments, a complete microbial sequence of each microbial strain is 81% - 95%, 82% - 95%, 83% - 95%, 84% - 95%, 85% - 95%, 86% - 95%, 87% - 95%, 88% - 95%, 89% - 95%, 90% - 95%, 91% - 95%, 92% - 95%, 93% - 95%, 94% - 95%, 80% - 94%, 81% - 94%, 82% - 94%, 83% - 94%, 84% - 94%, 85% - 94%, 86% - 94%, 87% - 94%, 88% - 94%, 89% - 94%, 90% - 94%, 91% - 94%, 92% - 94%, 93% - 94%, 81% - 93%, 82% - 93%, 83% - 93%, 84% - 93%, 85% - 93%, 86% - 93%, 87% - 93%, 88% - 93%, 89% - 93%, 90% - 93%, 91% - 93%, 92% - 93%, 80% - 92%, 81% - 92%, 82% - 92%, 83% - 93%, 84% - 93%, 85% - 93%, 86% - 93%, 87% - 93%, 88% - 93%, 89% - 93%, 90% - 93%, 91% - 93%, 92% - 93%, 80% - 92%, 81% - 92%, 82% - 92% 83% - 92%, 84% - 92%, 85% - 92%, 86% - 92%, 87% - 92%, 88% - 92%, 89% - 92%, 90% - 92%, 91% - 92%, 80% - 91%, 81% - 90%, 82% - 90%, 83% - 90%, 84% - 90%, 85% - 90%, 86% - 90%, 87% - 90%, 88% - 90%, 89% - 90%, 80% - 89%, 81% - 89%, 82% - 89%, 83% - 89%, 84% - 89%, 85% - 89%, 86% - 89%, 87% - 89%, 88% - 89%, 80% - 88%, 81% - 88%, 82% - 88%, 83% - 88%, 84% - 88%, 85% - 88%, 86% - 88%, 87% - 88%, 80% - 87%, 81% - 87%, 82% - 87%, 83% - 87%, 84% - 87%, 85% - 87%, 86% - 87%, 80% - 86%, 81% - 86%, 82% - 86%, 83% - 86%, 84% - 86%, 85% - 86%, 80% - 85%, 81% - 85%, 82% - 85%, 83% - 85%, 84% - 85%, 80% - 84%, 81% - 84%, 82% - 84%, 83% - 84%, 80% - 83%, 81% - 83%, 82% - 83%, 80% - 82%, 81% - 82%, 80% - 81%. In some embodiments, a complete microbial sequence of each microbial strain is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the known microbial sequence.

[0064] Lysing cells

[0065] Methods provided herein include lysing cells (e.g., human cells) in a clinical sample (e.g., blood, urine, or bronchoalveolar lavage). The term “lysing”, as used herein, refers to the breaking down or rupturing of a cell’s membranes to release its contents (e.g., DNA, RNA, proteins). In some embodiments, the subject (e.g., human) and microbial (e.g., bacteria, virus, or fungus) cells can be differentially lysed and fractioned based on their specific properties, such as size, density, or other chemical properties, including cell membrane or cell wall composition. There are several methods for performing cell lysis known in the art, such as mechanical cell lysis (e.g., homogenization, bead beating), chemical cell lysis (e.g., detergent lysis, such as with Triton X-100 or sodium dodecyl sulfate (SDS)), enzymatic cell lysis (e.g., using lysozyme), freeze-thaw lysis, ultrasonication, high-pressure homogenization, osmotic shock, or electroporation.

[0066] In some embodiments, methods provided herein include lysing cells (e.g., human cells). In some embodiments, methods provided herein include lysing cells (e.g., human cells) in a subsample (e.g., a first subsample or a second subsample) in the presence of a first detergent. Detergents are chemical compounds containing amphiphilic molecules that enable the disruption of cell membranes due to their ability to interact with and solubilize molecules or structures that are not water soluble. In some embodiments, the detergent is a nonionic detergent. A nonionic detergent is a detergent that does not carry a net electrical charge and is typically milder than ionic detergents. Milder means that a nonionic detergent is not as strong and will not disrupt cell membranes to the same degree as an ionic detergent. Nonlimiting examples of nonionic detergents include Triton X-100 and NP-40. In some embodiments, the detergent is an ionic detergent. Ionic detergents are generally harsher than nonionic detergents. This class of detergents can be further subdivided into anionic detergents (e.g., sodium dodecyl sulfate (SDS), ammonium dodecyl sulfate (ADS), sodium lauryl sulfate (SLS), cetylpyridinium chloride), cationic detergents (e.g., cetyltrimethylammonium bromide (CTAB), benzyldimethyloctylammonium chloride), and zwitterionic detergents (e.g., CHAPS, pentaerythrityl palmitate). Other detergents known in the art are contemplated.

[0067] In some embodiments, methods provided herein include lysing cells (e.g., human cells) in a subsample (e.g., a first subsample or a second subsample) in the presence of a second detergent and a surfactant. A surfactant is a compound used to alter the surface properties of liquids and can facilitate the disruption of cell membranes due to their ability to solubilize lipids and prevent the clumping and / or aggregation of cellular material. In some embodiments, the presence of a surfactant protects non-human cells (e.g., cells of one or more microbial species) from lysis. In some embodiments, the presence of a surfactant does not protect non-human cells (e.g., cells of one or more microbial species) from lysis. Nonlimiting examples of surfactants include Tween-20 (polysorbate-20), Tween-80 (polysorbate- 80), NP-40, and Triton X-100. Other surfactants known in the art are contemplated. In some embodiments, when a detergent and a surfactant are used in a sample (a clinical subsample), the detergent and the surfactant are different.

[0068] In some embodiments, methods provided herein include lysing cells (e.g., human cells) in a subsample (e.g., a first subsample or a second subsample) in the presence of one or more detergents. In some embodiments, the one or more detergents is 1, 2, 3, 4, 5, or more different detergents. In some embodiments, the one or more detergents is 1-2, 1-3, 1-4, 1-5,

[0069] 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 different detergents. In some embodiments, the one or more detergents is equal volumes and / or weight of 2, 3, 4, 5, or more different detergents. In some embodiments, the one or more detergents is equal volumes and / or weight of , 2-3, 2-4, 2-5, 3- 4, 3-5, or 4-5 different detergents. In some embodiments, the one or more detergents is unequal volumes and / or weight of 2, 3, 4, 5, or more different detergents. In some embodiments, the one or more detergents is unequal volumes and / or weight of 2-3, 2-4, 2-5,

[0070] 3-4, 3-5, or 4-5 different detergents.

[0071] In some embodiments, where a subsample comprises 2 or more detergents, one detergent is more concentrated (higher in concentration) than the other. In some embodiments, where a subsample comprises more than 2 detergents, one detergent is more concentrated than the other detergents.

[0072] In some embodiments, methods provided herein include lysing cells (e.g., human cells) in a subsample (e.g., a first subsample or a second sample) in the presence of one or more surfactants. In some embodiments, the one or more surfactants are 1, 2, 3, 4, 5, or more different surfactants. In some embodiments, the one or more surfactants are 2-3, 2-4, 2-5, 3-

[0073] 4, 3-5, or 4-5 different surfactants. In some embodiments, the one or more surfactants are equal volumes and / or weight of 2, 3, 4, 5, or more different surfactants. In some embodiments, the one or more surfactants are equal volumes and / or weight of 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 different surfactants. In some embodiments, the one or more surfactants are unequal volumes and / or weight of 2, 3, 4, 5, or more different surfactants. In some embodiments, the one or more surfactants are unequal volumes and / or weight of 2-3, 2-4, 2-

[0074] 5, 3-4, 3-5, or 4-5 different surfactants.

[0075] In some embodiments, where a subsample comprises 2 or more surfactants, one surfactant is more concentrated (higher in concentration) than the other. In some embodiments, where a subsample comprises more than 2 surfactants, one surfactant is more concentrated than the other surfactants.

[0076] In some embodiments, methods provided herein include lysing cells (e.g., human cells) in a clinical sample in the presence of one or more detergents. In some embodiments, the one or more detergents is 2, 3, 4, 5, or more different detergents. In some embodiments, the one or more detergents is 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 different detergents. In some embodiments, the one or more detergents is equal volumes and / or weight of 2, 3, 4, 5, or more different detergents. In some embodiments, the one or more detergents is equal volumes and / or weight of 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 different detergents. In some embodiments, the one or more detergents is unequal volumes and / or weight of 2, 3, 4, 5, or more different detergents. In some embodiments, the one or more detergents is unequal volumes and / or weight of 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 different detergents.

[0077] In some embodiments, methods provided herein include lysing cells (e.g., human cells) in a clinical sample in the presence of one or more surfactants. In some embodiments, the one or more surfactants are 2, 3, 4, 5, or more different surfactants. In some embodiments, the one or more surfactants are 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 different surfactants. In some embodiments, the one or more surfactants are equal volumes and / or weight of 2, 3, 4, 5, or more different surfactants. In some embodiments, the one or more surfactants are equal volumes and / or weight of 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 different surfactants. In some embodiments, the one or more surfactants are unequal volumes and / or weight of 2, 3, 4, 5, or more different surfactants. In some embodiments, the one or more surfactants are unequal volumes and / or weight of 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 different surfactants.

[0078] In some embodiments, the detergent is 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% SDS. In some embodiments, the detergent is 0.1% - 0.5%, 0.1% -1%, 0.1% - 3%, 0.1% - 5%, 0.1% - 10%, 0.5% - 1%, 0.5% - 3%, 0.5% - 5%, 0.5% - 10%, 1% - 3%, 1% - 5%, 1% - 10%, 3% - 5%, 3% - 10%, or a 5% - 10% SDS.

[0079] In some embodiments, the detergent is 1 pL - 1000 mL, 1 pL - 950 mL, 1 pL - 900 mL, 1 pL - 850 mL, 1 pL - 800 mL, 1 pL - 750 mL, 1 pL - 700 mL, 1 pL - 650 mL, 1 pL - 600 mL, 1 pL - 550 mL, 1 pL - 500 mL, 1 pL - 450 mL, 1 pL - 400 mL, 1 pL - 350 mL, IpL - 300 mL, 1 pL - 250 mL, 1 pL - 200 mL, 1 pL - 150 mL, 1 pL - 100 mL, 1 pL - 500 mL SDS.

[0080] In some embodiments, the surfactant is 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% Tween. In some embodiments, the surfactant is 0.1% - 0.5%, 0.1% -1%, 0.1% - 3%, 0.1% - 5%, 0.1% - 10%, 0.5% - 1%, 0.5% - 3%, 0.5% - 5%, 0.5% - 10%, 1% - 3%, 1% - 5%, 1% - 10%, 3% - 5%, 3% - 10%, or 5% - 10% Tween.

[0081] In some embodiments, the detergent is 1 pL - 1000 mL, 1 pL - 950 mL, 1 pL - 900 mL, 1 pL - 850 mL, 1 pL - 800 mL, 1 pL - 750 mL, 1 pL - 700 mL, 1 pL - 650 mL, 1 pL - 600 mL, 1 pL - 550 mL, 1 pL - 500 mL, 1 pL - 450 mL, 1 pL - 400 mL, 1 pL - 350 mL, IpL - 300 mL, 1 pL - 250 mL, 1 pL - 200 mL, 1 pL - 150 mL, 1 pL - 100 mL, 1 pL - 500 mL Tween.

[0082] In some embodiments, methods provided herein include removing human DNA from lysed cells in a subsample (e.g., a first subsample or a second subsample). The term “removing”, as used herein, refers to the process of physically separating two or more substances (e.g., human DNA from lysed cells). In some embodiments, removing may include centrifugation (e.g., velocity sedimentation), separating supernatant (e.g., from a pellet), and moving the supernatant to a separate tube (e.g., from the pellet), filtration, nucleic acid (e.g., DNA or RNA) isolation, or any combination thereof.

[0083] In some embodiments, removing may include one or more centrifugation steps, such as velocity sedimentation. Velocity sedimentation, also known as rate-zonal centrifugation, is a technique used to separate particles in a solution based on their size and shape, where larger molecules accumulate in a pellet at the bottom of the container (e.g., tube) being centrifuged and smaller molecules accumulate in the supernatant above the pellet. This method can be employed to fractionate macromolecules or particles (e.g., proteins, nucleic acids, or subcellular organelles, cells) according to their sedimentation rates in a centrifugal field. In the present disclosure, centrifugation may be used to compact human cells at the bottom of a tube. In some embodiments, where centrifugation is used to compact human cells at the bottom of a tube, a method of the present disclosure may further comprise removing supernatant from above the compacted human cells and placing it into a separate (e.g., a different) tube.

[0084] In some embodiments, removing may include separating a supernatant (e.g., from a pellet). Separating a supernatant typically involves the separation of a liquid (e.g., the supernatant containing one or more microbial species) from solid particles (e.g., the pellet containing human cells and macromolecular aggregates) that have settled at the bottom of a container. The supernatant can be separated by decanting the supernatant into a new container or by pipetting (e.g., using a Pasteur pipette or a micropipette). In some embodiments, the supernatant is moved forward with additional processing.

[0085] In some embodiments, removing may include filtration, whereby a sample is passed through a filter with a specific pore size that retains one or more microbial species (e.g., bacteria, virus, fungus, or parasite) while allowing other materials to pass through. In some embodiments, removing may be performed using a combination of centrifugation, filtration, and / or separating a supernatant. In some embodiments, removing may include nucleic acid (e.g., DNA or RNA) isolation. Nucleic acid isolation, (also known as nucleic acid extraction), is a technique used to obtain DNA / RNA from a biological sample. Any DNA / RNA that is isolated can be derived from the subject (e.g., a human subject) or from microbial species (e.g., bacteria, viruses, fungi, or parasites). Methods of performing nucleic acid isolation are known to those of skill in the art and can include, but are not limited to, phenol-chloroform extraction, silica- based spin columns, magnetic bead-based purification, organic solvent precipitation, solidphase extraction, and ultracentrifugation.

[0086] Methods provided herein include, in some embodiments, enriching an array of microbes (e.g., one or more microbes) in a clinical sample. Such methods include, but are not limited to: (i) obtaining a clinical sample (e.g., blood, urine, or bronchoalveolar lavage) from a subject (e.g. a human subject) comprising subject cells (e.g. human cells) and microbial cells; (ii) dividing the clinical sample into at least a first subsample and a second subsample; (iii) lysing subject cells (e.g., human cells) in the first subsample in the presence of a first detergent and subject cells (e.g. human cells) in the second subsample in the presence of a second detergent and a surfactant; and (iv) removing subject DNA (e.g., human DNA) from the lysed cells in the first subsample and the second subsample. In other embodiments, methods provided herein include simultaneously identifying and quantifying one or more microbial species (e.g., bacteria, viruses, fungi, or parasites) in a clinical sample (e.g., blood, urine, or bronchoalveolar lavage). Such methods include, but are not limited to: (i) obtaining a clinical sample (e.g., blood, urine, or bronchoalveolar lavage) from a subject (e.g., a human subject); (ii) dividing the clinical sample into at least a first subsample and a second subsample; (iii) amplifying microbial DNA in the first subsample to achieve whole genome amplification; (iv) sequencing microbial DNA in the first subsample and the second subsample; (v) identifying one or more microbial species present in the first subsample and the second subsample based on the sequenced microbial DNA; and (vi) quantifying the relative clinical abundance of the one or more microbial species in the second subsample.

[0087] Identifying and quantifying

[0088] Methods provided herein include simultaneously identifying and quantifying one or more microbial species in a clinical sample. The term “identifying”, as used herein, refers to the process of determining the identity of one or more microbial species present in a clinical sample (e.g., a blood, urine, or bronchoalveolar lavage sample). As used herein, the term “quantifying” refers to the process of measuring the quantity or concentration of one or more microbial species in a clinical sample (e.g., a blood, urine, or bronchoalveolar lavage sample). Quantifying one or more microbial species requires identifying the one or more microbial species that are to be quantified. Identifying the one or more microbial species to be quantified may be by any method provided herein or any other known to those of skill in the art (e.g., using open-source software tools such as BLAST, MetaPhlAn, Kraken, or Kaiju). One or more microbial species to be quantified may be amplified (e.g., by any method provided herein) or may not be amplified. It may be preferable to not amplify one or more microbial species to be quantified because then the proportion of the one or more microbial species in the sample (or subsample) more faithfully recapitulates the proportion of the one or more microbial species in the subject (e.g., human subject). In some embodiments, the one or more microbial species to be quantified are not amplified.

[0089] Quantification may be by any method known in the art. Non-limiting examples of quantifying one or more microbial species in a clinical sample include: measuring microbial genome coverage, measuring the number of times a target sequence (e.g., in a microbial genome) appears in a sample, and measuring colony-forming units (CFUs). In some embodiments, microbial genome coverage is measured to quantify one or more microbial species in a clinical sample. Measuring microbial genome coverage may be by any method provided herein (e.g., manually, using open-source software tools such as Samtools, Bedtools, Qualimap, or GATK). Quantification of one or more microbial species (e.g., in a clinical sample) may be expressed as a total number (e.g., total number of microbial cells, total number of CFUs, etc.) or as a relative number. If the quantification of one or more microbial species is expressed as a relative number, it may be relative to human or microbe (e.g., human cells, microbial cells, human DNA reads, microbial DNA reads). In some embodiments, quantification of one or more microbial species is by whole genome equivalents.

[0090] In some embodiments, the abundance of microbial species is quantified from an unamplified clinical subsample (e.g., second subsample) based on complete sequences from an amplified clinical sample (e.g., first subsample). Specifically, complete sequences may be used as a mapping reference for quantifying abundance of one or more microbial species in an unamplified clinical subsample. A mapping reference means that the complete sequences from the amplified clinical subsample may be used to align the sequences from the unamplified clinical subsample in order quantify the abundance of each microbial species in the unamplified clinical sample. Accurate quantification of one or more microbial species in a clinical sample can be achieved using a known concentration of a nucleic acid standard control (e.g., carrier nucleic acid) that has consistent sequencing output. In particular, a nucleic acid standard control is desirable in an unamplified clinical sample (e.g., microbial DNA), where the total level of nucleic acids may be low. Low means that a microbial species may be present in a clinical sample, but it’s present at a concentration that is below the limit of detection for a reaction (e.g., identification reaction, quantification reaction, DNA library preparation reaction, DNA sequencing reaction etc.). If the total level of nucleic acid is low in a clinical sample and a nucleic acid standard control is used to perform microbial identification, microbial quantification, or some combination thereof, technical failure of the reaction(s) can be eliminated during analysis. That is, if a known concentration of a nucleic acid standard control is added to a microbial identification reaction, a microbial quantification, or some combination thereof and no microbial species other than nucleic acid standard control is identified and / or quantified, technical failure of the reaction(s) can be eliminated as a cause of another microbial species not being identified.

[0091] Sequencing data from clinical samples is analyzed to measure the breadth of coverage of a microbial species genome within the sequencing data. This approach is particularly advantageous in sequencing data obtained from clinical samples, which may include human DNA and other confounding DNA sources in addition to DNA from one or more microbial species. By measuring the breadth of coverage of a microbial species genome, contaminating DNA (e.g., human DNA, confounding DNA) may be removed from microbial DNA for processing. In some embodiments, only microbial species with a 0.5x - lOx microbial genome coverage are identified in the present disclosure. In some embodiments, only microbial species with a 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, l.Ox, l.lx, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2. Ox, 2. lx, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3. Ox, 3. lx, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4. Ox, 4. lx, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x,

[0092] 4.9x, 5. Ox, 5. lx, 5.2x, 5.3x, 5.4x, 5.5x, 5.6x, 5.7x, 5.8x, 5.9x, 6. Ox, 6. lx, 6.2x, 6.3x, 6.4x,

[0093] 6.5x, 6.6x, 6.7x, 6.8x, 6.9x, 7.Ox, 7. lx, 7.2x, 7.3x, 7.4x, 7.5x, 7.6x, 7.7x, 7.8x, 7.9x, 8.0x,

[0094] 8. lx, 8.2x, 8.3x, 8.4x, 8.5x, 8.6x, 8.7x, 8.8x, 8.9x, 9. Ox, 9. lx, 9.2x, 9.3x, 9.4x, 9.5x, 9.6x,

[0095] 9.7x, 9.8x, 9.9x, or 10. Ox or more microbial genome coverage are identified in the present disclosure.

[0096] Sequencing reads following processing of a clinical sample are aligned to reference genomes from one or more microbial species. This alignment may be performed manually or by using an open-source software tool (e.g., bwa-mem, rhat, GraphMap, minimap2). The breadth of coverage of a microbial species may be calculated by simply counting sequencing reads for each microbial species or by reconstructing the whole genome of the one or more microbial species from sequencing reads in the clinical sample. Reconstructing the whole genome of one or more microbial species may be performed using an open-source software tool (e.g., flye, Canu, Raven, Shasta, Miniasm) or manually. Once a whole genome of one or more microbial species is reconstructed, the one or more microbial species may be identified using an open-source software tool (e.g., Kraken, BLAST) or manually.

[0097] In some embodiments, where amplified microbial DNA is detected using a sequencing modality (e.g., Illumina sequencing or Nanopore sequencing), the genome coverage of the amplified microbial DNA is at least 0. lx, at least 0.2x, at least 0.3x, at least 0.4x, at least 0.5x, at least 0.6x, at least 0.7x, at least 0.8x, at least 0.9x, at least l.Ox, at least 1.5x, at least 2. Ox, at least 2.5x, at least 3. Ox, at least 3.5x, at least 4. Ox, at least 4.5x, or at least 5. Ox or more. In some embodiments, where amplified microbial DNA is detected using a sequencing modality (e.g., Illumina sequencing or Nanopore sequencing), the genome coverage of the amplified microbial DNA is 0. lx - 0.2x, 0. lx - 0.3x, 0. lx - 0.4x, 0. lx - 0.5x, O.lx - 0.6x, O.lx - 0.7x, O.lx - 0.8x, O.lx - 0.9x, O.lx - l.Ox, O.lx - 1.5x, O.lx - 2. Ox, O. lx - 2.5x, O. lx - 3. Ox, O. lx - 3.5x, O. lx - 4. Ox, O. lx - 4.5x, O. lx - 5. Ox, 0.2x - 0.3x, 0.2x - 0.4x, 0.2x - 0.5x, 0.2x - 0.6x, 0.2x - 0.7x, 0.2x - 0.8x, 0.2x - 0.9x, 0.2x - l.Ox, 0.2x - 1.5x, 0.2x - 2. Ox, 0.2x - 2.5x, 0.2x - 3. Ox, 0.2x - 3.5x, 0.2x - 4. Ox, 0.2x - 4.5x, 0.2x - 5. Ox, 0.3x - 0.4x, 0.3x - 0.5x, 0.3x - 0.6x, 0.3x - 0.7x, 0.3x - 0.8x, 0.3x - 0.9x, 0.3x - l.Ox, 0.3x - 1.5x, 0.3x - 2. Ox, 0.3x - 2.5x, 0.3x - 3. Ox, 0.3x - 3.5x, 0.3x - 4. Ox, 0.3x - 4.5x, 0.3x - 5. Ox, 0.4x - 0.5x, 0.4x - 0.6x, 0.4x - 0.7x, 0.4x - 0.8x, 0.4x - 0.9x, 0.4x - l.Ox, 0.4x - 1.5x, 0.4x - 2. Ox, 0.4x - 2.5x, 0.4x - 3. Ox, 0.4x - 3.5x, 0.4x - 4. Ox, 0.4x - 4.5x, 0.4x - 5. Ox, 0.5x - 0.6x, 0.5x - 0.7x, 0.5x - 0.8x, 0.5x - 0.9x, 0.5x - l.Ox, 0.5x - 1.5x, 0.5x - 2. Ox, 0.5x - 2.5x, 0.5x - 3. Ox, 0.5x - 3.5x, 0.5x - 4. Ox, 0.5x - 4.5x, 0.5x - 5. Ox, 0.6x - 0.7x, 0.6x - 0.8x, 0.6x - 0.9x, 0.6x - l.Ox, 0.6x - 1.5x, 0.6x - 2. Ox, 0.6x - 2.5x, 0.6x - 3. Ox, 0.6x - 3.5x, 0.6x - 4. Ox, 0.6x - 4.5x, 0.6x - 5. Ox, 0.7x - 0.8x, 0.7x - 0.9x, 0.7x - l.Ox, 0.7x - 1.5x, 0.7x - 2. Ox, 0.7x - 2.5x, 0.7x - 3. Ox, 0.7x - 3.5x, 0.7x - 4. Ox, 0.7x - 4.5x, 0.7x - 5. Ox, 0.8x - 0.9x, 0.8x - l.Ox, 0.8x -1.5x, 0.8x - 2. Ox, 0.8x - 2.5x, 0.8x - 3. Ox, 0.8x - 3.5x, 0.8x - 4. Ox, 0.8x - 4.5x, 0.8x - 5. Ox, 0.9x - l.Ox, 0.9x - 1.5x, 0.9x - 2.0x, 0.9x - 2.5x, 0.9x - 3.0x, 0.9x - 3.5x, 0.9x - 4.0x, 0.9x - 4.5x, 0.9x - 5. Ox, l.Ox - 1.5x, l.Ox - 2. Ox, l.Ox - 2.5x, l.Ox - 3. Ox, l.Ox - 3.5x, l.Ox - 4. Ox, l.Ox - 4.5x, l.Ox - 5. Ox, 1.5x - 2.0x, 1.5x - 2.5x, 1.5x - 3.0x, 1.5x - 3.5x, 1.5x - 4.0x, 1.5x - 4.5x, 1.5x - 5. Ox, 2. Ox - 2.5x, 2. Ox - 3. Ox, 2. Ox - 3.5x, 2. Ox - 4. Ox, 2. Ox - 4.5x, 2. Ox - 5. Ox, 2.5x - 3.0x,2.5x - 3.5x, 2.5x - 4. Ox, 2.5x - 4.5x, 2.5x - 5. Ox, 3. Ox - 3.5x, 3. Ox - 4. Ox, 3. Ox - 4.5x, 3. Ox - 5. Ox, 3.5x - 4. Ox, 3.5x - 4.5x, 3.5x - 5. Ox, 4. Ox - 4.5x, 4. Ox - 5. Ox, or 4.5x - 5. Ox.

[0098] In some embodiments, where amplified microbial DNA is detected using a sequencing modality (e.g., Illumina sequencing or Nanopore sequencing), the genome coverage of the amplified microbial DNA is at least 0. lx, at least 0.2x, at least 0.3x, at least 0.4x, at least 0.5x, at least 0.6x, at least 0.7x, at least 0.8x, at least 0.9x, at least l.Ox, at least 1.5x, at least 2. Ox, at least 2.5x, at least 3. Ox, at least 3.5x, at least 4. Ox, at least 4.5x, or at least 5. Ox or more. In some embodiments, where amplified microbial DNA is detected using a sequencing modality (e.g., Illumina sequencing or Nanopore sequencing), the genome coverage of the amplified microbial DNA is 0. lx - 0.2x, 0. lx - 0.3x, 0. lx - 0.4x, 0. lx - 0.5x, O.lx - 0.6x, O.lx - 0.7x, O.lx - 0.8x, O.lx - 0.9x, O.lx - l.Ox, O.lx - 1.5x, O.lx - 2. Ox, O. lx - 2.5x, O. lx - 3. Ox, O. lx - 3.5x, O. lx - 4. Ox, O. lx - 4.5x, O. lx - 5. Ox, 0.2x - 0.3x, 0.2x - 0.4x, 0.2x - 0.5x, 0.2x - 0.6x, 0.2x - 0.7x, 0.2x - 0.8x, 0.2x - 0.9x, 0.2x - l.Ox, 0.2x - 1.5x, 0.2x - 2. Ox, 0.2x - 2.5x, 0.2x - 3. Ox, 0.2x - 3.5x, 0.2x - 4. Ox, 0.2x - 4.5x, 0.2x - 5. Ox, 0.3x - 0.4x, 0.3x - 0.5x, 0.3x - 0.6x, 0.3x - 0.7x, 0.3x - 0.8x, 0.3x - 0.9x, 0.3x - l.Ox, 0.3x - 1.5x, 0.3x - 2. Ox, 0.3x - 2.5x, 0.3x - 3. Ox, 0.3x - 3.5x, 0.3x - 4. Ox, 0.3x - 4.5x, 0.3x - 5. Ox, 0.4x - 0.5x, 0.4x - 0.6x, 0.4x - 0.7x, 0.4x - 0.8x, 0.4x - 0.9x, 0.4x - l.Ox, 0.4x - 1.5x, 0.4x - 2. Ox, 0.4x - 2.5x, 0.4x - 3. Ox, 0.4x - 3.5x, 0.4x - 4. Ox, 0.4x - 4.5x, 0.4x - 5. Ox, 0.5x - 0.6x, 0.5x - 0.7x, 0.5x - 0.8x, 0.5x - 0.9x, 0.5x - l.Ox, 0.5x - 1.5x, 0.5x - 2. Ox, 0.5x - 2.5x, 0.5x - 3. Ox, 0.5x - 3.5x, 0.5x - 4. Ox, 0.5x - 4.5x, 0.5x - 5. Ox, 0.6x - 0.7x, 0.6x - 0.8x, 0.6x - 0.9x, 0.6x - l.Ox, 0.6x - 1.5x, 0.6x - 2. Ox, 0.6x - 2.5x, 0.6x - 3. Ox, 0.6x - 3.5x, 0.6x - 4. Ox, 0.6x - 4.5x, 0.6x - 5. Ox, 0.7x - 0.8x, 0.7x - 0.9x, 0.7x - l.Ox, 0.7x - 1.5x, 0.7x - 2. Ox, 0.7x - 2.5x, 0.7x - 3. Ox, 0.7x - 3.5x, 0.7x - 4. Ox, 0.7x - 4.5x, 0.7x - 5. Ox, 0.8x - 0.9x, 0.8x - l.Ox, 0.8x -1.5x, 0.8x - 2. Ox, 0.8x - 2.5x, 0.8x - 3. Ox, 0.8x - 3.5x, 0.8x - 4. Ox, 0.8x - 4.5x, 0.8x - 5. Ox, 0.9x - l.Ox, 0.9x - 1.5x, 0.9x - 2.0x, 0.9x - 2.5x, 0.9x - 3.0x, 0.9x - 3.5x, 0.9x - 4.0x, 0.9x - 4.5x, 0.9x - 5. Ox, l.Ox - 1.5x, l.Ox - 2. Ox, l.Ox - 2.5x, l.Ox - 3. Ox, l.Ox - 3.5x, l.Ox - 4. Ox, l.Ox - 4.5x, l.Ox - 5. Ox, 1.5x - 2.0x, 1.5x - 2.5x, 1.5x - 3.0x, 1.5x - 3.5x, 1.5x - 4.0x, 1.5x - 4.5x, 1.5x - 5. Ox, 2. Ox - 2.5x, 2. Ox - 3. Ox, 2. Ox - 3.5x, 2. Ox - 4. Ox, 2. Ox - 4.5x, 2. Ox - 5. Ox, 2.5x - 3.0x,2.5x - 3.5x, 2.5x - 4. Ox, 2.5x - 4.5x, 2.5x - 5. Ox, 3. Ox - 3.5x, 3. Ox - 4. Ox, 3. Ox - 4.5x, 3. Ox - 5. Ox, 3.5x - 4. Ox, 3.5x - 4.5x, 3.5x - 5. Ox, 4. Ox - 4.5x, 4. Ox - 5. Ox, or 4.5x - 5. Ox.

[0099] Nucleic acid standard control

[0100] In some embodiments, a nucleic acid standard control (e.g., carrier nucleic acid) is a nucleic acid sequence from a bacterium. A bacterium is a single-celled microorganism that belongs to the domain Bacteria. They are prokaryotic organisms, meaning that they lack a cell nucleus and other membrane-bound organelles found in eukaryotic cells. In some embodiments, a nucleic acid standard control is a nucleic acid from a Gram-positive bacterium. A Gram-positive bacterium has a thick peptidoglycan cell wall that retains the crystal violet stain used in the Gram staining process and has no outer membrane. Nonlimiting examples of Gram-positive bacterial genera include Actinomyces, Aeromicrobium, Agrococcus, Agromyces, Alkalilimnicola, Arenimonas, Auraticoccus, Azoarcus, Azospira, Bacillus, Brachybacterium, Castellaniella, Cellulomonas, Clostridium, Corynebacterium, Deinococcus, Enterococcus, Georgenia, Gephyromycinifex, Ilumatobacter, Isoptericola, Isoptericola, Lactobacillus, Leucobacter, Limnochorda, Listeria, Luteimicrobium, Luteimonas, Lysobacter, Marinicauda, Marmoricola, Melaminivora, Microcella, Miltoncostaea, Mycobacterium, Paraoerskovia, Protaetiibacter, Pulveribacter, Rathayibacter, Sanguibacter, Serinicoccus, Staphylococcus, Starkeya, Streptococcus Tessaracoccus, Thauera, and Thermomonas. In some embodiments, a nucleic acid standard control is from a bacterium in the genus Leucobacter .

[0101] In some embodiments, a nucleic acid standard control is from a species selected from Leucobacter muris, Leucobacter triazinivorans, Cellulomonas fimi, Cellulomonas flavigena, Cellulomonas iranensis, Serinicoccus chungangensis, Serinicoccus hydrothermalis, Georgenia wutianyii, Georgenia yuyongxinii, or Agrococcus jejuensis. In some embodiments, a nucleic acid standard control is from a bacterial species selected from Aeromicrobium choanae, Aeromicrobium marinum, Agromyces archimandritae, Alkalilimnicola ehrlichii, Arenimonas daejeonensis, Auraticoccus monumenti, Azoarcus olearius, Azospira restricta, Brachybacterium faecium, Brachybacterium ginsengisoli, Castellaniella defragrans, Deinococcus maricopensis, Deinococcus radiodurans, Deinococcus radiopugnans, Gephyromycinifex aptenodytis, Ilumatobacter coccineus, Isoptericola dokdonensis, Isoptericola variabilis, Limnochorda pilosa, Luteimicrobium xylanilyticum, Luteimonas chenhongjianii, Luteimonas granuli, Lysobacter caseinilyticus, Lysobacter ciconiae, Lysobacter helvus, Lysobacter maris, Lysobacter soli, Lysobacter solisilvae, Marinicauda algicola, Marmoricola scoriae, Melaminivora jejuensis, Microcella jlavibacter, Paraoerskovia marina, Protaetiibacter larvae, Pulveribacter suum, Rathayibacter iranicus, Rathayibacter rathayi, Rathayibacter tritici, Sanguibacter keddieii, Starkeya novella, Tessaracoccus lapidicaptus, Thauera chlorobenzoica, or Thermomonas brevis.

[0102] In some embodiments, a nucleic acid standard control is from a Gram-negative bacterium. A Gram-negative bacterium has a thin peptidoglycan cell wall, an outer membrane composed of lipopolysaccharides, and does not retain the crystal violet stain used in the Gram staining process. Non-limiting examples of Gram-negative bacterial genera include Acinetobacter, Bdellovibrio, Bordetella, Brucella, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pasturella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Stenotrophomonas, and Vibrio.

[0103] In some embodiments, a nucleic acid standard control is from a Gram-positive bacterium. A Gram-positive bacterium has a thick peptidoglycan cell wall, no outer membrane, and retains the crystal violet stain used in the Gram staining process. Nonlimiting examples of Gram-positive bacterial genera include Staphylococcus, Streptococcus, Bacillus, and Clostridium.

[0104] In some embodiments, a nucleic acid standard control is from a virus. A virus is a microscopic infectious agent that lacks the ability to carry out metabolic processes, grow, or reproduce independently. A virus can be a DNA virus (e.g., Adenoviruses, Herpesviruses, Poxviruses, Parvoviruses') or an RNA virus (e.g., Reoviruses, Picornaviruses, Togaviruses, Orthomyxoviruses, Rhabdoviruses, Retroviruses).

[0105] An RNA virus contains RNA as its genetic material, and a DNA virus contains DNA as its genetic material. In some embodiments where a genetic material is RNA, a method provided herein comprises a reverse transcription step. Reverse transcription is synthesis of a new DNA molecule by the enzyme reverse transcriptase.

[0106] In embodiments of the present disclosure where a pathogenic microbial species is a virus, a method provided herein comprises lysing the virus. Lysing the virus may be by any method known in the art including, but not limited to: chemical lysis (e.g., sodium dodecyl sulfate (SDS) treatment, EDTA treatment, surfactant treatment), temperature lysis (e.g., repeated freeze / thaw cycles), mechanical lysis (e.g., homogenizer, bead beating), or some combination thereof.

[0107] In some embodiments, the RNA virus is a single-stranded RNA virus or a doublestranded RNA virus. In some embodiments, the single-stranded RNA virus is a singlestranded positive-sense RNA virus that can be translated into protein by a host cell (e.g., a cell in a subject). In some embodiments, the single-stranded RNA virus is a single-stranded negative-sense RNA virus that must be converted to positive-sense RNA prior to being translated into protein by a host cell (e.g., a cell in a subject). In some embodiments, the DNA virus is a single-stranded DNA virus, a partially double-stranded DNA virus, or a doublestranded DNA virus. in some embodiments, a nucleic acid standard control is from a fungus. A fungus is a eukaryotic organism belonging to the Kingdom Fungi. Non-limiting examples of fungal genera include Agaricus, Amanita, Aspergillus, Candida, Claviceps, Cryptococcus, Fusarium, Morchella, Penicillium, Puccinia, Rhizopus, Saccharomyces, and Trichoderma.

[0108] In some embodiments, one or more nucleic acid standard controls are bacteria (e.g., Gram-positive bacterium and / or Gram-negative bacteria), viruses, fungi, or any combination thereof. In some embodiments, nucleic acid standard controls are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 from different nucleic acid standard controls. In some embodiments, one or more nucleic acid standard controls are 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 5-15, 10-20, 1-20, 2-19, 3-18, 4-17, 5-16, 6-15, 7-14, 8-13, or 9-12 different nucleic acid standard controls.

[0109] In some embodiments, one or more nucleic acid standard controls are synthetic nucleic acids. A synthetic nucleic acid, as used herein, is a polynucleotide that is created in vitro chemically or enzymatically. In some embodiments, one or more nucleic acid controls are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different synthetic nucleic acids.

[0110] Relative abundance

[0111] The abundance of a microbial species (e.g., bacteria, virus, fungus, or parasite) can be an important factor in determining its clinical relevance. For example, in some embodiments, the abundance of a microbial species can be used as a diagnostic criterion. The term “clinically relevant”, as used herein, refers to a microbial species (e.g., bacteria, virus, fungus, or parasite) that has the potential to cause disease or infection in humans or other subjects. In some embodiments, a clinically relevant microbial species is a pathogenic microbial species.

[0112] In some embodiments, the abundance of a microbial species is at least 1 CFU / mL, at least 5 CFU / mL, at least 10 CFU / mL, at least 50 CFU / mL, at least 100 CFU / mL, at least 500 CFU / mL, at least 1,000 CFU / mL, at least 5,000 CFU / mL, at least 10,000 CFU / mL, at least 50,000 CFU / mL, or at least 100,000 CFU / mL. In some embodiments, the abundance of a microbial species is 1 CFU / mL - 5 CFU / mL, 1 CFU / mL - 10 CFU / mL, 1 CFU / mL - 50 CFU / mL, 1 CFU / mL - 100 CFU / mL, 1 CFU / mL - 500 CFU / mL, 1 CFU / mL - 1,000 CFU / mL, 1 CFU / mL - 5,000 CFU / mL, 1 CFU / mL - 10,000 CFU / mL, 1 CFU / mL - 50,000 CFU / mL, 1 CFU / mL - 100,000 CFU / mL, 5 CFU / mL - 10 CFU / mL, 5 CFU / mL - 50 CFU / mL, 5 CFU / mL - 100 CFU / mL, 5 CFU / mL - 500 CFU / mL, 5 CFU / mL - 1,000

[0113] - Il CFU / mL, 5 CFU / mL - 5,000 CFU / mL, 5 CFU / mL - 10,000 CFU / mL, 5 CFU / mL - 50,000 CFU / mL, 5 CFU / mL - 100,000 CFU / mL, 10 CFU / mL - 50 CFU / mL, 10 CFU / mL - 100 CFU / mL, 10 CFU / mL - 500 CFU / mL, 10 CFU / mL - 1,000 CFU / mL, 10 CFU / mL - 5,000 CFU / mL, 10 CFU / mL - 10,000 CFU / mL, 10 CFU / mL - 50,000 CFU / mL, 10 CFU / mL - 100,000 CFU / mL, 50 CFU / mL - 100 CFU / mL, 50 CFU / mL - 500 CFU / mL, 10 CFU / mL - 1,000 CFU / mL, 10 CFU / mL - 5,000 CFU / mL, 10 CFU / mL - 10,000 CFU / mL, 10 CFU / mL

[0114] - 50,000 CFU / mL, 10 CFU / mL - 100,000 CFU / mL, 50 CFU / mL - 100 CFU / mL, 50 CFU / mL - 500 CFU / mL, 50 CFU / mL - 1,000 CFU / mL, 50 CFU / mL - 5,000 CFU / mL, 50 CFU / mL - 10,000 CFU / mL, 50 CFU / mL - 50,000 CFU / mL, 50 CFU / mL - 100,000 CFU / mL, 100 CFU / mL - 500 CFU / mL, 100 CFU / mL - 1,000 CFU / mL, 100 CFU / mL - 5,000 CFU / mL, 100 CFU / mL - 10,000 CFU / mL, 100 CFU / mL - 50,000 CFU / mL, 100 CFU / mL - 100,000 CFU / mL, 500 CFU / mL - 1,000 CFU / mL, 500 CFU / mL - 5,000 CFU / mL, 500 CFU / mL - 10,000 CFU / mL, 500 CFU / mL - 50,000 CFU / mL, 500 CFU / mL - 100,000 CFU / mL, 1,000 CFU / mL - 5,000 CFU / mL, 1,000 CFU / mL - 10,000 CFU / mL, 1,000 CFU / mL - 50,000 CFU / mL, 1,000 CFU / mL - 100,000 CFU / mL, 5,000 CFU / mL - 10,000 CFU / mL, 5,000 CFU / mL - 50,000 CFU / mL, 5,000 CFU / mL - 100,000 CFU / mL, 10,000 CFU / mL - 50,000 CFU / mL, 10,000 CFU / mL - 100,000 CFU / mL, or 50,000 CFU / mL - 100,000 CFU / mL.

[0115] In some embodiments, the total abundance of microbial species in a clinical sample is at least 1 CFU / mL, at least 5 CFU / mL, at least 10 CFU / mL, at least 50 CFU / mL, at least 100 CFU / mL, at least 500 CFU / mL, at least 1,000 CFU / mL, at least 5,000 CFU / mL, at least 10,000 CFU / mL, at least 50,000 CFU / mL, or at least 100,000 CFU / mL. In some embodiments, the total abundance of microbial species in a clinical sample is 1 CFU / mL - 5 CFU / mL, 1 CFU / mL - 10 CFU / mL, 1 CFU / mL - 50 CFU / mL, 1 CFU / mL - 100 CFU / mL, 1 CFU / mL - 500 CFU / mL, 1 CFU / mL - 1,000 CFU / mL, 1 CFU / mL - 5,000 CFU / mL, 1 CFU / mL - 10,000 CFU / mL, 1 CFU / mL - 50,000 CFU / mL, 1 CFU / mL - 100,000 CFU / mL, 5 CFU / mL - 10 CFU / mL, 5 CFU / mL - 50 CFU / mL, 5 CFU / mL - 100 CFU / mL, 5 CFU / mL

[0116] - 500 CFU / mL, 5 CFU / mL - 1,000 CFU / mL, 5 CFU / mL - 5,000 CFU / mL, 5 CFU / mL - 10,000 CFU / mL, 5 CFU / mL - 50,000 CFU / mL, 5 CFU / mL - 100,000 CFU / mL, 10 CFU / mL

[0117] - 50 CFU / mL, 10 CFU / mL - 100 CFU / mL, 10 CFU / mL - 500 CFU / mL, 10 CFU / mL - 1,000 CFU / mL, 10 CFU / mL - 5,000 CFU / mL, 10 CFU / mL - 10,000 CFU / mL, 10 CFU / mL

[0118] - 50,000 CFU / mL, 10 CFU / mL - 100,000 CFU / mL, 50 CFU / mL - 100 CFU / mL, 50 CFU / mL - 500 CFU / mL, 10 CFU / mL - 1,000 CFU / mL, 10 CFU / mL - 5,000 CFU / mL, 10 CFU / mL - 10,000 CFU / mL, 10 CFU / mL - 50,000 CFU / mL, 10 CFU / mL - 100,000 CFU / mL, 50 CFU / mL - 100 CFU / mL, 50 CFU / mL - 500 CFU / mL, 50 CFU / mL - 1,000 CFU / mL, 50 CFU / mL - 5,000 CFU / mL, 50 CFU / mL - 10,000 CFU / mL, 50 CFU / mL - 50,000 CFU / mL, 50 CFU / mL - 100,000 CFU / mL, 100 CFU / mL - 500 CFU / mL, 100 CFU / mL - 1,000 CFU / mL, 100 CFU / mL - 5,000 CFU / mL, 100 CFU / mL - 10,000 CFU / mL, 100 CFU / mL - 50,000 CFU / mL, 100 CFU / mL - 100,000 CFU / mL, 500 CFU / mL - 1,000 CFU / mL, 500 CFU / mL - 5,000 CFU / mL, 500 CFU / mL - 10,000 CFU / mL, 500 CFU / mL - 50,000 CFU / mL, 500 CFU / mL - 100,000 CFU / mL, 1,000 CFU / mL - 5,000 CFU / mL, 1,000 CFU / mL - 10,000 CFU / mL, 1,000 CFU / mL - 50,000 CFU / mL, 1,000 CFU / mL - 100,000 CFU / mL, 5,000 CFU / mL - 10,000 CFU / mL, 5,000 CFU / mL - 50,000 CFU / mL, 5,000 CFU / mL - 100,000 CFU / mL, 10,000 CFU / mL - 50,000 CFU / mL, 10,000 CFU / mL - 100,000 CFU / mL, or 50,000 CFU / mL - 100,000 CFU / mL.

[0119] In some embodiments, the relative abundance of a microbial species in a clinical sample (e.g., relative to a non-microbial subject’s cell abundance, such as a human subject’s cell abundance) can be used as a diagnostic criterion. In some embodiments, the relative abundance of a microbial species relative to a subject’s cell abundance (e.g., a human subject’s cell abundance) can be used as a diagnostic criterion. In some embodiments, the abundance of a microbial species is at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least lOx, at least 20x, at least 3 Ox, at least 40x, at least 50x, at least 60x, at least 70x, at least 80x, at least 90x, at least lOOx, at least 150x, at least 200x, at least 250x, at least 300x, at least 350x, at least 400x or more than the abundance of a subject’s cell abundance (e.g., a human subject’s cell abundance) in a clinical sample. In some embodiments, the abundance of a microbial species is 2x - 3x, 2x - 4x, 2x - 5x, 2x - 6x, 2x - 7x, 2x - 8x, 2x - 9x, 2x - lOx, 2x - 20x, 2x - 3 Ox, 2x - 40x, 2x - 5 Ox, 2x - 60x, 2x

[0120] - 70x, 2x - 80x, 2x - 90x, 2x - lOOx, 2x - 150x, 2x - 200x, 2x - 250x, 2x - 300x, 2x - 350x, 2x - 400x, 3x - 4x, 3x - 5x, 3x - 6x, 3x - 7x, 3x - 8x, 3x - 9x, 3x - lOx, 3x - 20x, 3x - 3 Ox, 3x - 40x, 3x - 50x, 3x - 60x, 3x - 70x, 3x - 80x, 3x - 90x, 3x - lOOx, 3x - 150x, 3x - 200x, 3x - 250x, 3x - 300x, 3x - 350x, 3x - 400x, 4x - 5x, 4x - 6x, 4x - 7x, 4x - 8x, 4x - 9x, 4x - lOx, 4x - 20x, 4x - 30x, 4x - 40x, 4x - 50x, 4x - 60x, 4x - 70x, 4x - 80x, 4x - 90x, 4x - lOOx, 4x - 150x, 4x - 200x, 4x - 250x, 4x - 300x, 4x - 350x, 4x - 400x, 5x - 6x, 5x - 7x, 5x

[0121] - 8x, 5x - 9x, 5x - lOx, 5x - 20x, 5x - 30x, 5x - 40x, 5x - 50x, 5x - 60x, 5x - 70x, 5x - 80x, 5x - 90x, 5x - lOOx, 5x - 150x, 5x - 200x, 5x - 250x, 5x - 300x, 5x - 350x, 5x - 400x, 6x - 7x, 6x - 8x, 6x - 9x, 6x - lOx, 6x - 20x, 6x - 3 Ox, 6x - 40x, 6x - 5 Ox, 6x - 60x, 6x - 70x, 6x

[0122] - 80x, 6x - 90x, 6x - lOOx, 6x - 150x, 6x - 200x, 6x - 250x, 6x - 300x, 6x - 350x, 6x - 400x, 7x - 8x, 7x - 9x, 7x - lOx, 7x - 20x, 7x - 30x, 7x - 40x, 7x - 50x, 7x - 60x, 7x - 70x, 7x - 80x, 7x - 90x, 7x - lOOx, 7x - 150x, 7x - 200x, 7x - 250x, 7x - 300x, 7x - 350x, 7x - 400x, 8x - 9x, 8x - lOx, 8x - 20x, 8x - 3 Ox, 8x - 40x, 8x - 5 Ox, 8x - 60x, 8x - 70x, 8x - 80x, 8x - 90x, 8x - lOOx, 8x - 150x, 8x - 200x, 8x - 250x, 8x - 300x, 8x - 350x, 8x - 400x, 9x - lOx, 9x - 20x, 9x - 30x, 9x - 40x, 9x - 50x, 9x - 60x, 9x - 70x, 9x - 80x, 9x - 90x, 9x

[0123] - lOOx, 9x - 150x, 9x - 200x, 9x - 250x, 9x - 300x, 9x - 350x, 9x - 400x, lOx - 20x, lOx - 30x, lOx - 40x, lOx - 50x, lOx - 60x, lOx - 70x, lOx - 80x, lOx - 90x, lOx - lOOx, lOx - 150x, lOx - 200x, lOx - 250x, lOx - 300x, lOx - 350x, lOx - 400x, 20x - 30x, 20x - 40x, 20x - 50x, 20x - 60x, 20x - 70x, 20x - 80x, 20x - 90x, 20x - lOOx, 20x - 150x, 20x - 200x, 20x - 250x, 20x - 300x, 20x - 350x, 20x - 400x, 30x - 40x, 30x - 50x, 30x - 60x, 30x - 70x, 30x - 80x, 30x - 90x, 30x - lOOx, 30x - 150x, 30x - 200x, 30x - 250x, 30x - 300x, 30x

[0124] - 350x, 30x - 400x, 40x - 50x, 40x - 60x, 40x - 70x, 40x - 80x, 40x - 90x, 40x - lOOx, 40x

[0125] - 150x, 40x - 200x, 40x - 250x, 40x - 300x, 40x - 350x, 40x - 400x, 50x - 60x, 50x - 70x, 50x - 80x, 50x - 90x, 50x - lOOx, 50x - 150x, 50x - 200x, 50x - 250x, 50x - 300x, , 50x - 350x, 50x - 400x, 60x - 70x, 60x - 80x, 60x - 90x, 60x - lOOx, 60x - 150x, 60x - 200x, 60x

[0126] - 250x, 60x - 300x, , 60x - 350x, 60x - 400x, 70x - 80x, 70x - 90x, 70x - lOOx, 70x - 150x, 70x - 200x, 70x - 250x, 70x - 300x, 70x - 350x, 70x - 400x, 80x - 90x, 80x - lOOx, 80x - 150x, 80x - 200x, 80x - 250x, 80x - 300x, 80x - 350x, 80x - 400x, 90x - lOOx, 90x - 150x, 90x - 200x, 90x - 250x, 90x - 300x, 90x - 350x, 90x - 400x, lOOx - 150x, lOOx - 200x, 100x - 250x, 100x - 300x, 100x - 350x, 100x - 400x, 150x - 200x, 150x - 250x, 150x - 300x, 150x - 350x, 150x - 400x, 200x - 250x, 200x - 300x, 200x - 350x, 200x - 400x, 250x

[0127] - 300x, 250x - 350x, 250x - 400x, 300x - 350x, 300x - 400x, or 350x - 400x, more than the abundance of a subject’s cell abundance (e.g., a human subject’s cell abundance) in a clinical sample.

[0128] In some embodiments, the relative abundance of a microbial species relative to an internal control can be used as a diagnostic criterion. Non-limiting examples of internal controls include microbial species present in the sample (e.g., commensal microbial species), microbial species introduced into the sample, or synthetic controls (e.g., synthetic DNA sequences). An internal control may be introduced at any point during sample processing, including, but not limited to, after obtaining a sample but before any processing steps, after some processing steps but before other processing steps, or after all the processing steps but before any identification or quantification is performed. In some embodiments, an internal control may be added to one subsample but not to another subsample. In some embodiments, an internal control may be added to multiple subsamples. In some embodiments, an internal control may be added to all subsamples. In some embodiments, an internal control is carrier DNA.

[0129] In some embodiments, the relative abundance of a microbial species relative to another microbial species (e.g., commensal microbial species) present in the clinical sample can be used as a diagnostic criterion. In some embodiments, the abundance of a microbial species is at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least lOx, at least 20x, at least 30x, at least 40x, at least 50x, at least 60x, at least 70x, at least 80x, at least 90x, at least lOOx, at least 150x, at least 200x, at least 250x, at least 300x, at least 350x, at least 400x or more than the abundance of another microbial species (e.g., commensal microbial species) present in the clinical sample. In some embodiments, the abundance of a microbial species is 2x - 3x, 2x - 4x, 2x - 5x, 2x - 6x, 2x - 7x, 2x - 8x, 2x - 9x, 2x - lOx, 2x - 20x, 2x - 30x, 2x - 40x, 2x - 50x, 2x - 60x, 2x - 70x, 2x - 80x, 2x - 90x, 2x - lOOx, 2x - 150x, 2x - 200x, 2x - 250x, 2x - 300x, 2x - 350x, 2x - 400x, 3x - 4x, 3x - 5x, 3x - 6x, 3x - 7x, 3x - 8x, 3x - 9x, 3x - lOx, 3x - 20x, 3x - 30x, 3x - 40x, 3x - 50x, 3x - 60x, 3x - 70x, 3x - 80x, 3x - 90x, 3x - lOOx, 3x - 150x, 3x - 200x, 3x - 250x, 3x - 300x, 3x

[0130] - 350x, 3x - 400x, 4x - 5x, 4x - 6x, 4x - 7x, 4x - 8x, 4x - 9x, 4x - lOx, 4x - 20x, 4x - 30x, 4x - 40x, 4x - 50x, 4x - 60x, 4x - 70x, 4x - 80x, 4x - 90x, 4x - lOOx, 4x - 150x, 4x - 200x, 4x - 250x, 4x - 300x, 4x - 350x, 4x - 400x, 5x - 6x, 5x - 7x, 5x - 8x, 5x - 9x, 5x - lOx, 5x

[0131] - 20x, 5x - 30x, 5x - 40x, 5x - 50x, 5x - 60x, 5x - 70x, 5x - 80x, 5x - 90x, 5x - lOOx, 5x - 150x, 5x - 200x, 5x - 250x, 5x - 300x, 5x - 350x, 5x - 400x, 6x - 7x, 6x - 8x, 6x - 9x, 6x - lOx, 6x - 20x, 6x - 30x, 6x - 40x, 6x - 50x, 6x - 60x, 6x - 70x, 6x - 80x, 6x - 90x, 6x - lOOx, 6x - 150x, 6x - 200x, 6x - 250x, 6x - 300x, 6x - 350x, 6x - 400x, 7x - 8x, 7x - 9x, 7x

[0132] - lOx, 7x - 20x, 7x - 30x, 7x - 40x, 7x - 50x, 7x - 60x, 7x - 70x, 7x - 80x, 7x - 90x, 7x - lOOx, 7x - 150x, 7x - 200x, 7x - 250x, 7x - 300x, 7x - 350x, 7x - 400x, 8x - 9x, 8x - lOx, 8x - 20x, 8x - 30x, 8x - 40x, 8x - 50x, 8x - 60x, 8x - 70x, 8x - 80x, 8x - 90x, 8x - lOOx, 8x

[0133] - 150x, 8x - 200x, 8x - 250x, 8x - 300x, 8x - 350x, 8x - 400x, 9x - lOx, 9x - 20x, 9x - 30x, 9x - 40x, 9x - 50x, 9x - 60x, 9x - 70x, 9x - 80x, 9x - 90x, 9x - lOOx, 9x - 150x, 9x - 200x, 9x - 250x, 9x - 300x, 9x - 350x, 9x - 400x, lOx - 20x, lOx - 3Ox, lOx - 40x, lOx - 5Ox, lOx

[0134] - 60x, lOx - 70x, lOx - 8Ox, lOx - 90x, lOx - lOOx, lOx - 15Ox, lOx - 200x, lOx - 250x, lOx - 3OOx, lOx - 35 Ox, lOx - 400x, 20x - 3 Ox, 20x - 40x, 20x - 5 Ox, 20x - 60x, 20x - 70x, 20x - 8Ox, 20x - 90x, 20x - lOOx, 20x - 15Ox, 20x - 200x, 20x - 250x, 20x - 3OOx, 20x - 35Ox, 20x - 400x, 3Ox - 40x, 3Ox - 5Ox, 3Ox - 60x, 3Ox - 70x, 3Ox - 8Ox, 3Ox - 90x, 3Ox - lOOx, 3Ox - 15Ox, 3Ox - 200x, 3Ox - 250x, 3Ox - 3OOx, 3Ox - 35Ox, 3Ox - 400x, 40x - 5Ox, 40x - 60x, 40x - 70x, 40x - 8Ox, 40x - 90x, 40x - lOOx, 40x - 15Ox, 40x - 200x, 40x - 250x, 40x - 3OOx, 40x - 35Ox, 40x - 400x, 5Ox - 60x, 5Ox - 70x, 5Ox - 8Ox, 5Ox - 90x, 5Ox - lOOx, 5Ox - 15Ox, 5Ox - 200x, 5Ox - 250x, 5Ox - 3OOx, 5Ox - 35Ox, 5Ox - 400x, 60x -70x, 60x - 8Ox, 60x - 90x, 60x - lOOx, 60x - 15Ox, 60x - 200x, 60x - 250x, 60x - 3OOx, 60x - 35Ox, 60x - 400x, 70x - 8Ox, 70x - 90x, 70x - lOOx, 70x - 15Ox, 70x - 200x, 70x -250x, 70x - 3OOx, 70x - 35Ox, 70x - 400x, 8Ox - 90x, 8Ox - lOOx, 8Ox - 15Ox, 8Ox - 200x, 8Ox - 250x, 8Ox - 3OOx, 8Ox - 35Ox, 8Ox - 400x, 90x - lOOx, 90x - 15Ox, 90x - 200x, 90x -250x, 90x - 3OOx, 90x - 35Ox, 90x - 400x, lOOx - 15Ox, lOOx - 200x, lOOx - 250x, lOOx -3OOx, 100x - 350x, 100x - 400x, 150x - 200x, 150x - 250x, 150x - 300x, 150x - 350x, 15Ox

[0135] - 400x, 200x - 250x, 200x - 3OOx, 200x - 35Ox, 200x - 400x, 250x - 3OOx, 250x - 35Ox, 250x - 400x, 3OOx - 35Ox, 3OOx - 400x, or 35Ox - 400x, more than the abundance of another microbial species (e.g., commensal microbial species) present in the clinical sample.

[0136] In some embodiments, the relative abundance of a microbial species relative to a microbial species introduced into a clinical sample (e.g., carrier nucleic acid) can be used as a diagnostic criterion. In some embodiments, the abundance of a microbial species is at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least lOx, at least 20x, at least 30x, at least 40x, at least 50x, at least 60x, at least 70x, at least 80x, at least 90x, at least lOOx, at least 150x, at least 200x, at least 250x, at least 3OOx, at least 35Ox, at least 400x or more than the abundance of a microbial species introduced into a clinical sample. In some embodiments, the abundance of a microbial species is 2x - 3x, 2x - 4x, 2x - 5x, 2x - 6x, 2x - 7x, 2x - 8x, 2x - 9x, 2x - lOx, 2x - 20x, 2x - 3 Ox, 2x - 40x, 2x - 50x, 2x - 60x, 2x - 70x, 2x - 80x, 2x - 90x, 2x - lOOx, 2x - 150x, 2x - 200x, 2x - 250x, 2x

[0137] - 300x, 2x - 350x, 2x - 400x, 3x - 4x, 3x - 5x, 3x - 6x, 3x - 7x, 3x - 8x, 3x - 9x, 3x - lOx, 3x - 20x, 3x - 30x, 3x - 40x, 3x - 50x, 3x - 60x, 3x - 70x, 3x - 80x, 3x - 90x, 3x - lOOx, 3x

[0138] - 150x, 3x - 200x, 3x - 250x, 3x - 300x, 3x - 350x, 3x - 400x, 4x - 5x, 4x - 6x, 4x - 7x, 4x

[0139] - 8x, 4x - 9x, 4x - lOx, 4x - 20x, 4x - 30x, 4x - 40x, 4x - 50x, 4x - 60x, 4x - 70x, 4x - 80x, 4x - 90x, 4x - lOOx, 4x - 15Ox, 4x - 200x, 4x - 250x, 4x - 300x, 4x - 350x, 4x - 400x, 5x - 6x, 5x - 7x, 5x - 8x, 5x - 9x, 5x - lOx, 5x - 20x, 5x - 30x, 5x - 40x, 5x - 50x, 5x - 60x, 5x

[0140] - 70x, 5x - 80x, 5x - 90x, 5x - lOOx, 5x - 15Ox, 5x - 200x, 5x - 250x, 5x - 3OOx, 5x - 35Ox, 5x - 400x, 6x - 7x, 6x - 8x, 6x - 9x, 6x - lOx, 6x - 20x, 6x - 3 Ox, 6x - 40x, 6x - 5 Ox, 6x - 60x, 6x - 70x, 6x - 8Ox, 6x - 90x, 6x - lOOx, 6x - 15Ox, 6x - 200x, 6x - 250x, 6x - 3OOx, 6x

[0141] - 35Ox, 6x - 400x, 7x - 8x, 7x - 9x, 7x - lOx, 7x - 20x, 7x - 3Ox, 7x - 40x, 7x - 5Ox, 7x - 60x, 7x - 70x, 7x - 8Ox, 7x - 90x, 7x - lOOx, 7x - 15Ox, 7x - 200x, 7x - 250x, 7x - 3OOx, 7x

[0142] - 35 Ox, 7x - 400x, 8x - 9x, 8x - lOx, 8x - 20x, 8x - 3 Ox, 8x - 40x, 8x - 5 Ox, 8x - 60x, 8x - 70x, 8x - 8Ox, 8x - 90x, 8x - lOOx, 8x - 15Ox, 8x - 200x, 8x - 250x, 8x - 3OOx, 8x - 35Ox, 8x - 400x, 9x - lOx, 9x - 20x, 9x - 3Ox, 9x - 40x, 9x - 5Ox, 9x - 60x, 9x - 70x, 9x - 8Ox, 9x - 90x, 9x - lOOx, 9x - 15Ox, 9x - 200x, 9x - 250x, 9x - 3OOx, 9x - 35Ox, 9x - 400x, lOx - 20x, lOx - 3Ox, lOx - 40x, lOx - 5Ox, lOx - 60x, lOx - 70x, lOx - 8Ox, lOx - 90x, lOx - lOOx, lOx - 15Ox, lOx - 200x, lOx - 250x, lOx - 3OOx, lOx - 35Ox, lOx - 400x, 20x - 3Ox, 20x - 40x, 20x - 5Ox, 20x - 60x, 20x - 70x, 20x - 8Ox, 20x - 90x, 20x - lOOx, 20x - 15Ox, 20x - 200x, 20x - 250x, 20x - 3OOx, 20x - 35Ox, 20x - 400x, 3Ox - 40x, 3Ox - 5Ox, 3Ox - 60x, 3Ox - 70x, 3Ox - 8Ox, 3Ox - 90x, 3Ox - lOOx, 3Ox - 15Ox, 3Ox - 200x, 3Ox - 250x, 3Ox

[0143] - 3OOx, 3Ox - 35Ox, 3Ox - 400x, 40x - 5Ox, 40x - 60x, 40x - 70x, 40x - 8Ox, 40x - 90x, 40x

[0144] - lOOx, 40x - 15Ox, 40x - 200x, 40x - 250x, 40x - 3OOx, 40x - 35Ox, 40x - 400x, 5Ox - 60x, 5Ox - 70x, 5Ox - 8Ox, 5Ox - 90x, 5Ox - lOOx, 5Ox - 15Ox, 5Ox - 200x, 5Ox - 250x, 5Ox - 3OOx, 5Ox - 35Ox, 5Ox - 400x, 60x - 70x, 60x - 8Ox, 60x - 90x, 60x - lOOx, 60x - 15Ox, 60x

[0145] - 200x, 60x - 250x, 60x - 3OOx, 60x - 35Ox, 60x - 400x, 70x - 8Ox, 70x - 90x, 70x -lOOx, 70x - 15Ox, 70x - 200x, 70x - 250x, 70x - 3OOx, 70x - 35Ox, 70x - 400x, 8Ox - 90x, 8Ox - lOOx, 8Ox - 15Ox, 8Ox - 200x, 8Ox - 250x, 8Ox - 3OOx, 8Ox - 35Ox, 8Ox - 400x, 90x -lOOx, 90x - 15Ox, 90x - 200x, 90x - 250x, 90x - 3OOx, 90x - 35Ox, 90x - 400x, lOOx -15Ox, lOOx

[0146] - 200x, 100x - 250x, 100x - 300x, 100x - 350x, 100x - 400x, 150x - 200x, 15Ox

[0147] - 250x, 15Ox - 3OOx, 15Ox - 35Ox, 15Ox - 400x, 200x - 250x, 200x - 3OOx, 200x - 35Ox, 200x - 400x, 250x - 3OOx, 250x - 35Ox, 250x - 400x, 3OOx - 35Ox, 3OOx - 400x, or 35Ox - 400x, more than the abundance of a microbial species introduced into the clinical sample.

[0148] In some embodiments, the relative abundance of a microbial species relative to a synthetic control (e.g., synthetic DNA sequence) can be used as a diagnostic criterion. In some embodiments, the abundance of a microbial species is at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least lOx, at least 20x, at least 3Ox, at least 40x, at least 5Ox, at least 60x, at least 70x, at least 8Ox, at least 90x, at least lOOx, at least 15Ox, at least 200x, at least 250x, at least 3OOx, at least 35Ox, at least 400x or more than the abundance of a synthetic control (e.g., synthetic DNA sequence) in a clinical sample. In some embodiments, the abundance of a microbial species is 2x - 3x, 2x - 4x, 2x - 5x, 2x - 6x, 2x - 7x, 2x - 8x, 2x - 9x, 2x - lOx, 2x - 20x, 2x - 3 Ox, 2x - 40x, 2x - 5 Ox, 2x - 60x, 2x - 70x, 2x - 8Ox, 2x - 90x, 2x - lOOx, 2x - 15Ox, 2x - 200x, 2x - 250x, 2x - 3OOx, 2x

[0149] - 35Ox, 2x - 400x, 3x - 4x, 3x - 5x, 3x - 6x, 3x - 7x, 3x - 8x, 3x - 9x, 3x - lOx, 3x - 20x, 3x - 3Ox, 3x - 40x, 3x - 5Ox, 3x - 60x, 3x - 70x, 3x - 8Ox, 3x - 90x, 3x - lOOx, 3x - 15Ox, 3x - 200x, 3x - 250x, 3x - 3OOx, 3x - 35Ox, 3x - 400x, 4x - 5x, 4x - 6x, 4x - 7x, 4x - 8x, 4x

[0150] - 9x, 4x - lOx, 4x - 20x, 4x - 3Ox, 4x - 40x, 4x - 5Ox, 4x - 60x, 4x - 70x, 4x - 8Ox, 4x - 90x, 4x - lOOx, 4x - 15Ox, 4x - 200x, 4x - 250x, 4x - 3OOx, 4x - 35Ox, 4x - 400x, 5x - 6x, 5x - 7x, 5x - 8x, 5x - 9x, 5x - lOx, 5x - 20x, 5x - 3Ox, 5x - 40x, 5x - 5Ox, 5x - 60x, 5x - 70x, 5x - 80x, 5x - 90x, 5x - lOOx, 5x - 15Ox, 5x - 200x, 5x - 250x, 5x - 3OOx, 5x - 35Ox, 5x - 400x, 6x - 7x, 6x - 8x, 6x - 9x, 6x - lOx, 6x - 20x, 6x - 3 Ox, 6x - 40x, 6x - 5 Ox, 6x - 60x, 6x - 70x, 6x - 8Ox, 6x - 90x, 6x - lOOx, 6x - 15Ox, 6x - 200x, 6x - 250x, 6x - 3OOx, 6x

[0151] - 35Ox, 6x - 400x, 7x - 8x, 7x - 9x, 7x - lOx, 7x - 20x, 7x - 3Ox, 7x - 40x, 7x - 5Ox, 7x - 60x, 7x - 70x, 7x - 8Ox, 7x - 90x, 7x - lOOx, 7x - 15Ox, 7x - 200x, 7x - 250x, 7x - 3OOx, 7x

[0152] - 35 Ox, 7x - 400x, 8x - 9x, 8x - lOx, 8x - 20x, 8x - 3 Ox, 8x - 40x, 8x - 5 Ox, 8x - 60x, 8x - 70x, 8x - 8Ox, 8x - 90x, 8x - lOOx, 8x - 15Ox, 8x - 200x, 8x - 250x, 8x - 3OOx, 8x - 35Ox, 8x - 400x, 9x - lOx, 9x - 20x, 9x - 3Ox, 9x - 40x, 9x - 5Ox, 9x - 60x, 9x - 70x, 9x - 8Ox, 9x

[0153] - 90x, 9x - lOOx, 9x - 15Ox, 9x - 200x, 9x - 250x, 9x - 3OOx, 9x - 35Ox, 9x - 400x, lOx - 20x, lOx - 3Ox, lOx - 40x, lOx - 5Ox, lOx - 60x, lOx - 70x, lOx - 8Ox, lOx - 90x, lOx - lOOx, lOx - 15Ox, lOx - 200x, lOx - 250x, lOx - 3OOx, lOx - 35Ox, lOx - 400x, 20x - 3Ox, 20x - 40x, 20x - 5Ox, 20x - 60x, 20x - 70x, 20x - 8Ox, 20x - 90x, 20x - lOOx, 20x - 15Ox, 20x - 200x, 20x - 250x, 20x - 3OOx, 20x - 35Ox, 20x - 400x, 3Ox - 40x, 3Ox - 5Ox, 3Ox - 60x, 3Ox - 70x, 3Ox - 8Ox, 3Ox - 90x, 3Ox - lOOx, 3Ox - 15Ox, 3Ox - 200x, 3Ox - 250x, 3Ox

[0154] - 3OOx, 3Ox - 35Ox, 3Ox - 400x, 40x - 5Ox, 40x - 60x, 40x - 70x, 40x - 8Ox, 40x - 90x, 40x

[0155] - lOOx, 40x - 15Ox, 40x - 200x, 40x - 250x, 40x - 3OOx, 40x - 35Ox, 40x - 400x, 5Ox - 60x, 5Ox - 70x, 5Ox - 8Ox, 5Ox - 90x, 5Ox - lOOx, 5Ox - 15Ox, 5Ox - 200x, 5Ox - 250x, 5Ox - 3OOx, 5Ox - 35Ox, 5Ox - 400x, 60x - 70x, 60x - 8Ox, 60x - 90x, 60x - lOOx, 60x - 15Ox, 60x

[0156] - 200x, 60x - 250x, 60x - 3OOx, 60x - 35Ox, 60x - 400x, 70x - 8Ox, 70x - 90x, 70x -lOOx, 70x - 15Ox, 70x - 200x, 70x - 250x, 70x - 3OOx, 70x - 35Ox, 70x - 400x, 8Ox - 90x, 8Ox - lOOx, 8Ox - 15Ox, 8Ox - 200x, 8Ox - 250x, 8Ox - 3OOx, 8Ox - 35Ox, 8Ox - 400x, 90x -lOOx, 90x - 15Ox, 90x - 200x, 90x - 250x, 90x - 3OOx, 90x - 35Ox, 90x - 400x, lOOx -15Ox, lOOx

[0157] - 200x, 100x - 250x, 100x - 300x, 100x - 350x, 100x - 400x, 150x - 200x, 15Ox

[0158] - 250x, 15Ox - 3OOx, 15Ox - 35Ox, 15Ox - 400x, 200x - 250x, 200x - 3OOx, 200x - 35Ox, 200x - 400x, 250x - 3OOx, 250x - 35Ox, 250x - 400x, 3OOx - 35Ox, 3OOx - 400x, or 35Ox - 400x, more than the abundance of a synthetic control (e.g., synthetic DNA sequence) in a clinical sample.

[0159] Microbial Species

[0160] Methods provided herein include enriching, identifying and / or quantifying one or more microbial species in a clinical sample. The term “microbial species”, as used herein, is any microorganism that is present in a sample (e.g., a clinical sample). In some embodiments, the microbial species is a bacteria, a virus, a fungus, a parasite, or any combination thereof. The microbial species present in a sample may be a commensal organism, which is a microorganism (e.g., bacteria, virus, fungus, or parasite) that lives in or on a subject without causing harm to the host or providing any significant benefits, or may be a pathogenic organism, which is a microorganism (e.g., bacteria, virus, fungus, mold, or parasite) that has disease-causing potential. The terms “pathogenic organism” or “pathogenic microbial species”, are used interchangeably herein and refer to a microorganism (e.g., bacteria, virus, fungus, or parasite), that has the ability to cause disease in a subject by invading, colonizing, and / or multiplying within the subject, leading to various illnesses or infections. In some embodiments, a commensal organism can turn into a pathogenic organism in a process referred to as “commensal-to-pathogen transition”. Non-limiting factors contributing to this shift include: i) genetic mutations that may result in the acquisition of new genes or the loss of genes that regulate virulence, increasing the organism’s pathogenic potential; ii) horizontal gene transfer, in which a commensal organism acquires virulence factors or antibiotic resistance genes from another microorganism; iii) changes in environmental conditions, such as antibiotic use or immune system suppression; and, iv) microbiota dysbiosis, in which alterations in a host’s microbiome composition and diversity creates opportunities for commensal organisms to complete with or displace other microorganisms.

[0161] In some embodiments, one or more microbial species in a clinical sample provided herein is a pathogenic microbial species. One microbial species or multiple microbial species in a clinical sample may be pathogenic. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more microbial species are pathogenic microbial species. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more pathogenic microbial species are enriched, identified and / or quantified.

[0162] In some embodiments, a pathogenic microbial species is a bacterium. A bacterium is a prokaryote that lacks a true cell nucleus and membrane-bound organelles. Non-limiting examples of pathogenic bacteria include Abiotrophia defective, Acetobacter nitrogenifigens, Achromobacter denitrificans, Achromobacter insolitus, Achromobacter ruhlandii, Achromobacter xylosoxidans, Acidaminococcus intestini, Acidovorax citrulli, Acinetobacter baumannii, Acinetobacter bereziniae, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter pittii, Acinetobacter radioresistens, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter ursingii, Actinobacillus suis, Actinobacillus ureae, Actinomadura latina, Actinomadura madurae, Actinomyces gerencseriae, Actinomyces graevenitzii, Actinomyces israelii, Actinomyces massiliensis, Actinomyces oris, Actinomyces timonensis, Actinomyces urogenitalis, Actinomyces viscosus, Aerococcus christensenii, Aerococcus sanguinicola, Aerococcus urinae, Aerococcus urinaehominis, Aerococcus viridans, Aeromonas caviae, Aeromonas enteropelogenes, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas schubertii, Aeromonas veronii, Afipia felis, Aggregatibacter actinomycetemcomitans, Aggregatibacter aphrophilus, Aggregatibacter segnis, Agrobacterium tumefaciens, Alcaligenes faecalis, Alloiococcus otitis, Alloscardovia omnicolens, Alysiella crassa, Anaerobiospirillum succiniciproducens, Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus prevotii, Anaerococcus tetradius, Anaeroglobus geminatus, Anaerostipes caccae, Anaplasma phagocytophilum, Arcanobacterium haemolyticum, Aliarcobacter butzleri (Arcobacter butzleri), Aliarcobacter cryaerophilus (Arcobacter cryaer ophilus), Aliarcobacter skirrowii (Arcobacter skirrowii), Atlantibacter hermannii (Escherichia hermannii), Lancefieldella parvula (Atopobium parvulum), Lancefieldella rimae (Atopobium rimae), Fannyhessea vaginae (Atopobium vaginae), Aureimonas altamirensis, Azospirillum brasilense (Roseomonas fauriae), Bacillus anthracis, Bacillus cereus, Niallia circulans (Bacillus circulans), Weizmannia coagulans (Bacillus coagulans), Bacillus glycinifermentans, Bacillus licheniformis, Priestia megaterium (Bacillus megaterium), Bacillus mycoides, Bacillus paralicheniformis, Bacillus pumilus, Bacillus safensis, Bacillus subtilis, Bacillus thuringiensis, Bacteroides caccae, Bacteroides eggerthii, Bacteroides faecis, Bacteroides fragilis, Bacteroides ovatus, Bacteroides pyogenes, Bacteroides salyersiae, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Phocaeicola vulgatus (Bacteroides vulgatus), Bartonella alsatica, Bartonella ancashensis, Bartonella bacilliformis, Bartonella birtlesii, Bartonella bovis, Bartonella clarridgeiae, Bartonella doshiae, Bartonella elizabethae, Bartonella grahamii, Bartonella henselae, Bartonella koehlerae, Bartonella quintana, Bartonella rattaustraliani, Bartonella rochalimae, Bartonella schoenbuchensis, Bartonella taylorii, Bartonella tribocorum, Bartonella vinsonii, Bergeyella zoohelcum, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium longum, Bifidobacterium psychraerophilum, Bifidobacterium scardovii, Bordetella avium, Bordetella bronchialis, Bordetella bronchiseptica, Bordetella flabilis, Bordetella hinzii, Bordetella holmesii, Bordetella parapertussis, Bordetella pertussis, Bordetella petrii, Bordetella trematum, Borrelia crocidurae, Borrelia duttonii, Borrelia hermsii, Borrelia hispanica, Borreliella mayonii (Borrelia mayonii), Borrelia miyamotoi, Borrelia parkeri, Borrelia persica, Borrelia recurrentis, Borrelia turicatae, Borreliella afzelii (Borrelia afzelii), Borreliella burgdorferi (Borrelia burgdorferi), Borreliella garinii (Borrelia garinii), Brachyspira pilosicoli, Brevibacillus brevis, Brevibacillus laterosporus, Brevibacterium casei, Brevundimonas vesicularis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Budvicia aquatica, Bulleidia extructa, Burkholderia cepacia complex, Burkholderia gladioli, Burkholderia glumae, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia thailandensis, Buttiauxella brennerae, Buttiauxella ferragutiae, Buttiauxella gaviniae, Campylobacter coli, Campylobacter concisus, Campylobacter corcagiensis, Campylobacter cuniculorum, Campylobacter curvus, Campylobacter fetus, Campylobacter gracilis, Campylobacter hominis, Campylobacter hyointestinalis, Campylobacter iguaniorum, Campylobacter jejuni, Campylobacter lari, Campylobacter mucosalis, Campylobacter showae, Campylobacter sputorum, Campylobacter upsaliensis, Campylobacter ureolyticus, Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Capnocytophaga gingivalis, Capnocytophaga granulosa, Capnocytophaga haemolytica, Capnocytophaga ochracea, Capnocytophaga sputigena, Cardiobacterium hominis, Cardiobacterium valvarum, Christensenella hongkongensis (Catabacter hongkongensis), Cedecea davisae, Cedecea neteri, Cellulomonas jlavigena, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium haemolyticum, Chromobacterium violaceum, Chryseobacterium gleum, Chryseobacterium indologenes, Citrobacter amalonaticus, Citrobacter braakii, Citrobacter farmeri, Citrobacter freundii, Citrobacter koseri, Citrobacter sedlakii, Clostridioides difficile (Clostridium difficile), Clostridium baratii, Clostridium butyricum, Clostridium cadaveris, Clostridium haemolyticum, Clostridium hylemonae, Clostridium innocuum, Clostridium neonatale, Clostridium novyi, Clostridium paraputrificum, Clostridium perfringens, Clostridium tetani, Sarcina ventriculi (Clostridium ventriculi), Comamonas kerstersii, Comamonas terrigena, Corynebacterium accolens, Corynebacterium afermentans, Corynebacterium amycolatum, Corynebacterium argentoratense, Corynebacterium aurimucosum, Corynebacterium diphtheriae, Corynebacterium falsenii, Corynebacterium freiburgense, Corynebacterium freneyi, Corynebacterium glucuronolyticum, Corynebacterium halotolerans, Corynebacterium jeikeium, Corynebacterium kroppenstedtii, Corynebacterium kutscheri, Corynebacterium lipophiloflavum, Corynebacterium massiliense, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium otitidis (Turicella otitidis), Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum, Corynebacterium pseudotuberculosis, Corynebacterium renale, Corynebacterium riegelii, Corynebacterium simulans, Corynebacterium stationis, Corynebacterium striatum, Corynebacterium timonense, Corynebacterium tuscaniense, Corynebacterium ulcerans, Corynebacterium urealyticum, Corynebacterium ureicelerivorans, Corynebacterium xerosis, Coxiella burnetii, Cronobacter sakazakii (Enterobacter sakazakii), Cupriavidus gilardii, Cupriavidus metallidurans, Cupriavidus pauculus, Cutibacterium granulosum (Propionibacterium granulosum), Delftia acidovorans, Dermabacter hominis, Dermacoccus nishinomiyaensis, Dermatophilus congolensis, Desulfomicrobium orale, Dialister micraerophilus, Dielma fastidiosa, Dolosigranulum pigrum, Dysgonomonas capnocytophagoides, Dysgonomonas gadei, Dysgonomonas hofstadii, Dysgonomonas mossii, Edwardsiella hoshinae, Edwardsiella tarda, Eggerthella lenta, Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia muris, Eikenella corrodens, Elizabethkingia anophelis, Elizabethkingia meningoseptica, Elizabethkingia miricola, Empedobacter brevis, Empedobacter falsenii, Enterobacter cloacae complex, Enterobacter mori, Enterocloster clostridioformis (Clostridium clostridioforme), Enterococcus asini, Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus gilvus, Enterococcus haemoper oxidus, Enterococcus hirae, Enterococcus italicus, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pallens, Enterococcus phoeniculicola, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus sulfureus, Enterococcus thailandicus, Erwinia billingiae, Erwinia gerundensis, Erysipelothrix rhusiopathiae, Escherichia albertii, Escherichia coli, Escherichia fergusonii, Eubacterium limosum, Eubacterium nodatum, Facklamia hominis, Facklamia sourekii, Faecalicoccus pleomorphus, Fenollaria massiliensis, Filifactor alocis, Finegoldia magna, Fluoribacter bozemanae (Legionella bozemanae), Fluoribacter dumoffii (Legionella dumoffii), Francisella hispaniensis, Francisella noatunensis, Francisella philomiragia, Francisella tularensis, Franconibacter helveticus, Fusobacterium mortiferum, Fusobacterium necrophorum, Fusobacterium nucleatum, Fusobacterium periodonticum, Fusobacterium russii, Fusobacterium ulcerans, Fusobacterium varium, Gardnerella vaginalis, Gemella bergeri, Gemella haemolysans, Gemella morbillorum, Gemella sanguinis, Gleimia europaea (Actinomyces europaeus), Globicatella sanguinis, Gordonia aichiensis, Gordonia bronchialis, Gordonia otitidis, Gordonia rubripertincta, Gordonia terrae, Gordonibacter pamelaeae, Granulibacter bethesdensis, Granulicatella adiacens, Granulicatella elegans, Grimontia hollisae, Haemophilus aegyptius, Haemophilus ducreyi, Haemophilus haemolyticus, Haemophilus influenzae, Haemophilus parahaemolyticus, Haemophilus parainfluenzae, Haemophilus paraphrohaemolyticus, Haemophilus quentini, Haemophilus sputorum, Hafnia alvei, Hafnia paralvei, Helcococcus kunzii, Helicobacter bills, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter felis, Helicobacter fennelliae, Helicobacter heilmannii, Helicobacter magdeburgensis, Helicobacter pylori, Isoptericola variabilis, Janibacter indicus, Janibacter melonis, Johnsonella ignava, Jonesia denitrificans, Kerstersia gyiorum, Kingella denitrificans, Kingella kingae, Kingella oralis, Klebsiella aerogenes (Enterobacter aerogenes), Klebsiella michiganensis, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola, Kluyvera ascorbata, Kluyvera cryocrescens, Kluyvera intermedia, Kocuria rhizophila, Kurthia huakuii, Kurthia massiliensis, Kytococcus sedentarius, Lactobacillus acidophilus, Limosilactobacillus antri (Lactobacillus antri), Lacticaseibacillus casei (Lactobacillus casei), Lactobacillus crispatus, Limosilactobacillus fermentum (Lactobacillus fermentum), Lactobacillus gasseri, Lactobacillus iners, Lactobacillus jensenii, Lactiplantibacillus paraplantarum (Lactobacillus paraplantarum), Lactiplantibacillus plantarum (Lactobacillus plantarum), Limosilactobacillus pontis (Lactobacillus pontis), Lacticaseibacillus rhamnosus (Lactobacillus rhamnosus), Ligilactobacillus saerimneri (Lactobacillus saerimneri), Latilactobacillus sakei (Lactobacillus sakei), Lactobacillus ultunensis, Lactococcus garvieae, Laribacter hongkongensis, Lawsonella clevelandensis, Lawsonia intracellularis, Leclercia adecarboxylata, Legionella adelaidensis, Legionella anisa, Legionella birminghamensis, Legionella brunensis, Legionella cherrii, Legionella cincinnatiensis, Legionella clemsonensis, Legionella drancourtii, Legionella drozanskii, Legionella erythra, Legionella fairfieldensis, Legionella fallonii, Legionella feeleii, Legionella geestiana, Legionella hackeliae, Legionella israelensis, Legionella jamestowniensis, Legionella jordanis, Legionella lansingensis, Legionella londiniensis, Legionella longbeachae, Legionella maceachernii, Legionella massiliensis, Legionella moravica, Legionella nautarum, Legionella norrlandica, Legionella oakridgensis, Legionella parisiensis, Legionella pneumophila, Legionella quateirensis, Legionella quinlivanii, Legionella rubrilucens, Legionella sainthelensi, Legionella santicrucis, Legionella shakespearei, Legionella spiritensis, Legionella steelei, Legionella tucsonensis, Legionella tunisiensis, Legionella wadsworthii, Legionella waltersii, Legionella worsleiensis, Leifsonia aquatica, Lelliottia amnigena (Enterobacter amnigenus), Leminorella grimontii, Leptospira alexanderi, Leptospira alstonii, Leptospira biflexa, Leptospira borgpetersenii, Leptospira broomii, Leptospira fainei, Leptospira inadai, Leptospira interrogans, Leptospira kirschneri, Leptospira kmetyi, Leptospira licerasiae, Leptospira mayottensis, Leptospira meyeri, Leptospira noguchii, Leptospira santarosai, Leptospira terpstrae, Leptospira vanthielii, Leptospira weilii, Leptospira wolbachii, Leptospira yanagawae, Leptotrichia buccalis, Pseudoleptotrichia goodfellowii (Leptotrichia goodfellow ii), Leptotrichia shahii, Leptotrichia wadei, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeria seeligeri, Listeria welshimeri, Lysinibacillus sphaericus (Bacillus sphaericus), Macrococcus caseolyticus (Staphylococcus caseolyticus), Mannheimia haemolytica, Megasphaera micronuciformis, Microbacterium foliorum, Microbacterium maritypicum, Microbacterium oxydans, Microbacterium paraoxydans, Microbacterium testaceum, Micrococcus luteus, Micrococcus lylae, Mitsuokella multacida, Mobiluncus curtisii, Mobiluncus mulieris, Moeller ella wisconsensis, Mogibacterium timidum, Moraxella atlantae, Moraxella catarrhalis, Moraxella lacunata, Moraxella lincolnii, Moraxella nonliquefaciens, Morganella morganii, Morococcus cerebrosus, Mycobacterium asiaticum, Mycobacterium avium complex (MAC), Mycobacterium celatum, Mycobacterium chimaera, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium grossiae, Mycobacterium haemophilum, Mycobacterium heckeshornense, Mycobacterium intermedium, Mycobacterium kansasii, Mycobacterium kyorinense, Mycobacterium leprae, Mycobacterium lepromatosis, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium nebraskense, Mycobacterium paraffmicum, Mycobacterium parascrofulaceum, Mycobacterium scrofulaceum, Mycobacterium sherrisii, Mycobacterium shigaense, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium talmoniae, Mycobacterium triplex, Mycobacterium tuberculosis complex, Mycobacterium xenopi, Mycobacteroides abscessus (Mycobacterium abscessus), Mycobacteroides chelonae (Mycobacterium chelonae), Mycobacteroides franklinii (Mycobacterium franklinii), Mycobacteroides immunogenum (Mycobacterium immunogenum), Mycobacteroides saopaulense (Mycobacterium saopaulense), Mycolicibacillus koreensis (Mycobacterium koreense), Mycolicibacter arupensis (Mycobacterium arupense), Mycolicibacter heraklionensis (Mycobacterium heraklionense), Mycolicibacter kumamotonensis (Mycobacterium kumamotonense), Mycolicibacterium aurum, Mycolicibacterium brisbanense (Mycobacterium brisbanense), Mycolicibacterium canariasense (Mycobacterium canariasense), Mycolicibacterium chlorophenolicum, Mycolicibacterium chubuense, Mycolicibacterium conceptionense (Mycobacterium conceptionense), Mycolicibacterium cosmeticum (Mycobacterium cosmeticum), Mycolicibacterium diernhoferi (Mycobacterium diernhoferi), Mycolicibacterium elephantis (Mycobacterium elephantis), Mycolicibacterium jlavescens (Mycobacterium jlavescens), Mycolicibacterium fortuitum (Mycobacterium fortuitum), Mycolicibacterium goodii (Mycobacterium goodii), Mycolicibacterium hassiacum, Mycolicibacterium holsaticum (Mycobacterium holsaticum), Mycolicibacterium iranicum (Mycobacterium iranicum), Mycolicibacterium llatzerense (Mycobacterium llatzerense), Mycolicibacterium mageritense (Mycobacterium mageritense), Mycolicibacterium mucogenicum (Mycobacterium mucogenicum), Mycolicibacterium neoaurum (Mycobacterium neoaurum), Mycolicibacterium novocastrense (Mycobacterium novocastrense), Mycolicibacterium obuense (Mycobacterium obuense), Mycolicibacterium peregrinum (Mycobacterium peregrinum), Mycolicibacterium phlei (Mycobacterium phlei), Mycolicibacterium septicum (Mycobacterium septicum), Mycolicibacterium setense (Mycobacterium setense), Mycolicibacterium smegmatis (Mycobacterium smegmatis), Mycolicibacterium thermoresistibile (Mycobacterium thermoresistibile), Mycolicibacterium tusciae (Mycobacterium tusciae), Mycolicibacterium vaccae (Mycobacterium vaccae), Mycolicibacterium wolinskyi (Mycobacterium wolinskyi), Mycoplasmopsis arginini (Mycoplasma arginini), Mycoplasma capricolum, Mycoplasmopsis cynos (Mycoplasma cynos), Mycoplasmopsis fermentans (Mycoplasma fermentans), Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma hyopneumoniae, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasmopsis pulmonis (Mycoplasma pulmonis), Myroides marinus, Myroides odoratimimus, Myroides odoratus, Neisseria animaloris, Neisseria bacilliformis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria meningitidis, Neisseria mucosa, Neisseria polysaccharea, Neisseria sicca, Neisseria wadsworthii, Neisseria weaveri, Neorickettsia helminthoeca, Neorickettsia sennetsu, Nocardia abscessus, Nocardia acidivorans, Nocardia africana, Nocardia alba, Nocardia amamiensis, Nocardia anaemiae, Nocardia aobensis, Nocardia araoensis, Nocardia arizonensis, Nocardia arthritidis, Nocardia asiatica, Nocardia beijingensis, Nocardia brasiliensis, Nocardia brevicatena, Nocardia caishijiensis, Nocardia carnea, Nocardia cerradoensis, Nocardia concava, Nocardia coubleae, Nocardia crassostreae, Nocardia cummidelens, Nocardia cyriacigeorgica, Nocardia elegans, Nocardia exalbida, Nocardia farcinica, Nocardia flavor osea, Nocardia fusca, Nocardia gamkensis, Nocardia grenadensis, Nocardia harenae, Nocardia higoensis, Nocardia ignorata, Nocardia inohanensis, Nocardia jejuensis, Nocardia jiangxiensis, Nocardia kruczakiae, Nocardia lijiangensis, Nocardia mexicana, Nocardia mikamii, Nocardia miyunensis, Nocardia niigatensis, Nocardia niwae, Nocardia nova, Nocardia otitidiscaviarum, Nocardia paucivorans, Nocardia pneumoniae, Nocardia pseudobrasiliensis, Nocardia pseudovaccinii, Nocardia puris, Nocardia rhamnosiphila, Nocardia salmonicida, Nocardia seriolae, Nocardia shimofusensis, Nocardia sienata, Nocardia soli, Nocardia spehmcae, Nocardia takedensis, Nocardia tenerifensis, Nocardia terpenica, Nocardia testacea, Nocardia thailandica, Nocardia transvalensis, Nocardia uniformis, Nocardia vaccinii, Nocardia vermiculata, Nocardia veterana, Nocardia vinacea, Nocardia violaceofusca, Nocardia vulneris, Nocardia xishanensis, Nocardia yamanashiensis, Nocardiopsis dassonvillei (Nocardia dassonvillei), Obesumbacterium proteus, Brucella anthropi (Ochrobactrum anthropi), Brucella intermedia (Ochrobactrum intermedium), Brucella oryzae (Ochrobactrum oryzae), Odoribacter laneus, Odoribacter splanchnicus, Oerskovia turbata, Oligella ureolytica, Oligella urethralis, Olsenella uli, Oribacterium sinus, Orientia tsutsugamushi, Oscillibacter ruminantium, Paenalcaligenes hominis, Paenibacillus alvei, Paeniclostridium sordellii (Clostridium sordellii), Pandoraea apista, Pandoraea pulmonicola, Pandoraea sputorum, Pannonibacter phragmitetus, Pantoea agglomerans, Pantoea ananatis, Parabacteroides distasonis (Bacteroides distasonis), Parabacteroides goldsteinii, Parabacteroides gordonii, Parabacteroides johnsonii, Parabacteroides merdae (Bacteroides merdae), Paraburkholderia fungorum, Parachlamydia acanthamoebae, Paraclostridium bifermentans (Clostridium bifermentans), Paracoccus sanguinis, Paracoccus yeei, Parvimonas micra, Pasteurella bettyae, Pasteur ella multocida, Pediococcus acidilactici, Pediococcus pentosaceus, Pelobacter propionicus, Peptoniphilus coxii, Peptoniphilus duerdenii, Peptoniphilus harei, Peptoniphilus indolicus, Peptoniphilus lacrimalis, Peptoniphilus rhinitidis, Peptoniphilus senegalensis, Peptostreptococcus anaerobius, Peptostreptococcus stomatis, Photobacterium damselae, Photorhabdus asymbiotica, Photorhabdus luminescens, Plesiomonas shigelloides, Pluralibacter gergoviae, Porphyromonas asaccharolytica, Porphyromonas gingivalis, Porphyromonas gingivicanis, Prevotella bivia, Prevotella buccae, Prevotella buccalis, Prevotella corporis, Prevotella denticola, Prevotella disiens, Prevotella intermedia, Prevotella loescheii, Prevotella melaninogenica, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Propionibacterium acidifaciens, Cutibacterium namnetense (Propionibacterium namnetense), Propionimicrobium lymphophilum (Corynebacterium lymphophilum), Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Pseudescherichia vulneris (Escherichia vulneris), Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas cannabina, Pseudomonas citronellolis, Pseudomonas fluorescens, Pseudomonas fulva, Pseudomonas luteola, Pseudomonas mendocina, Pseudomonas mosselii, Pseudomonas oryzihabitans, Pseudomonas poae, Pseudomonas protegens, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas veronii, Arachnia propionica (Propionibacterium propionicum), Pseudoramibacter alactolyticus, Psychrobacter cryohalolentis, Psychrobacter phenylpyruvicus (Moraxella phenylpyruvica), Rahnella aquatilis, Ralstonia insidiosa, Ralstonia mannitolilytica, Raoultella ornithinolytica, Raoultella planticola, Rhodococcus erythropolis, Rhodococcus fascians, Rhodococcus hoagii (Rhodococcus equi), Rhodococcus rhodochrous, Rickettsia akari, Rickettsia amblyommatis, Rickettsia australis, Rickettsia canadensis, Rickettsia conorii, Rickettsia felis, Rickettsia Helvetica, Rickettsia honei, Rickettsia japonica, Rickettsia massiliae, Rickettsia monacensis, Rickettsia parkeri, Rickettsia prowazekii, Rickettsia raoultii, Rickettsia rickettsii, Rickettsia sibirica, Rickettsia slovaca, Rickettsia typhi, Riemerella anatipestifer, Robinsoniella peoriensis, Rodentibacter pneumotropicus (Pasteurella pneumotropica), Roseobacter denitrificans, Roseomonas cervicalis, Roseomonas gilardii, Roseomonas mucosa, Rothia aeria, Rothia dentocariosa, Rothia kristinae (Kocuria kristinae), Rothia mucilaginosa, Rouxiella chamberiensis, Saccharopolyspora rectivirgula (Micropolyspora faeni), Salmonella bongori, Salmonella enterica, Sanguibacteroides justesenii, Scardovia wiggsiae, Schaalia cardiffensis (Actinomyces cardiffensis), Schaalia georgiae (Actinomyces georgiae), Schaalia meyeri (Actinomyces meyeri), Schaalia odontolytica (Actinomyces odontolyticus), Schaalia turicensis (Actinomyces turicensis), Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Serratia odorifera, Serratia plymuthica, Serratia rubidaea, Serratia ureilytica, Shewanella algae, Shewanella putrefaciens, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shimwellia blattae (Escherichia blattae), Siccibacter turicensis, Slackia exigua, Sneathia vaginalis (Leptotrichia amnionii), Sneathia sanguinegens, Solobacterium moorei, Sphingobacterium spiritivorum, Staphylococcus agnetis, Staphylococcus argenteus, Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcus condimenti, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Mammaliicoccus lentus (Staphylococcus lentus), Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus pettenkoferi, Staphylococcus pseudintermedius, Staphylococcus saprophyticus, Staphylococcus schleiferi, Mammaliicoccus sciuri (Staphylococcus sciuri), Staphylococcus simiae, Staphylococcus simulans, Staphylococcus succinus, Mammaliicoccus vitulinus (Staphylococcus vitulinus), Staphylococcus warneri, Staphylococcus xylosus, Stenotrophomonas acidaminiphila, Stenotrophomonas maltophilia, Streptobacillus moniliformis, Streptococcus acidominimus, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus canis, Streptococcus constellatus, Streptococcus criceti, Streptococcus cristatus, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus ferus, Streptococcus gallolyticus, Streptococcus gordonii, Streptococcus hyovaginalis, Streptococcus infantarius, Streptococcus infantis, Streptococcus iniae, Streptococcus intermedins, Streptococcus lutetiensis, Streptococcus macacae, Streptococcus macedonicus, Streptococcus massiliensis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus oralis subsp. dentisani (Streptococcus dentisani), Streptococcus oralis subsp. tigurinus (Streptococcus tigurinus), Streptococcus parasanguinis, Streptococcus pasteurianus, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus porcinus, Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus thermophilus, Streptococcus uberis, Streptococcus vestibularis, Streptomyces cattleya, Streptomyces somaliensis, Sutterella wadsworthensis, Tannerella forsythia (Bacteroides forsythus), Tatlockia micdadei (Legionella micdadei), Tatumella ptyseos, Taylorella asinigenitalis, Taylorella equigenitalis, Terrisporobacter othiniensis, Treponema pallidum, Tropheryma whipplei, Trueperella bernardiae (Arcanobacterium bernardiae), Trueperella pyogenes (Arcanobacterium pyogenes), Tsukamurella paurometabola, Tsukamurella pulmonis, Tsukamurella tyrosinosolvens, Ureaplasma parvum, Ureaplasma urealyticum, Veillonella dispar, Veillonella montpellierensis, Veillonella parvula, Veillonella seminalis, Vibrio albensis, Vibrio alginolyticus, Vibrio cholerae, Vibrio fluvialis, Vibrio furnissii, Vibrio harveyi, Vibrio metschnikovii, Vibrio mimicus, Vibrio navarrensis, Vibrio parahaemolyticus, Vibrio vulnificus, Waddlia chondrophila, Weeksella virosa, Weissella confusa, Weissella par ame senter oides, Weissella viridescens, Winkia neuii (Actinomyces neuii), Wohlfahrtiimonas chitiniclastica, Wolbachia pipientis, Xanthomonas axonopodis, Xylanimonas cellulosilytica, Yersinia enter ocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia ruckeri, Yokenella regensburgei, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium canettii, Mycobacterium tuberculosis, Burkholderia ambifaria, Burkholderia anthina, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia contaminans, Burkholderia diffusa, Burkholderia dolosa, Burkholderia lata, Burkholderia latens, Burkholderia multivorans, Burkholderia pseudomultivorans, Burkholderia pyrrocinia, Burkholderia seminalis, Burkholderia stabilis, Burkholderia stagnalis, Burkholderia territorii, Burkholderia vietnamiensis, Mycobacterium avium, Mycobacterium colombiense, Mycobacterium indicus pranii, Mycobacterium intr acellular e, Mycobacterium vulneris, Mycobacterium yongonense, Enter obacter asburiae, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter cloacae complex 'Hoffmann cluster III', Enterobacter cloacae complex 'Hoffmann cluster IV', Enterobacter hormaechei, Enterobacter kobei, Enterobacter lignolyticus, Enterobacter ludwigii, and Enterobacter xiangfangensis .

[0163] In some embodiments, microbial species are bacterial species Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus lugdunesis, Enterococcus faecium, Staphylococcus haemolyticus, Proteus mirabilis, Staphylococcus saprophyticus, Proteus vulgaris, Streptococcus agalactiae, Pseudomonas aeruginosa, Streptococcus pyogenes, Enterobacter cloacae complex, Streptococcus dysgalactiae, Klebsiella oxytoca, Streptococcus anginosus, Citrobacter freundii complex, Streptococcus pseudoporcinus, Citrobacter koser, Aerococcus urinae, Klebsiella aerogenes, Aerococcus viridans, Morganella morganii, Acinetobacter baumannii, Serratia marcescens, Stenotrophomonas maltophilia, Providencia rettget, and Panotea agglomerans.

[0164] In some embodiments, a pathogenic microbial species is a virus. A virus is a microscopic infectious agent that contains genetic material (e.g., DNA or RNA) and a protein coat (e.g., capsid). In some embodiments, the virus is an RNA virus or a DNA virus. In some embodiments, the RNA virus is a single-stranded RNA virus or a double-stranded RNA virus. In some embodiments, the single-stranded RNA virus is a single-stranded positivesense RNA virus that can be immediately translated into protein by a host cell (e.g., a cell in a subject). In some embodiments, the single-stranded RNA virus is a single-stranded negativesense RNA virus that must be converted to positive-sense RNA prior to being translated into protein by a host cell (e.g., a cell in a subject). In some embodiments, the DNA virus is a single-stranded DNA virus, a partially double-stranded DNA virus, or a double-stranded DNA virus. Non-limiting examples of pathogenic viruses include Adeno-associated dependoparvovirus A, Adeno-associated dependoparvovirus B, Alphapapillomavirus 9, BK polyomavirus, Betapapillomavirus 1, Betapapillomavirus 2, Cowpox virus, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Gammapapillomavirus 1, Gammapapillomavirus 10, Gammapapillomavirus 11, Gammapapillomavirus 13, Gammapapillomavirus 14, Gammapapillomavirus 15, Gammapapillomavirus 16, Gammapapillomavirus 17, Gammapapillomavirus 19, Gammapapillomavirus 2, Gammapapillomavirus 3, Gammapapillomavirus 4, Gammapapillomavirus 5, Gammapapillomavirus 6, Gammapapillomavirus 7, Gammapapillomavirus 8, Gammapapillomavirus 9, Herpes B virus, Herpes simplex virus type 1 (HSV-1), Herpes simplex virus type 2 (HSV-2), Human adenovirus A, Human adenovirus B, Human adenovirus C, Human adenovirus D, Human adenovirus E, Human adenovirus F, Human bocavirus, Human herpesvirus 6A, Human herpesvirus 6B, Human herpesvirus 7, Human papillomavirus, Human papillomavirus 1, Human papillomavirus 10, Human papillomavirus 132, Human papillomavirus 136, Human papillomavirus 140, Human papillomavirus 154, Human papillomavirus 167, Human papillomavirus 18, Human papillomavirus 2, Human papillomavirus 26, Human papillomavirus 32, Human papillomavirus 34, Human papillomavirus 41, Human papillomavirus 49, Human papillomavirus 53, Human papillomavirus 6, Human papillomavirus 61, Human papillomavirus 63, Human papillomavirus 7, Human papillomavirus 90, Human papillomavirus 92, Human papillomavirus 96, Human parvovirus Bl 9, Human polyomavirus 6, Human polyomavirus 7, JC polyomavirus, KI polyomavirus, Kaposi sarcoma-associated herpesvirus, MW polyomavirus, Merkel cell polyomavirus, Molluscum contagiosum virus, Monkeypox virus, Orf virus, Porcine circovirus 1, Porcine circovirus 2, Primate bocaparvovirus 1, Primate bocaparvovirus 2, Primate tetraparvovirus 1 (human PARV-4), Pseudocowpox virus, STL polyomavirus, Tanapox virus, Torque teno virus, Torque teno virus 1, Torque teno virus 10, Torque teno virus 12, Torque teno virus 14, Torque teno virus 15, Torque teno virus 16, Torque teno virus 19, Torque teno virus 2, Torque teno virus 25, Torque teno virus 26, Torque teno virus 27, Torque teno virus 28, Torque teno virus 3, Torque teno virus 4, Torque teno virus 6, Torque teno virus 7, Torque teno virus 8, Trichodysplasia spinulosa-associated polyomavirus, Vaccinia virus, Varicellazoster virus (VZV), Variola virus, WU Polyomavirus, and Yaba monkey tumor virus.

[0165] In some embodiments, a pathogenic microbial species is a fungus. In some embodiments, the fungus is a mold. A fungus is a eukaryotic microorganism, with a defined nucleus and membrane-bound organelles. Non-limiting examples of pathogenic fungi include Absidia glauca, Absidia repens, Acremonium chrysogenum, Fur caster igmium furcatum (Acremonium furcatum), Actinomucor elegans, Alternaria alternata, Alternaria arborescens, Alternaria brassicicola, Anncaliia algerae, Apiotrichum porosum (Trichosporon porosum), Apophysomyces elegans, Apophysomyces trapeziformis, Apophysomyces variabilis, Aspergillus aculeatus, Aspergillus arachidicola, Aspergillus bombycis, Aspergillus brasiliensis, Aspergillus calidoustus, Aspergillus campestris, Aspergillus candidus, Aspergillus carbonarius, Aspergillus chevalieri, Aspergillus clavatus, Aspergillus cristatus, Aspergillus fischeri, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus hancockii, Aspergillus lentulus, Aspergillus luchuensis, Aspergillus nidulans, Aspergillus niger, Aspergillus nomiae, Aspergillus novofumigatus, Aspergillus ochraceoroseus, Aspergillus oryzae, Aspergillus parasiticus, Aspergillus persii, Aspergillus pseudoterreus, Aspergillus rambellii, Aspergillus ruber, Aspergillus sclerotiorum, Aspergillus sojae, Aspergillus steynii, Aspergillus sydowii, Aspergillus taichungensis, Aspergillus terreus, Aspergillus thermomutatus, Aspergillus tubingensis, Aspergillus turcosus, Aspergillus udagawae, Aspergillus ustus, Aspergillus versicolor, Aspergillus wentii, Aspergillus w ester dijkiae, Aureobasidium melanogenum, Aureobasidium namibiae, Aureobasidium pullulans, Aureobasidium subglaciale, Basidiobolus meristosporus, Beauveria bassiana, Beauveria rudraprayagi, Blastomyces dermatitidis, Blastomyces percursus, Paecilomyces variotii (Byssochlamys spectabilis), Candida aaseri, Candida albicans, Candida arabinofermentans, Candidozyma auria, Candida boidinii, Candida bracarensis, Candida castellii, Candida dubliniensis, Candida duobushaemulonis, Candida ethanolica, Candida glabrata, Candida haemulonis, Candida intermedia, Candida ipomoeae, Candida nivariensis, Candida orthopsilosis, Candida parapsilosis, Candida pseudohaemulonis, Candida psychrophila, Candida sojae, Candida sorboxylosa, Candida succiphila, Yamadazyma tenuis (Candida tenuis), Candida tropicalis, Ceratocystis adiposa, Ceratocystis albifundus, Ceratocystis eucalypticola, Ceratocystis fimbriata, Ceratocystis manginecans, Ceratocystis platani, Chaetomium globosum, Chaetomium thermophilum, Chrysosporium queenslandicum, Cladophialophora bantiana, Cladophialophora carrionii, Cladophialophora immunda, Cladophialophora psammophila, Cladophialophora yegresii, Cladosporium cladosporioides, Clavispora lusitaniae (Candida lusitaniae), Coccidioides immitis, Coccidioides posadasii, Cokeromyces recurvatus, Colletotrichum acutatum, Colletotrichum falcatum, Colletotrichum fioriniae, Colletotrichum gloeosporioides, Colletotrichum godetiae, Colletotrichum graminicola, Colletotrichum higginsianum, Colletotrichum incanum, Colletotrichum nymphaeae, Colletotrichum orbiculare, Colletotrichum salicis, Colletotrichum simmondsii, Colletotrichum sublineola, Colletotrichum tofieldiae, Conidiobolus coronatus, Conidiobolus incongruus, Coniosporium apollinis, Corynespora cassiicola, Cryptococcus gattii VGI (Cryptococcus gattii), Cryptococcus gattii VGII (Cryptococcus deuterogattii), Cryptococcus gattii VGIII (Cryptococcus bacillisporus), Cryptococcus gattii VGIV (Cryptococcus tetragattii), Cryptococcus neoformans, Cunninghamella, Curvularia lunata, Curvularia papendorfii (Bipolaris papendorfii), Cutaneotrichosporon cutaneum (Trichosporon cutaneum), Cutaneotrichosporon oleaginosum (Trichosporon oleaginosus), Cyberlindnera fabianii (Hansenula fabianii), Cyberlindnera jadinii (Candida utilis), Cyphellophora europaea, Debaryomyces fabryi, Debaryomyces hansenii (Candida famata), Diaporthe ampelina, Diaporthe aspalathi, Diaporthe longicolla, Emmonsia crescens, Blastomyces silverae (Emmonsia parva), Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Encephalitozoon romaleae, Enterocytozoon bieneusi, Exophiala alcalophila, Exophiala aquamarina, Exophiala calicioides, Exophiala dermatitidis, Exophiala mesophila, Exophiala oligosperma, Exophiala sideris, Exophiala spinifera, Exophiala xenobiotica, Filobasidium wieringae, Fonsecaea erecta, Fonsecaea monophora, Fonsecaea multimorphosa, Fonsecaea nubica, Fonsecaea pedrosoi, Fusarium agapanthi, Fusarium asiaticum, Fusarium avenaceum, Fusarium circinatum, Fusarium culmorum, Fusarium euwallaceae, Fusarium fujikuroi, Fusarium graminearum, Fusarium hostae, Fusarium langsethiae, Fusarium mangiferae, Fusarium meridionale, Fusarium nygamai, Fusarium oxysporum, Fusarium pininemorale, Fusarium poae, Fusarium praegraminearum, Fusarium proliferatum, Fusarium pseudograminearum, Fusarium sambucinum, Fusarium solani, Fusarium temperatum, Fusarium udum, Fusarium verticillioides, Geotrichum candidum, Graphilbum fragrans, Hanseniaspora uvarum, Histoplasma capsulatum, Hortaea werneckii, Hyphopichia homilentoma (Candida homilentoma), Kluyveromyces lactis, Kluyveromyces marxianus (Candida kefyr), Kwoniella bestiolae (Cryptococcus bestiolae), Kwoniella dejecticola (Cryptococcus dejecticola), Kwoniella pini (Cryptococcus pinus), Lachancea kluyveri, Lachancea lanzarotensis, Lachancea thermotolerans, Lachancea waltii, Leptosphaeria maculans, Lichtheimia corymbifera, Lichtheimia ramosa, Lodderomyces elongisporus, Lomentospora prolificans, Macrophomina phaseolina, Madurella mycetomatis, Malassezia caprae, Malassezia cuniculi, Malassezia dermatis, Malassezia equina, Malassezia furfur, Malassezia globosa, Malassezia japonica, Malassezia nana, Malassezia obtusa, Malassezia pachydermatis, Malassezia sloofftae, Malassezia sympodialis, Malassezia yamatoensis, Memnoniella echinata, Metarhizium acridum, Metarhizium album, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium guizhouense, Metarhizium majus, Metarhizium rileyi, Metarhizium robertsii, Metschnikowia bicuspidata, Metschnikowia fructicola, Metschnikowia kipukae (Candida kipukae), Meyer ozyma carpophila (Candida carpophila), Microsporum canis (Arthroderma otae), Mortierella alpina, Linnemannia elongata (Mortierella elongata), Podila verticillata (Mortierella verticillata), Mucor ambiguus, Mucor circinelloides, Mucor indicus, Mucor irregularis, Mucor velutinosus, Nakaseomyces bacillisporus, Nakaseomyces delphensis, Nakazawaea peltata, Nannizzia gypsea (Microsporum gypseum), Naumovozyma dairenensis, Fusarium vanettenii (Nectria haematococca), Neofusicoccum parvum, Nigrograna mackinnonii (Biatriospora mackinnonii), Nosema apis, Nosema bombycis, Nosema ceranae, Ochroconis constricta, Ogataea methanolica, Ogataea parapolymorpha, Ogataea polymorpha, Ophiostoma novo- ulmi, Ophiostoma piceae, Samsoniella hepiali (Paecilomyces hepiali), Paracoccidioides brasiliensis, Paracoccidioides lutzii, Pascua guehoae (Trichosporon guehoae), Penicillium antarcticum, Penicillium brasilianum, Penicillium capsulatum, Penicillium carneum, Penicillium coprophilum, Penicillium decumbens, Penicillium digitatum, Penicillium expansum, Penicillium flavigenum, Penicillium freii, Penicillium griseofulvum, Penicillium italicum, Penicillium janthinellum, Penicillium nalgiovense, Penicillium nordicum, Penicillium occitanis, Penicillium oxalicum, Penicillium paneum, Penicillium paxilli, Penicillium roqueforti, Penicillium sclerotiorum, Penicillium steckii, Penicillium subrubescens, Penicillium vulpinum, Phaeoacremonium minimum, Phaeotremella fagi (Cryptococcus fagi), Phaeotremella skinneri (Cryptococcus skinneri), Phanerochaete carnosa, Phanerodontia chrysosporium (Phanerochaete chrysosporium), Pyrrhoderma noxium (Phellinus noxius), Phialophora americana (Capronia semi-immersa), Phialophora attinorum (Phialophora attae), Phoma herbarum, Phycomyces blakesleeanus, Pichia kudriavzevii (Candida krusei), Pneumocystis carinii, Pneumocystis jirovecii, Pneumocystis murina, Pseudocercospora fijiensis (Cercospora fijiensis), Pseudopyr enochaeta ly coper sici (Pyrenochaeta lycopersici), Pseudozyma hubeiensis, Purpureocillium lilacinum, Rasamsonia emersonii, Rhinocladiella mackenziei (Ramichloridium mackenziei), Rhizoctonia solani, Rhizomucor miehei, Rhizomucor pusillus, Rhizomucor variabilis, Rhizopus delemar, Rhizopus microsporus, Rhizopus arrhizus (Rhizopus oryzae), Rhizopus stolonifer, Rhodotorula graminis, Rhodotorula mucilaginosa, Rhodotorula toruloides, Rhytidhysteron rufulum, Saccharomyces cerevisiae, Saksenaea oblongispora, Saksenaea vasiformis, Scedosporium apiospermum, Scedosporium aurantiacum, Scedosporium boydii, Scedosporium dehoogii, Schizophyllum commune, Sporopachydermia quercuum, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix insectorum, Sporothrix pallida, Sporothrix schenckii, Stachybotrys chartarum, Stachybotrys chlorohalonata, Starmerella apicola (Candida apicola), Starmerella bacillaris (Candida zemplininia), Stemphylium lycopersici, Suhomyces tanzawaensis (Candida tanzawaensis), Syncephalastrum monosporum, Syncephalastrum racemosum, Talaromyces amestolkiae, Talaromyces atroroseus, Talaromyces cellulolyticus, Talaromyces islandicus (Penicillium islandicum), Evansstolkia leycettana (T alaromyces leycettanus), Talaromyces marneffei (Penicillium marneffei), Talaromyces piceae (Penicillium piceum), Talaromyces pinophilus (Penicillium pinophilum), Talaromyces purpureogenus (Penicillium purpur ogenum), Talaromyces stipitatus, Talaromyces verruculosus (Penicillium verruculosum), Talaromyces wortmannii, Thermoascus crustaceus, Thermomyces lanuginosus, Thermothelomyces thermophilus (Myceliophthora thermophila), Thermothielavioides terrestris (Thielavia terrestris), Torulaspora delbrueckii, Trachipleistophora hominis, Trichoderma asperellum, Trichoderma atroviride, Trichoderma gamsii, Trichoderma hamatum, Trichoderma harzianum, Trichoderma longibrachiatum, Trichoderma parareesei, Trichoderma reesei, Trichoderma virens, Trichophyton, Trichophyton benhamiae (Arthroderma benhamiae), Trichosporon asahii, Trichosporon coremiiforme, Trichosporon faecale, Trichosporon inkin, Trichosporon ovoides, Ustilago cynodontis, Ustilago esculenta, Ustilago hordei, Ustilago maydis, Ustilago trichophora, Valsa mali, Verruconis gallopava (Ochroconis gallopava), Verticillium alfalfae, Verticillium dahliae, Verticillium longisporum, Verticillium tricorpus, Vittaforma corneae, Volvariella volvacea, Wallemia ichthyophaga, Wallemia mellicola, Wickerhamiella sorbophila (Candida sorbophila), Wickerhamiella versatilis (Candida versatilis), Wicker hamomyces anomalus (Pichia anomala), Wickerhamomyces ciferrii, Yarrowia deformans, Yarrowia keelungensis, Yarrowia lipolytica, Cunninghamella bertholletiae, Cunninghamella, Trichophyton equinum, Trichophyton inter digitale, Trichophyton rubrum, Trichophyton soudanense, Trichophyton tonsurans, Trichophyton verrucosum, and Trichophyton violaceum.

[0166] In some embodiments, a pathogenic microbial species is a parasite. In some embodiments, a parasite is a protozoa. Protozoa are single-celled eukaryotic microorganisms belonging to the Kingdom Protist. Helminths are a group of parasitic, multicellular, eukaryotic worms that can be classified into three main groups based on their morphology: 1) trematodes (flukes), which are flatworms that commonly infect the internal organs of their host; 2) cestodes (tapeworms), which typically attach to a host’s intestine; and, 3) nematodes (roundworms), which can infect various body tissues including the digestive tract, respiratory system, and muscles. Arthropods are invertebrate animals that can transmit disease-causing pathogens (e.g., bacteria, viruses, protozoa, or helminths) to humans and other animals. Nonlimiting examples of pathogenic parasites include Acanthamoeba, Ancylostoma ceylanicum, Ancylostoma duodenale, Angiostrongylus cantonensis, Angiostrongylus costaricensis, Anisakis simplex, Ascaris, Babesia divergens, Babesia microti, Balamuthia mandrillaris, Blastocystis hominis, Brugia malayi, Clonorchis sinensis, Cryptosporidium hominis, Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium parvum, Cryptosporidium ubiquitum, Cyclospora cayetanensis, Dictyostelium discoideum, Dirofdaria immitis, Dracunculus medinensis, Echinococcus granulosus, Echinococcus multilocularis, Echinostoma caproni, Entamoeba dispar, Entamoeba histolytica, Enter obius vermicularis, Fasciola hepatica, Giardia intestinalis (Giardia lamblia), Hymenolepis diminuta, Leishmania aethiopica, Leishmania amazonensis, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania panamensis, Leishmania tropica, Loa loa, Naegleria fowleri, Necator americanus, Onchocerca volvulus, Opisthorchis viverrini, Plasmodium cynomolgi, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Pythium insidiosum, Rodentolepis nana (Hymenolepis nana), Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Strongyloides stercoralis, Taenia asiatica, Taenia saginata, Taenia solium, Toxocara canis, Toxoplasma gondii, Trichinella, Trichomonas vaginalis, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Wuchereria bancrofti, Acanthamoeba astronyxis, Acanthamoeba castellanii, Acanthamoeba comandoni, Acanthamoeba culbertsoni, Acanthamoeba divionensis, Acanthamoeba healyi, Acanthamoeba lenticulata, Acanthamoeba lugdunensis, Acanthamoeba mauritaniensis, Acanthamoeba pearcei, Acanthamoeba polyphaga, Acanthamoeba quina, Acanthamoeba rhysodes, Acanthamoeba royreba, Ascaris lumbricoides, Ascaris suum, Trichinella britovi, Trichinella murrelli, Trichinella nativa, Trichinella nelsoni, Trichinella papuae, Trichinella patagoniensis, Trichinella pseudospiralis, Trichinella spiralis, and Trichinella zimbabwensis .

[0167] One or more microbial species

[0168] In some embodiments, the one or more different microbial species (e.g. the one or more different microbial species present in a sample, enriched, identified and / or quantified) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more microbial species. In some embodiments, the one or more different microbial species (e.g. the one or more different microbial species present in a sample, enriched, identified and / or quantified) is greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 15, greater than 20, greater than 25, greater than 30, greater than 35, greater than 40, greater than 45, or greater than 50, or more different microbial species. In some embodiments, the one or more different microbial species (e.g. the one or more different microbial species present in a sample, enriched, identified and / or quantified) is 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 45-50, 1-30, 2-29, 3-28, 4-27, 5-26, 6-25, 7-24, 8-23, 9- 22, 10-21, 11-20, 12-19, 13-18, or 14-17 different microbial species. One or more different microbial species in a clinical sample may all be from the same microbe type (e.g., bacteria, virus, fungus, parasite) or from different microbe types. In some embodiments, one or more different microbial species in a clinical sample are all bacteria, all viruses, all fungi, or all parasites. In some embodiments, one or more different microbial species are bacteria, viruses, fungi, parasites, or some combination thereof.

[0169] Sample

[0170] In some embodiments, a method for identifying and / or quantifying one or more microbial species in a clinical sample comprises obtaining a clinical sample comprising one or more microbial species from a subject. The term “obtaining”, as used herein, refers to collecting a clinical sample (e.g., blood, urine, or bronchoalveolar lavage) from a subject at a designated time. A clinical sample is a biological sample obtained or previously obtained from a subject. Non-limiting examples of clinical samples include a blood sample (e.g., whole blood, plasma, or serum), a urine sample, a saliva sample, a stool sample, a cerebrospinal fluid sample, a throat swab sample, an oral swab sample, a bronchial lavage sample, an endotracheal aspirate, and / or and a lower respiratory tract infection (LRTI) sample. In some embodiments, a clinical sample is a blood sample, wherein the blood sample is a whole blood sample, a plasma sample, or a serum sample. In some embodiments, the blood sample is obtained from a subject by venipuncture or finger prick. In some embodiments, a clinical sample is a urine sample, wherein the urine sample is obtained by midstream urine collection, catheterization (e.g., using a Foley catheter), or bladder puncture. A clinical sample may, in some embodiments, contain low levels of commensal bacteria from the skin and / or urinary tract microbiome.

[0171] Subjects

[0172] Methods provided herein include obtaining a clinical sample from a subject. A subject may be any organism that can be infected by a microbial species. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human primate (e.g., a laboratory animal such as a rhesus monkey). In some embodiments, the subject is a rodent, such as a rat or mouse. In some embodiments, the subject is a goat, rabbit, sheep, or pig. In some embodiments, the subject is a human.

[0173] Methods of Use A method of the present disclosure may be used in determining drug susceptibility of one or more microbial species in a subsample (e.g., a first subsample). Determining drug susceptibility may be performed using antimicrobial susceptibility testing (AST), a laboratory procedure that encompasses several different methods. AST has become increasingly essential to improve clinical outcomes as antimicrobial resistance rates continue to rise. Exemplary methods of performing AST are known to those of ordinary skill in the art and include, but are not limited to, culture (e.g., surveillance cultures), broth dilution tests (e.g., to determine minimum inhibitory concentrations (MIC)), antimicrobial gradient diffusion, disk diffusion, molecular assays (e.g., PCR), and genome-based analysis (e.g., sequencing).

[0174] A method of the present disclosure may be used in predicting appropriate treatment for infection with one or more microbial species. A microbe may be susceptible to a potential treatment (e.g., a drug), resistant to a potential treatment, or neither susceptible nor resistant to a potential treatment. A microbe is susceptible to a potential treatment if contacting the microbe (e.g., in a human subject) with the potential treatment causes reduced survival of the microbe, reduced proliferation of the microbe, or some combination thereof. A microbe is resistant to a potential treatment if contacting the microbe with the potential treatment does not cause reduced survival of the microbe, reduced proliferation of the microbe, or some combination thereof. A microbe may be susceptible to a potential treatment under certain circumstances (e.g., environmental conditions, co-infections with other microbes, etc.) and resistant to the same potential treatment under other circumstances. In some embodiments, a subject (e.g., a human subject) may be an individual that has been treated with, is being treated with, or will be treated with a treatment (e.g. an antibiotic) that one or more microbial species are resistant to. In some embodiments, a subject (e.g., a human subject) may be an individual that has been treated with, is being treated with, or will be treated with a treatment (e.g. an antibiotic) that one or more microbial species are susceptible to. In some embodiments, a subject (e.g., a human subject) may be an individual that has been treated with, is being treated with, or will be treated with an antibiotic that one or more microbial species are neither susceptible to nor resistant to.

[0175] Predicting appropriate treatment for infection requires identifying one or more microbial species, which may be accomplished by any method provided herein. In some embodiments complete sequences of the one or more microbial species whose nucleic acid has been amplified (e.g., in a first subsample) may be used to predict comprehensive genomic antimicrobial susceptibility testing (gAST). Comprehensive gAST means that the resistance of one or more microbial species and the susceptibility of the one or more microbial species to an antimicrobial drug is predicted based on the complete sequences for each microbial species identified in the amplified sample for which a complete sequence is generated.

[0176] Appropriate treatment may be predicted by any method known in the art. Nonlimiting methods of predicting appropriate treatment include: utilizing sequencing data and testing antimicrobial agents that the one or more microbial species are known to be susceptible to. In some embodiments, methods of predicting appropriate antimicrobial treatment includes utilizing sequencing data. Sequencing data may be whole genome sequencing data or partial genome sequencing data. When microbial genome (e.g. whole genome) sequencing data is used to predict appropriate antimicrobial treatment, the genome sequences for a microbe may be compared against a reference database of sequences known to confer antimicrobial agent resistance or susceptibility. Non-limiting examples reference databases include: Comprehensive Antibiotic Resistance Database (CARD), National Database of Antibiotic Resistant Organisms (NARDO), AMRFinder Plus, ResFinder, ResFinder Plus, and Resistance Map.

[0177] For example, when microbial genome sequencing data is used to predict appropriate antimicrobial treatment in Staphylococcus aureus (S. aureus), the whole genome sequencing data may be compared to sequences known to confer resistance to antibiotics such as vancomycin, methicillin, or any other antibiotic used to treat S. aureus infection. Alternatively, whole genome sequencing data may be used to create a de novo reference database that draws on known antimicrobial susceptibility or resistance sequences.

[0178] An antimicrobial agent may be an antibacterial agent, an antifungal agent, an antiviral agent, an anti-parasitic agent, or a combination thereof. Non-limiting examples of antimicrobial agents include: penicillin, methicillin, oxacillin, ampicillin, piperacillin, amoxicillin, cephazolin, cephalexin, cefuroxime, cefoxitin, cefotaxime, ceftriaxone, cefepime, vancomycin, teicoplanin, bleomycin, imipenem, meropenem, gentamicin, tobramycin, amikacin, erythromycin, clarithromycin, azithromycin, tetracycline, doxycycline, chloramphenicol, clindamycin, sulfisoxazole, sulfamethoxazole, metronidazole, trimethoprim, rifampicin, quinolones, clotrimazole, econazole, miconazole, terbinafine, fluconazole, ketoconazole, nystatin, amphotericin, candicidin, fllipin, hamycin, natamycin, rimocidin, bifonazole, butoconazole, econazole, fenti conazole, isoconazole, luliconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole, epoxiconazole, isavuconazole, itraconazole, posaconazole, propi conazole, ravuconazole, terconazole, voriconazole, abafungin, anidulafungin, caspfungin, micafungin, ibrexafungerp, acrisorcin, amorolfme, aurones, benzoic acid, abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir, umifenovir, atazanavir, tenofovir, baloxavir marboxil, boceprevir, bulevirtide, cidofovir, cobicistat, combivir, daclatasvir, daclatasvir, darunavir, delaviridine, didanosine, docosanol, dolutegravir, doravirine, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, ensitrelvir, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, ganciclovir, ibacitabine, ibalizumab, idoxuridine, imiquimoid, inosine pranobex, idinavir, lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nitazoxanide, norvir, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, remdesivir, ribavirin, rilpivirine, rimantadine, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, taribavirin, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, Truvada, umifenovir, valaciclovir, valganciclovir, vicriviroc, vidarabine, zalcitabine, zanamivir, zidovudine, ivermectin, pyrimethamine, dapsone, mefloquine, sulfadiazine, quinine, nitazoxanide, albendazole, piperazine, amodiaquine, furazolidone, mebendazole, sulfametopyrazine, diethylcarbamazine, thiabendazole, pentamidine, levamisole, tinidazole, praziquantel, primaquine, oxamniquine, atovaquone, proguanil, halofantrine, poscaconazole, sulfadoxine, paromomycin, hydroxychloroquine, sinefungin 5-S-methyl-5’-thioadenosine, fumagillin, benzimidazole, diminazene, radicicol, geneticin, halofuginone, oxibendazole, lucanthone, andrographolide, spiramycin, eflornithine, lonidamine, tafenoquine, niclosamide, anisomycin, chlortetracycline, artesunate, pyrantel, hexylresorcinol, dichlorvos, doramectin, moxidectin, phenothiazine, selamectin, salinomycin, artenimol, nifurtimox, triclabendazole, secnidazole, carbendazim, piperaquine, chloroquine, quinacrine, suramin, sodium stibogluconate, artemether, hycanthone, tetrandrine, and lotilaner.

[0179] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0180] EXAMPLES

[0181] Example 1 - Urine Sample Processing

[0182] The present disclosure provides a urine sample processing protocol that meets the critical need for appropriate antibiotic therapy for complicated urinary tract infections (cUTIs). Importantly, this protocol does not require a urine culture, and instead takes a urine sample from a human patient and amplifies bacterial cellular DNA for whole genome sequencing (WGS) using a next-generation sequencing modality (e.g., Nanopore).

[0183] In this protocol, (1) a clinical urine specimen is divided into at least 2 sub-samples for processing, (2) size-based velocity sedimentation separates smaller microbial cells from larger human cells, (3) human cells are selectively lysed, and (4) released human DNA is removed (FIG. 1). This produces enriched bacterial subsamples because the bulk of the human cells and human DNA have been removed. The enriched bacterial subsamples are then subjected to microbial lysis. Microbial DNA in subsample 1 is amplified via isothermal amplification (e.g., LAMP), resulting in enrichment of microbial DNA (relative to human DNA) by a factor of up to one billion (109) or more. Subsample 2 is processed without a DNA amplification step, allowing for quantification of microbial DNA relative to human DNA. Both subsamples move forward with WGS, and the sequencing data is analyzed to determine species identification (ID) and antibiotic sensitivity (AST) (subsample 1) and microbial abundance (subsample 2). This novel split approach delivers a comprehensive clinical result to drive rapid and appropriate treatment of infections (e.g., urinary tract infections).

[0184] Beginning with an existing sample processing protocol, baseline data was corrected for two target bacterial species: Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis). Bacteria were spiked into commercially sourced human urine (from a single donor) at IxlO5CFU / mL (n=4 for each target species) and compared to a negative control (n=4 nonspiked human urine). The bacterial DNA to human DNA ratio from subsample 1 was >lxl04for both E. coli and E. faecalis, confirming enrichment of each target species. Isolated bacterial genomic DNA was sequenced using next-generation sequencing (e.g., Nanopore sequencing), and data was analyzed for bacterial species identification. >99% genome coverage was achieved for each target species, and the bacterial genome coverage value for each sample was found to be above the previously defined threshold for the species, resulting in 100% accuracy in species identification in all 8 samples. The bacterial genome coverage value associated with each negative control was determined to be well below the threshold of any bacterial species, as expected. Importantly, the low-level presence of a common skin flora species, Corynebacterium tuber culostearicum, was not detected in each of the four negative control samples.

[0185] Protocol Development Since rapid processing of clinical urine samples is critical to identify and treat cUTIs, two variables that are likely to reduce turn-around time were examined. Firstly, a rapid transposon-based protocol (0.5 hours) was utilized instead of a library preparation protocol (6 hours). In addition, different sequencing modalities were tested to identify potential increases in the speed of sequencing data generation. The rigor of the experimental approach was also increased by using urine from three donors (both male and female), for a total of n=12 for each target species (i.e., E. coli, E. faecalis) and negative controls. Rigor was maintained by blinding the investigators to the sample type. The data demonstrated >99% genome coverage for both E. coli and E. faecalis, confirming the accuracy and efficiency of the workflow (FIG. 2)

[0186] As standard DNA amplification takes approximately two hours and typically yields a limit of detection < 10 CFUs / mL, it was anticipated that the amplification time could be reduced to detect a 4-log higher bacterial load of IxlO5CFU / mL, which is clinically relevant in urine. To reduce amplification time, commercially sourced human urine samples were spiked with two target bacterial species, E. coli and E. fctecalis, at a clinically relevant bacterial load of IxlO5CFU / mL. Amplification was evaluated at four time points: 120 (control), 60, 30, and 0 minutes. Comparison of each amplification time point found no significant difference in percentage of genome coverage (FIG. 3A) or genome coverage breadth (FIG. 3B) for each target species.

[0187] Processing Accuracy

[0188] To demonstrate the accuracy of protocol, 7 different bacterial species representative of the pathogen diversity associated with cUTI and urosepsis were selected: Escherichia coli (E. coli), Proteus mirabilis (P. mirabilis), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Enterococcus faecalis (E. faecalis), Enterococcus faecium (E. faecium), and Staphylococcus aureus (S. aureus). These seven gram-negative and gram-positive pathogens of interest vary in morphology, membrane and surface structure and adhesion properties, and other genomic characteristics, such as genome guanosine / cytosine (G / C) content. Commercially sourced human urine samples from four different donors were tested. A total of 16 replicates of each of the 7 species (n=16 per species; 4 urine donors, 4 replicates each) were tested using the protocol. Only 1 sample (out of 112) was not identified, for a total accuracy of 99.1% (Table 1). Table 1: Species ID Accuracy

[0189] * Accuracy across 112 samples processed among 7 target pathogens

[0190] Antibiotic sensitivity (AST) determination for uropathogenic species

[0191] With growing antibiotic resistance, knowing the antimicrobial resistance / susceptibility (AMR / S) profile is critical to informing clinical treatment decisions. WGS data generated with the protocol was used to predict AMR / S for a clinically relevant range of bacteria-antibiotic (“bug-drug”) combinations. Antimicrobial susceptibility prediction models were generated for 4 urosepsis species (Enterococcus faecalis (E. faecalis). Enterococcus faecium (E. faecium). Escherichia coli (E. coli). and Klebsiella pneumoniae (K. pneumoniae ) using a machine learning (ML) model that uses a training dataset comprising >55,000 samples and identifies patterns in the whole genome that predict antibiotic susceptibility / resistance.

[0192] Using ML models, Very High (>99.9%) or High (>87%) accuracy of antimicrobial susceptibility prediction models were obtained for 27 bug-drug combinations. Additional bug / drug combinations are being analyzed for High (>87%) accuracy, including those associated with Pseudomonas aeruginosa (Table 2).

[0193] Table 2: gAST Determination

[0194] Bacterial enrichment in the presence of excess human white blood cells

[0195] It is recognized that a urine sample from a patient with a suspected cUTI is likely to be enriched in white blood cells (WBCs), a clinical sign of infection. To evaluate whether WBCs (containing human DNA) compromise the efficiency of enrichment for bacterial DNA, the recovery of bacterial DNA was measured in urine samples spiked with IxlO5CFU / mL bacteria (control), or urine samples that contained both IxlO5CFU / mL bacteria and a 5-fold excess of human WBCs (i.e., IxlO5E. coli + 5xl05WBCs). For these studies a velocity sedimentation step was evaluated in three different conditions: no velocity sedimentation (“Excluded”), one round (“Included lx”) or two rounds (“Included 2x”) of velocity sedimentation. The ratio of target microbial DNA to human DNA after sequencing was compared. In the control experiment, there was no difference observed between the three groups (FIG. 4A). In contrast, the WBC spike-in data demonstrated that one round of velocity sedimentation significantly outperformed a process that excluded a velocity sedimentation step. Moreover, adding an additional round of velocity sedimentation did not significantly improve assay robustness (FIG. 4B). These data demonstrate that velocity sedimentation is beneficial for recovering bacterial DNA when an infection (i.e., cUTI or urosepsis) is suspected and that only a single velocity sedimentation step was sufficient to achieve efficient recovery of bacterial DNA.

[0196] Because the time sample collection and data-driven diagnostic results is a key differentiator in the standard-of-care and protocols described herein, the amount of time each step in the workflow takes was measured, demonstrating the potential of a sample-to-answer turn-around time in less than 6 hours, well within the first round (8-12 hours) of empiric antibiotic therapy (Table 3).

[0197] Table 3: Turn-around Time

[0198] Increasing sensitivity and specificity In most clinical laboratories, the threshold criterion for diagnosing cUTI in adult urine samples is IxlO5CFU / mL of a single bacterial species. However, cUTI diagnostic guidelines from culture-based methods are variable, and depend on urine collection method (e.g., clean catch, suprapubic aspiration, urethral catheterization), organism, and laboratory. Further, some guidelines suggest that concentrations below IxlO5CFU are clinically relevant and lower bacterial titers 1X102-1X104CFU / mL should be considered positive.

[0199] To achieve diagnostic sensitivity of at least IxlO3CFU / mL, commercially sourced urine samples from 4 donors containing 5 xlO5WBCs / mL were spiked with 1X102-1X105CFU / mL E.coli or E. faecalis . Each sample containing a target species was evaluated at least four times (n=16 replicates per species per experimental concentration). Negative control samples were tested in parallel (4 urine samples in duplicates, n=8). Using the protocol described herein, the percentage of genome coverage, species identification, sequencing depth, and turnaround time were calculated. To evaluate sensitivity at lower bacterial loads, a few variables (e.g., sample volume, DNA amplification time) were iteratively modified to achieve accurate species identification with starting bacterial load of IxlO3CFU / mL or less in the sample.

[0200] Determining pathogen abundance

[0201] High sensitivity in detecting pathogens in urine samples can lead to results that are not clinically relevant. For example, urogenital tract commensal species are often present in urine samples, but detection of these lowly abundant commensal species would not necessarily be indicative of cUTI. Species abundance is a metric that clinicians use to guide cUTI diagnosis and treatment. To establish an internal control, a calibration curve plotting bacterial concentration (CFU / mL) versus the sequencing abundance was used to estimate pathogen abundance in an unknown sample. If the abundance of an internal control falls outside of the expected range after sequencing, then the sample could be discarded due to process failure. Data collected from duplicate E. coli samples showed a correlation between bacterial load and sequence abundance in the absence of sample amplification (FIG. 5).

[0202] Determination of species identification limit of detection (LOD)

[0203] Contrived (control) samples are generated for 30 target species known to be associated with cUTI and urosepsis (Table 4). This panel of target species includes the seven species that account for -93% of cUTI infections, and 23 additional bacterial species of interest. Table 4. Target Species. * Indicate species for inclusivity testing

[0204] The screen of the 30 target species is performed at spiked-in concentrations of IxlO2, IxlO3, and IxlO4CFU / mL, each in triplicate. After the initial target concentration is confirmed, the Limit of Detection (LOD) is defined as the lowest concentration at which all 30 target species are identified as present using genome coverage values in 19 of 20 replicates (95% accuracy) per species.

[0205] Determination of species abundance range

[0206] For each of the 30 bacterial species, abundance quantification is performed at spikedin concentrations of IxlO2, IxlO3, and IxlO4CFU / mL. Each concentration is tested in triplicate and overall accuracy is determined. Since abundance is reported as a discontinuous variable (e.g., rare, low, moderate, high), overall accuracy reflects the binary outcome of a correct or incorrect abundance classification. Therefore, 95% total accuracy allows for 18 incorrect classifications.

[0207] Assay precision with automated prototype To validate overall precision within an automated concept demonstrator prototype (CDP), target species (E coli and E.faecalis) spiked-in at a concentration of IxlO5CFU / mL are tested for 6 non-consecutive days, with at least 2 operators per sample, for at least 24 replicates per species (total n=48) with the automated system. Manual processing is performed in parallel as controls.

[0208] To validate reagent precision, the same 2 target species (E. coli, E.faecalis) are tested in triplicate at lxlO5CFU / mL using two different reagent lots: for amplification (Al, A2) and sequencing (SI, S2) according to the testing matrix in Table 5.

[0209] Table 5. Reagent testing matrix

[0210] Inclusivity testing

[0211] Five clinical strains of each top 10 priority species (Table 4) are selected to represent variations in phenotypical characteristics, including AST profiles. Samples include all 50 clinical strains at a concentration of IxlO5CFU / mL. These samples are tested in triplicates (n=150) to confirm accurate species identification, abundance, and AST.

[0212] Example 2 - Split Clinical Sample Processing

[0213] Human nuclei require harsh chemical conditions for lysis and release of genomic DNA (gDNA), a step required for enzymatic depletion of human DNA (huDNA). There is variation in how well different microbial species survive this lysis, as well as variation in how well different microbial species survive other aspects of human cell depletion and microbial cell retention in clinical sample processing (e.g., buffer conditions, time of processing, etc.). The present disclosure provides an innovative protocol for splitting a clinical sample into at least two subsamples, each of which may be simultaneously and differentially treated to retain target microbial species (FIG. 6A). The sample split protocol therefore enables differential enrichment of target microbial species.

[0214] Tween Promotes Gram-Negative Bacterial Cell Survival

[0215] Acinetobacter baumannii (A. baumannii), Morganella morganii (M. morganii), and Stenotrophomonas maltophilia (S. maltophilia') are gram-negative bacteria that can be lysed by detergents, such as sodium dodecyl sulfate (SDS), that are often used to lyse and release huDNA. This lysis of gram-negative bacteria often results in a reduction or loss of gramnegative bacterial gDNA signal in clinical samples.

[0216] Adding a surfactant such as Tween (polysorbate) to lysis buffer containing SDS protects gram -negative bacteria from lysis while enabling lysis and release of huDNA. A. baumannii, M. morganii, and S. maltophilia were spiked into human whole blood samples. These spiked whole blood samples were evaluated using the sample split protocol to determine the effect of surfactant on gram negative bacterial cell survival in the presence of SDS. Briefly, the spiked whole blood samples were split into two subsamples and treated with lysis buffer (DNAse Buffer, TBS, or DPBS) containing SDS or lysis buffer containing SDS and Tween (DNAse Buffer+Tween, TBS+Tween, DPBS+Tween). There was an appreciable increase in the total recovered A. baumannii, M. morganii, and S. maltophilia CFUs and A. baumannii, M. morganii, and S. maltophilia CFUs relative to spiked-in input (FIGs. 6B-6C).

[0217] Tween promotes detection of lowly abundant microbial species

[0218] To evaluate whether the addition of Tween would allow increased sensitivity of the assay, A. baumannii was spiked-in at a concentration of 6.82 CFU / mL (high) or 2.8 CFU / mL (low) into whole blood samples. The high concentration samples were treated with SDS, while the low concentration samples were treated with SDS and Tween. The results showed a significant increase in the number of sequenced megabases mapped in the low concentration samples (FIG. 7A), breadth of genome coverage (FIG. 7B), and the bacterial DNA to human DNA ratio (FIG. 7C). These results indicate that clinically-relevant species can be recovered from clinical samples (e.g., blood, urine, or bronchoalveolar lavage) when present at low concentrations by adding surfactant (e.g., Tween).

[0219] The split protocol enables detection of lowly abundant microbial species The use of Tween to promote detection of lowly abundant microbial species was incorporated into a split protocol workflow. Briefly, whole blood samples were spiked with either 0.9-1.4 CFU / mL P. mirabilis or 3.7-4.8 CFU / mL S. pneumoniae and subject to either a standard processing protocol (SOP) or a sample split protocol (Split). For the split-protocol, each sample was subdivided into two subsamples: Arm A and Arm B. Subsamples in Arm A were subject to lysis using SDS and Tween, while subsamples in Arm B were subject to lysis with SDS only. After DNAse treatment, both subsamples were combined for downstream processing and sequencing.

[0220] A split protocol incorporating Tween was demonstrated to improve capture of microbial species of interest. Whole blood samples were spiked with P. mirabilis (a Gramnegative bacteria) or S. pneumoniae (a Gram-positive bacteria) as previously described and subject to the split protocol, where subsamples in Arm A were lysed with SDS and Tween while subsamples in Arm B were lysed with SDS. Three conditions were tested: 1) all subsamples were included in Arm A (Both arms A); 2) all subsamples were included in Arm B (Both arms B); and, 3) half of the subsamples were included in Arm A and the other half were included in Arm B (Split: One arm A, one arm B). When all subsamples were subject to the lysis conditions of Arm A (SDS and Tween), only P. mirabilis DNA was recovered; when all subsamples were subject to the lysis conditions of Arm B (SDS), only S. pneumoniae DNA was recovered. However, when the subsamples were split into two arms, both P. mirabilis DNA and S. pneumonia DNA were recovered (FIGs. 8A-8B) and evenly represented (FIGs. 8C-8D).

[0221] Taken together, these results demonstrate specific lysis conditions applicable to a microbial species of interest and that a split protocol can incorporate said conditions and provide clinically relevant results in less than 6 hours with high sensitivity and accuracy.

[0222] Example 3 - Simultaneous Microbial Abundance, Microbial Species Identification, and Determining Drug Susceptibility from a Single Clinical Sample

[0223] A clinical sample processing protocol was developed to enable simultaneous microbial identification, quantification of microbial species abundance, and prediction of drug (e.g., antibiotic) susceptibility of the microbes simultaneously in a clinical sample (FIG. 9). In this protocol, a clinical sample is obtained from a human subject (Step 1), selective human lysis and bacterial enrichment is performed (Step 2), the clinical sample is divided into at least a first subsample and a second subsample (Step 3), microbial DNA is amplified in a subsample to achieve whole genome amplification (Step 4A), relative abundance of microbial species is quantified in an unamplified subsample (Step 4B), and microbial DNA is sequenced in the first subsample and the second subsample (Step 5). After this clinical sample processing: a) one or more microbial species present in the first subsample and the second subsample are identified based on the sequenced microbial DNA; b) complete sequences of each microbial strain identified are created based on the amplified DNA; c) the complete sequences created in (b) are used for comprehensive genomic drug (e.g., antibiotic) susceptibility predictions; and / or d) microbial abundance measurement may be performed for each microbial strain in the original clinical sample based on the complete sequences in step (b).

[0224] The clinical sample processing protocol in FIG. 9 was used to perform microbial (e.g., pathogen) identification and measure microbial abundance simultaneously from a single lower respiratory tract infection (LRTI) clinical sample. Using this protocol, five microbial species A. baumannii, K. pneumoniae, P. aeruginosa, S. aureus, and S. pneumoniae were accurately identified at both 104CFU / mL input and 105CFU / mL microbial input (FIGs. 10A-10B) As expected, there was also human DNA depletion (FIG. IOC) and full microbial genome coverage at both 104CFU / mL input and 105CFU / mL microbial input (FIGs. 10D- 10E).

[0225] A workflow was used to pair and process sequencing data from amplified and nonamplified (FIG. 9, Steps 4A and 4B) clinical samples (FIG. 12). Carrier DNA was integrated into the clinical sample processing protocol in to compute a normalized measurement for K. pneumoniae in the clinical sample from 3 donors. The measurement from each of the 3 donors falls closely to the normalized signal from carrier DNA (FIG. 11 A), indicating that the split sample processing protocol is robust. The normalized signal is compared to a standard curve to generate a semi -quantitative abundance ranges (FIG. 11B), indicating that the abundance measurements are also robust.

[0226] Taken together, this data indicates that the clinical sample processing protocol in FIG. 9 allows for simultaneous microbial identification, drug susceptibility testing, and measuring microbial abundance in a single clinical sample when the information produced in the clinical sample processing protocol analyzed by the workflow in FIG. 12. Although particular embodiments of the invention have been illustrated by the foregoing exemplary embodiments, it should be understood that the examples are illustrative only and not intended to be limiting. One of skill in the art will recognize that methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and numerous changes in the details of implementation of the disclosed subject matter may be made without departing from the spirit and scope of the disclosed subject matter. The scope of the invention is limited only by the claims.

Claims

CLAIMSWe claim:

1. A method for enriching an array of microbes in a clinical sample, comprising:(i) obtaining a clinical sample from a human subject comprising human cells and microbial cells;(ii) dividing the clinical sample into at least a first subsample and a second subsample;(iii) lysing human cells in the first subsample in the presence of a first detergent and human cells in the second subsample in the presence of a second detergent and a surfactant; and(iv) removing human DNA from the lysed cells in the first subsample and the second subsample; wherein the enrichment of the array of microbes in the first subsample and / or the second subsample is increased relative to a control.

2. The method of claim 1, further comprising (v) amplifying microbial DNA in the first subsample and / or the second subsample.

3. The method of claim 1 or claim 2, further comprising (vi) sequencing microbial DNA in the first subsample and the second subsample.

4. The method of claim 3, further comprising (vii) identifying one or more microbial species present in the first subsample and the second subsample based on the sequenced microbial DNA.

5. A method for simultaneously identifying and quantifying one or more microbial species in a clinical sample, comprising:(i) obtaining a clinical sample from a human subject;(ii) dividing the clinical sample into at least a first subsample and a second subsample;(iii) amplifying microbial DNA in the first subsample to achieve whole genome amplification;(iv) sequencing microbial DNA in the first subsample and the second subsample;(v) identifying one or more microbial species present in the first subsample and the second subsample based on the sequenced microbial DNA; and(vi) quantifying relative clinical abundance of the one or more microbial species in the second subsample.

6. The method of claim 5, wherein prior to step (ii), the clinical sample is processed to enrich microbial cells relative to human cells by steps comprising:(a) dividing the clinical sample into at least a first subsample and a second subsample;(b) lysing human cells in the first subsample in the presence of a first detergent and human cells in the second subsample in the presence of a second detergent and a surfactant; and(c) removing human DNA from the lysed cells in the first subsample and the second subsample; wherein the enrichment of the array of microbes in the first subsample and / or the second subsample is increased relative to a control.

7. The method of claim 5 or claim 6, further comprising (vii) determining drug susceptibility of the one or more microbial species in the first subsample.

8. The method of any one of claims 5-7, wherein the method is accomplished in 6 hours or less.

9. The method of claim 7 or claim 8, further comprising (viii) treating the human subject with a drug to which the one or more microbial species are susceptible.

10. The method of any one of claims 5-9, wherein the clinical sample is a urine sample.

11. The method of any one of claims 6-10, wherein processing comprises centrifuging the first subsample and the second subsample to compact human cells at the bottom of separate tubes containing the first subsample and the second subsample.

12. The method of claim 11, further comprising removing supernatant from above the compacted human cells and placing it into separate tubes.

13. The method of any one of claims 2-12, wherein the microbial DNA and human DNA in the first subsample are amplified by polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), quantitative PCR (qPCR), reverse-transcriptase PCR (RT- PCR), degenerate oligonucleotide PCR, primer extension pre-amplification, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HD A), transcription mediated amplification (TMA), or recombinase polymerase amplification (RPA).

14. The method of any one of claims 4-13, wherein identifying the one or more microbial species comprises aligning the microbial DNA to a reference microbial genome.

15. The method of any one of claims 1-14, wherein the one or more microbial species are bacteria, fungi, or a combination thereof.

16. The method of any one of claims 7-15, wherein determining drug sensitivity comprises identifying patterns in whole genomes of the one or more microbial species that are associated with drug sensitivity.

17. A method for simultaneously identifying and quantifying one or more microbial species in a clinical sample, comprising:(i) obtaining a clinical sample from a human subject;(ii) dividing the clinical sample into at least a first subsample and a second subsample;(iii) amplifying microbial DNA in the first subsample;(iv) sequencing microbial DNA in the first subsample and the second subsample;(v) identifying one or more microbial species present in the first subsample and the second subsample based on the sequenced microbial DNA; wherein the amplified DNA from the first subsample is used to create complete sequences for each microbial species in the clinical sample; wherein the complete sequences are used for:(a) antibiotic susceptibility testing (AST) predictions; and(b) as a mapping reference for abundance quantification in the second subsample.

18. A method for analyzing a clinical sample to measure microbial abundance and predict drug susceptibility, comprising:(i) using complete microbial genome sequences from a first clinical subsample whose microbial sequences have been amplified and sequenced to predict microbial drug susceptibility; and(ii) mapping microbial sequences from a second clinical subsample whose microbial sequences have not been amplified to measure microbial abundance, wherein the first clinical subsample and the second clinical subsample are subsamples from a clinical sample obtained from a human subject.

19. The method of claim 17 or claim 18, wherein amplifying microbial DNA in the first subsample is whole genome amplification (WGA).

20. The method of claim 18 or claim 19, wherein the microbial DNA in the first subsample and the second subsample are sequenced.

21. The method of any one of claims 17-20, wherein the amplified DNA from the first subsample is used to create complete sequences of each microbial strain found in the clinical sample.

22. A method for enriching an array of microbes in a clinical sample, comprising:(i) obtaining a clinical sample from a human subject comprising human cells and microbial cells;(ii) lysing human cells in the presence of a detergent;(iii) removing human DNA from the lysed cells;(iv) dividing the clinical sample into at least a first subsample and a second subsample; wherein the enrichment of the array of microbes in the first subsample and / or the second subsample is increased relative to a control.

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