Systems and methods for microbial DNA and RNA detection

Abstract

The present disclosure provides systems and methods for detection of microbial DNA and RNA. In an aspect, a system or method for detection of microbial DNA or RNA comprises metagenomic next-generation sequencing of clinical samples (e.g., cerebral spinal fluid) to detect the microbial composition of the sample to diagnose a microbial infection (e.g., of the central nervous system).

Description

WSGR Docket No. 63228-706.601SYSTEMS AND METHODS FOR MICROBIAL DNA AND RNA DETECTIONCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 721,269, filed November 15, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] The advancement of diagnostic tests in clinical microbiology has been aided by the advent of metagenomic sequencing, a technology that enables an assessment of microbial taxa present in a sample. Timely, low-cost, accurate, and sensitive detection of pathogenic microbes is crucial for effective treatment and management of infections. However, detection and diagnosis of microbial infections is often costly, inefficient, and unreliable.SUMMARY

[0003] In 2019, over 10 million people worldwide died from microbial infections. Microbial infection can be caused by a wide range of pathogenic microbes including bacteria, DNA viruses, RNA viruses, fungi, and / or parasites, and the standard diagnostic paradigm is costly and inefficient. Additionally, the sensitivity and accuracy of such tests can be negatively impacted by the presence of background signal (e.g., human material) in the sample. Therefore, methods of minimizing the impact of background signal on such testing is critical. Accurate and rapid detection of microbial presence in subject samples is critical to ensure that subjects receive tailored and targeted therapies specific to their diagnosed condition.

[0004] Diagnosis of microbial infection may require using laboratory tests, e.g., metagenomic next-generation sequencing (mNGS), of a subject sample to detect the presence of one or more pathogens. However, as these tests may be costly and inefficient and subject samples may have a high background signal, pathogens can go undetected, leading to delayed treatment and undesirable treatment outcomes (FIG. 2).

[0005] Recognized herein is the need for rapid, low-cost, accurate, and sensitive methods for interpreting the multi-faceted results of laboratory tests, e.g., mNGS, of subject samples to facilitate pathogen detection and infection diagnosis.

[0006] The present disclosure provides systems and methods that may advantageously provide evaluation of sample quality and microbial composition, enhancing the speed, sensitivity, and accuracy of diagnostic interpretations. Systems and methods provided herein can allow for the detection of a multitude of infections agents simultaneously, with a single test, without a prioriWSGR Docket No. 63228-706.601clinical suspicion, and focus on advancing assay sensitivity, sample stability, utility of internal and external controls, microbial cell extraction efficiency, clinical capacity, sequencing depth, and turnaround time. Systems and methods provided herein provide, in part, processes of modernizing and scaling the detection of pathogens to improve assay sensitivity. System and methods provided herein can pave the way for broader application and advancements in unbiased mNGS diagnostics.

[0007] In an aspect, provided herein is a method for detecting microbial nucleic acids in a mixture comprising human nucleic acids, comprising: (a) adding a contrived sample matrix to the mixture, and (b) performing a nucleic acid assay on the mixture to detect a presence or quantitative measure of microbial nucleic acids in the mixture, wherein the nucleic acid assay has an assay sensitivity that is calibrated based at least in part on analyzing a plurality of mixtures comprising contrived sample matrices to determine a simulated presence or quantitative measure of human nucleic acids in the plurality of mixtures. In some embodiments, the method (e.g., a method provided herein) further comprises performing the nucleic acid assay on a biological sample. In some embodiments, the nucleic acid assay is configured to process nucleic acids obtained or derived from the biological sample.

[0008] In some embodiments, the biological sample comprises whole blood, blood serum, blood plasma, bile, breath, urine, feces, sebum, tissue, breast milk, hair, saliva, sputum, sweat, tears, lymphatic fluid, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, cerebrospinal fluid, seminal fluid, vaginal secretion, amniotic fluid, nasal fluid, otic fluid, interstitial fluid, gastric fluid, intestinal fluid, transudates, exudates, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs, or biological washes. In some embodiments, the biological sample comprises cerebrospinal fluid (CSF), blood plasma, peritoneal fluid, pleural fluid, synovial fluid, or pericardial fluid.

[0009] In some embodiments, the method (e.g., a method provided herein) further comprises determining the nucleic acid assay sensitivity to optimize the nucleic acid assay. In some embodiments, optimizing the nucleic acid assay comprises one or more of decreasing the noise of the assay, increasing the signal to noise ratio of the assay, and increasing the sensitivity of the assay.

[0010] In some embodiments, performing the nucleic acid assay comprises sequencing. In some embodiments, the nucleic acid assay comprises a metagenomic next generation sequencing (mNGS) assay. In some embodiments, the sequencing comprises one or more of nucleic acid extraction, nucleic acid isolation, nucleic acid fragmentation, reverse transcription (e.g., of RNA), transcript fragmentation, adapter ligation, amplification, targeted enrichment, nucleicWSGR Docket No. 63228-706.601acid library preparation, bisulfite conversion, and methylation conversion. In some embodiments, the sequencing comprises one or more of targeted sequencing, single molecule real-time sequencing, exonor exome sequencing, intron sequencing, electron microscopy -based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole -genome sequencing, sequencing by hybridization, pyrosequencing, duplex sequencing, cycle sequencing, single -base extension sequencing, solid phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co -amplification at lower denaturation temperature -PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, long-read sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, and MS-PET sequencing. In some embodiments, the sequencing comprises a polymerase chain reaction (PCR) or isothermal amplification. In some embodiments, the amplification comprisesuse of amplification reagents, the amplification reagents comprising reagents for one or more of polymerase chain reaction (PCR), transcription mediated amplification (TMA), helicase dependent amplification (HD A), circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA).

[0011] In some embodiments, performing the nucleic acid assay comprises extracting the microbial nucleic acids or the human nucleic acid. In some embodiments, performing the nucleic acid assay comprises preparing a nucleic acid library. In some embodiments, performing the nucleic acid assay comprises (i) extracting the microbial nucleic acids or the human nucleic acids and (ii) preparing the nucleic acid library.

[0012] In some embodiments, performing the nucleic acid assay comprises adding two or more internal controls to the mixture.

[0013] In some embodiments, performing the nucleic acid assay comprises lysing a microbial or a human cell. In some embodiments, performing the lysing comprises adding a chemical reagent. In some embodiments, the lysing comprises bead bashing. In some embodiments, performing the nucleic acid assay comprises spinning down the mixture or the biological sampleWSGR Docket No. 63228-706.601to form a pellet comprising microbial nucleic acids. In some embodiments, performing the nucleic acid assay comprises removing a supernatant. In some embodiments, a volume of the supernatant removed is about 50%, 60%, 70%, 80%, 85%, 90%, or 95%, of an initial volume of the mixture. In some embodiments, a volume of the supernatant removed is about 50% -99%, 75% -95%, or 85-95% of an initial volume of the mixture.

[0014] In some embodiments, the contrived sample matrix comprises two or more internal controls. In some embodiments, the contrived sample matrix comprises three or more internal controls. In some embodiments, the contrived sample matrix comprises three or more internal controls. In some embodiments, the two or more internal controls comprise two or more internal deoxyribonucleic acid (DNA) controls. In some embodiments, the two or more internal controls comprise two or more internal ribonucleic acid (RNA) controls. In some embodiments, the mixture comprises (i) the two or more internal DNA controls or (ii) the two or more internal RNA controls. In some embodiments, the mixture comprises (i) a first internal DNA control and a second internal DNA control and (ii) a first internal RNA control and a second internal RNA control. In some embodiments, the two or more internal controls comprise two or more of T1 DNA phage, MS2 RNA phage, Lambda DNA phage, and an RNA standard (e.g., the External RNA Controls Consortium (ERCC) standard).

[0015] In some embodiments, the method (e.g., a method provided herein), further comprises adding the two or more internal controls to the mixture prior to the extracting the microbial nucleic acids or the human nucleic acid. In some embodiments, the method (e.g., a method provided herein), further comprises adding the two or more internal controls to the mixture after the extracting the microbial nucleic acids or the human nucleic acid. In some embodiments, the two or more internal controls are added to the mixture after the extracting the microbial nucleic acids or the human nucleic acid and prior to the preparing the nucleic acid library. In some embodiments, the first internal DNA control and the first internal RNA control are added to the mixture prior to the extracting the microbial nucleic acids or the human nucleic acid, and wherein the second internal DNA control and the second internal RNA control are added to the mixture after the extracting the microbial nucleic acids or the human nucleic acid.

[0016] In some embodiments, the microbial nucleic acids comprise microbial DNA. In some embodiments, the microbial nucleic acids comprise microbial RNA.

[0017] In some embodiments, the human nucleic acids comprise human DNA. In some embodiments, the contrived sample matrix has about 0.01 picograms (pg) to about 1 microgram (pg) of human DNA. In some embodiments, the contrived sample matrix has about 1,000 pg to about 100,000 pg of human DNA. In some embodiments, the contrived sample matrix has about 1,200, about 1,900 pg, or about 76,000 pg of human DNA.WSGR Docket No. 63228-706.601

[0018] In some embodiments, the human nucleic acids comprise human RNA. In some embodiments, the human nucleic acids comprise human ribosomal (rRNA). In some embodiments, the contrived sample matrix has aboutO.Ol pgto about 100 pg of human RNA. In some embodiments, the contrived sample matrix has about 1 pg to about 10,000 pg of human RNA. In some embodiments, the contrived sample matrix has about 7.9 pg, about 121 pg, or about 7,400 pg of human RNA.

[0019] In some embodiments, the mixture comprises at least 5 human cells per microliter, at least 250 human cells per microliter, or at least 500 human cells per microliter.

[0020] In some embodiments, the microbial nucleic acids comprise nucleic acids from one or more microbes. In some embodiments, the one or more microbes comprise one or more of a filamentous fungus, a gram negative bacteria, a gram positive bacteria, a yeast, a parasite, a DNA virus, and an RNA virus. In some embodiments, the one or more microbes comprise one or more of a filamentous fungus, a gram negative bacteria, a gram positive bacteria, a yeast, a parasite, a DNA virus, and an RNA virus.

[0021] In some embodiments, the filamentous fungus comprises one or more of a fungus of the genus Acre monium. Alternaria, Aspergillus, Cladosporium, Cryptococcus, Curvularia, Fusarium, Histoplasma, Lichtheimia, I.omenlospora, Mucor, Paecilomyces, Penicillium, Rhizomucor , Rhizopus, Scedosporium, Schizophyllum, and Trichoderma. In some embodiments, the filamentous fungus comprises one or more of Acremonium spp., Alternaria spp., Alternaria alternata, Alternaria infectoria, Aspergillus spp., Aspergillus carneus, Aspergillus clavatus, Aspergillus flavus, Aspergillus jumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor, Aspergillus parasiticus, Cladosporium spp., Cryptococcus spp., Cryptococcus neoformans, Curvularia spp., Fusarium spp., Fusarium avenaceum, Fusarium culmorum, Fusarium equiseti, Fusarium fujikuroi, Fusarium graminearum, Fusarium nivale, Fusarium proliferatum, Fusarium oxysporum, Fusarium roseum, Fusarium solani, Fusarium verticillioides, Histoplasma spp., Histoplasma capsulatum, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramose, Lomentospora spp., I.omenlospor prolificans, Mucor spp., Mucor circinelloides, Paecilomyces spp., Paecilomyces variotii, Penicillium spp., Penicillium aurantiogriseum, Penicillium brunneum, Penicillium citreoviride , Penicillium citrinin, Penicillium claviforme, Penicillium crustosum, Penicillium expansum, Penicillium griseojulvum, Penicillium hirsutum, Penicillium islandicum, Penicillium kloeckeri, Penicillium roqueforti, Penicillium rubrum, Penicillium rugulosum, Penicillium verrucossum, Penicillium viridicatum, Rhizomucor spp., Rhizomucor pusillus, Rhizopus spp., Rhizopus arrhizus, Rhizopus microspores, Scedosporium spp.,WSGR Docket No. 63228-706.601Scedosporium apiospermum, Schizophyllum spp., Schizophyllum commune, and Trichoderma spp.

[0022] In some embodiments, the gram negative bacteria comprises one or more of a bacterium of the genus Acinetobacter , Bacteroides, Cereibacter , Chlamydia, Citrobacter, Enterobacter , Escherichia, Haemophilus, Klebsiella, Moraxella, Morganella, Neisseria, Pantoea, Proteus, Pseudomonas, Shigella, Salmonella, and Yersinia. In some embodiments, the gram negative bacteria comprises one or more of Acinetobacter spp ., Acinetobacter baumannii, Acinetobacter johnsonii, Bacteroides sp., Bacteroides fragilis, Bacteroides thetaiotaomicron , Chlamydia spp., Chlamydia trachomatis, Citrobacter spp., Citrobacter freundii, Citrobacter koseri, Enterobacter spp., Enterobacter aerogenes, Enterococcus avium, Enterobacter cloacae complex, Escherichia coli, Escherichia coli Extended-Spectrum Beta-Lactamase (ESBL), Haemophilus spp., Haemophilus influenzae, Klebsiella spp., Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella pneumoniae Extended-Spectrum Beta -Lactamase (ESBL), Klebsiella variicola, Moraxella spp., Moraxella sp. K1664, Moraxella bovis, Moraxella canis, Moraxella catarrhalis, Moraxella lacunata, Moraxella nonliquefaciens, Moraxella osloensis, Moraxella phenylpyruvica, Morganella spp., Morganella mor ganii, Neisseria spp., Neisseria gonorrhoeae, Pantoea spp., Pantoea agglomerans, Proteus spp., Proteus mirabilis, Pseudomonas spp., Pseudomonas aeruginosa, Salmonella spp ., Salmonella enterica, Salmonella bongori, Shigella spp., Shigella dysenteriae , Shigella flexneri, Shigella boydii, Shigella sonnei, Yersinia spp., and Yersinia pestis.

[0023] In some embodiments, the gram positive bacteria comprises one or more of a bacterium of the genus Bacillus, Brevibacterium, Clostridium, Corynebacterium, Eggerthella, Enterococcus, Granulicatella, Micrococcus, Mycobacterium, Staphylococcus, and Streptococcus. In some embodiments, the gram positive bacteria comprises one or more of Bacillus sp., Brevibacterium sp., Clostridium spp., Clostridium difficile, Corynebacterium sp., Eggerthella sp., Eggerthella lenta, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Vancomycin resistant Enterococcus faecium (VRE), Enterococcus faecium, Vancomycin resistant Enterococcus faecium (VRE), Enterococcus gallinarum, Granulicatella spp., Granulicatella adiacens, Micrococcus spp ., Micrococcus luteus, Mycobacterium spp., and Mycobacterium tuberculosis Staphylococcus spp., Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA), Methicillin / Oxacillin Resistant Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus simulans, Staphylococcus warneri, Streptococcus spp., Streptococcus agalactiae, Streptococcus aginosus, Streptococcus canis, Streptococcus constellatus, Streptococcus intermedins, Streptococcus mitis,WSGR Docket No. 63228-706.601Streptococcus pneumoniae , Streptococcus pyogenes, Streptococcus salivarius, Streptococcus uberis, and Streptococcus warneri.

[0024] In some embodiments, the yeast comprises one or more of a yeast of the genus Candida or Cryptococcus . In some embodiments, the yeast comprises one or more of Candida auris, Candida albicans, Candida glabr ata, Candida parapsilosis , Candida tropicalis, Candida krusei, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, and Cryptococcus uniguttulatus .

[0025] In some embodiments, the parasite comprises one or more of a protozoa, a helminth, or an ectoparasite.

[0026] In some embodiments, the DNA virus comprises one or more of herpesvirus, cytomegalovirus (CMV), muromegalovirus, human papillomavirus (HPV), adenovirus, hepatitis B virus (HBV), poxvirus, and polyomavirus. In some embodiments, the herpesvirus comprises one or more of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Kaposi sarcoma-associated herpesvirus (gamma herpesvirus), varicella-zoster virus (VZV), Roseolovirus, and Epstein-Barr virus (EBV). In some embodiments, the polyomavirus comprises Human polyomavirus 1 (BK Virus).

[0027] In some embodiments, the RNA virus comprises one or more of influenza virus, respiratory syncytial virus (RSV), coronavirus (e.g., SARS-CoV-2), enterovirus, norovirus, rotavirus, human immune deficiency virus (HIV), hepatitis C virus (HCV), Rift Valley fever virus, morbilivirus Tick-borne encephalitis virus, Zika virus, Dengue virus, West Nile virus, Ebola virus, yellow fever virus, Saint Louis encephalitis virus (SLEV), Eastern Equine encephalitis virus (EEEV), La Crosse encephalitis virus (LCEV), and Japanese encephalitis virus.

[0028] In an aspect, provided herein is a method for calibrating a nucleic acid assay, comprising: (a) analyzing a plurality of mixtures comprising contrived sample matrices to determine a simulated presence or a quantitative measure of human nucleic acids in the plurality of mixtures; (b) determining an assay characteristic of the nucleic acid assay based at least in parton the analyzing the plurality of mixtures; and (c) calibrating the nucleic acid assay based at least in part on the assay characteristic determined in (b).INCORPORATION BY REFERENCE

[0029] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by referenceWSGR Docket No. 63228-706.601contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure are obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0031] FIG. 1 illustrates an example workflow of a metagenomic Next-Generation Sequencing (mNGS)-based test for central nervous system (CNS) infections.

[0032] FIG.2 illustrates the clinical and commercial landscape for diagnosis of suspected CNS infections.

[0033] FIG. 3 illustrates an example workflow of the wet lab and diagnostic operations of an mNGS-based test for CNS infection detection and diagnosis provided herein.

[0034] FIG. 4 illustrates an example wet lab workflow of a system or method of an mNGS-based test for CNS infection detection and diagnosis provided herein.

[0035] FIG. 5 illustrates an example dry lab operation of a system or method of an mNGS-based test for CNS infection detection and diagnosis provided herein.

[0036] FIG.6 illustrates an example bioinformatics pipeline comprising modules of a system or method of an mNGS-based test for CNS infection detection and diagnosis provided herein.

[0037] FIG.7 illustrates an example workflow of the dry lab operation of a system or method of an mNGS-based test for CNS infection detection and diagnosis provided herein.

[0038] FIG. 8 illustrates the taxonomic diversity of CNS infections.

[0039] FIG. 9A provides an example of a quantification of the amount of human deoxyribonucleic acid (DNA) biomass detected using an mNGS-based test for CNS infection detection and diagnosis provided herein.

[0040] FIG.9B provides an example of a quantification of the amount of human ribonucleic acid (RNA) biomass detected using an mNGS-based test for CNS infection detection and diagnosis provided herein.

[0041] FIGS. 10A-10G provide examples of the detection sensitivity of a system or method of detection of microbial DNA or RNA provided herein, as measured by the microbial signal in reads per million (rpM) for a clinically -relevant concentration of filamentous fungus (FIG.10A), gram negative bacteria (FIG. 10B), yeast (FIG. 10C), DNA virus (FIG. 10D), parasiteWSGR Docket No. 63228-706.601(FIG. 10E), gram positive bacteria (FIG. 10F), or RNA virus (FIG. 10G) spiked into a sample matrix.

[0042] FIGS. 11A-11C provide target organisms’ rpM ratio (relative to control with no target organisms) in synthetic CSF (sCSF) containing no human content (FIG. 11 A), low human content (5 human cells / uL, FIG. 11B) and high human content (500 human cell / uL, FIG.11C) at varying dilutions of internal controls.

[0043] FIGS. 12A-12C provide target organisms’ biomass (picograms, pg) in sCSF containing no human content (FIG. 12 A), low human content (5 human cells / uL, FIG. 12B) and high human content (500 human cell / uL, FIG. 12C) at varying dilutions of internal controls.

[0044] FIGS. 13A-13C provide measurements of the human deoxyribonucleic acid (DNA) biomass (FIG. 13A), human ribonucleic acid (RNA) biomass (FIG. 13B), and the percent of total human biomass that maps to ribosomal RNA (rRNA; FIG. 13C) in cerebrospinal fluid (CSF), plasma, ascites, pleural, synovial, and pericardial biological samples.

[0045] FIGS. 14A and 14B provide measurements of the amount of microbial DNA biomass (FIG. 14A) and microbial RNA biomass (FIG. 14B) in CSF, plasma, peritoneal, pleural, synovial, and pericardial biological samples.

[0046] FIGS. 15A and 15B provide quantifications of the concentration of DNA (FIG. 15A) and RNA (FIG. 15B) in CSF, plasma, peritoneal, pleural, synovial, and pericardial biological samples.

[0047] FIG. 16 provides example workflows for nucleic acid assays of methods of detecting microbial acids provided herein.

[0048] FIGS. 17A-17C provide example workflows for nucleic acid assays provided herein, including a baseline workflow (FIG. 17A), and modified workflows comprising (a) the use of a reagent to remove RNA during DNA synthesis (FIG. 17B) or (b) removal of a volume of supernatant from the sample (FIG. 17C).

[0049] FIG. 18 provides the percentage of human RNA in plasm, serum, whole blood, Peripheral Blood Mononuclear Cell (PBMC), and CSF samples.

[0050] FIG. 19 provides a diagram of a workflow for the removal of supernatant from a biological sample comprising: (a) pelleting a 1 mL sample and (b) removing 900 uL of the supernatant.

[0051] FIG.20 provides the effect of (a) pelleting a 1 mL sample and (b) removing 900 uL of the supernatant on the number of reads of nucleic acids corresponding to specific microbes in a positive control sample.WSGR Docket No. 63228-706.601DETAILED DESCRIPTION

[0052] While various embodiments of the invention have been shown and described herein, it is obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0053] The term “subject,” as used herein, generally refers to a human such as a patient. The subject may be a person (e.g., a patient) with a disease, disorder, or condition, or a person that has been treated for a disease, disorder, or condition, or a person that is being monitored for a disease, disorder, or condition, or a person that is suspected of having the disease, disorder, or condition, or a person that does not have or is not suspected of having the disease, disorder, or condition. The disease, disorder, or condition maybe an infectious disease, an immune disorder or disease, an injury, or a rare disease. The infectious disease may be caused by bacteria (e.g., gram negative or gram positive bacteria), viruses (e.g., DNA or RNA viruses), fungi (e.g., filamentous fungi), and / or parasites. For example, the disease or disorder may be bacterial, viral, fungal, or parasitic meningitis; encephalitis, cytomegalovirus, orbrain abscess.

[0054] Biological samples can be obtained or derived from a subject. Biological samples can contain one or more of cerebral spinal fluid (CSF), peritoneal fluid (e.g., ascites fluid or abdominal fluid), pericardial fluid, pleural fluid, synovial fluid (e.g., joint fluid), or ocular fluid (e.g., aqueous humor or vitreous humor).

[0055] Recognized herein is the need for methods and systems for the detection of microbial organisms, such as those implicatedin microbial infections, e.g., central nervous system (CNS) infections, in clinical samples, such as cerebral spinal fluid (CSF), using unbiased metagenomic next-generation sequencing (mNGS). With over 26,000 species of bacteria, over 10,000 viral species, over 16,000 fungal species, and over 15,000 parasitic species implicated in CNS infection alone (FIG. 8), such methods and systems for the detection of microbial organisms can offer a comprehensive overview of the microbial landscape in a clinical sample, such as cerebral spinal fluid (CSF), facilitating the detection of rare, novel, or unexpected pathogens critical for diagnosing microbial infections. Additionally, a method or systems for diagnosing an infection or detecting a microbe provided herein can increase the sensitivity of infectious disease workup on subject samples, e.g., cerebrospinal fluid samples with a high background of human material. For example, a system or method provided herein can have high sensitivity and accuracy in detecting viruses (e.g., DNA or RNA viruses), bacteria (e.g., gram negative or gram positive bacteria), fungi (e.g., filamentous fungi), and parasites at one time (simultaneously) from oneWSGR Docket No. 63228-706.601sample with a high level of background human nucleic acid signal and background microbial nucleic acid signal, e.g., deriving from environmental sources.

[0056] Systems and methods provided herein address this critical need with a solution for detection of microbial organisms in clinical samples with high levels of background microbial nucleic acid signal deriving from environmental sources and variable levels of human nucleic acid signal present in different clinical samples. The systems and methods provided herein provide evaluation of sample quality and microbial composition while incorporating features to increase accuracy of pathogen detection and diagnosis. Additionally, systems and methods provided herein provide evaluation and reporting of the sensitivity of mNGS for microbial organism detection. These features enhance the reliability of diagnostic interpretations, thereby supporting informed clinical decision-making and improving subject outcomes.

[0057] Systems and methods of detecting microbial DNA and RNA provided herein can comprise collection, processing, sequencing, and analysis of samples and reporting of analysis results (FIG. 1). In some embodiments, a samples, e.g., a CSF sample, is collected at a medical institution and mailed with a requisition form for processing, sequencing, and analysis. In some embodiments, after the sample is received, nucleic acid is extracted from the sample, a library is prepared, and the sample is sequenced. In some embodiments, after sequencing, the sample is analyzed with an informatic pipeline. In some embodiments, a report is generated, e.g., a report indicating the detection of microbial RNA or DNA in a sample. In some embodiments, the time from receiving the sample to delivery of the report is less than about 2 weeks. In some embodiments, the time from receiving the sample to delivery of the report is less than about 10 days. In some embodiments, the time from receiving the sample to delivery of the report is less than about 7 days. In some embodiments, the time from receiving the sample to delivery of the report is less than about 5 days. In some embodiments, the time from receiving the sample to delivery of the reportis less than about 4 days. In some embodiments, the time from receiving the sample to delivery of the report is less than about 3 days. In some embodiments, the time from receiving the sample to delivery of the report is less than about 2 days. In some embodiments, the time from receiving the sample to delivery of the report is less than about 1 day. In some embodiments, the time from receiving the sample to delivery of the report is less than about 12 hours.

[0058] In an aspect, provided herein are methods for detecting microbial nucleic acids in a mixture comprising human nucleic acids. In some embodiments, a method for detecting microbial nucleic acids in a mixture comprising human nucleic acids provided herein comprises adding a contrived sample matrix to the mixture. In some embodiments, a method for detecting microbial nucleic acids in a mixture comprising human nucleic acids provided herein comprisesWSGR Docket No. 63228-706.601performing a nucleic acid assay on the mixture to detect a presence or quantitative measure of microbial nucleic acids in the mixture, wherein the nucleic acid assay has an assay sensitivity that is calibrated based at least in part on analyzing a plurality of mixtures comprising contrived sample matrices to determine a simulated presence or quantitative measure of human nucleic acids in the plurality of mixtures.

[0059] In some embodiments, provided herein are methods for detecting microbial nucleic acids in a mixture comprising human nucleic acids, comprising: (a) adding a contrived sample matrix to the mixture; and (b) performing a nucleic acid assay on the mixture to detect a presence or quantitative measure of microbial nucleic acids in the mixture, wherein the nucleic acid assay has an assay sensitivity that is calibrated based at least in part on analyzing a plurality of mixtures comprising contrived sample matrices to determine a simulated presence or quantitative measure of human nucleic acids in the plurality of mixtures .

[0060] In some embodiments, provided herein is a method for detecting microbial nucleic acids comprising determining a nucleic acid assay sensitivity to optimize the nucleic acid assay. In some embodiments, optimizing the nucleic acid assay comprises decreasing the noise of the assay, increasing the signal to noise ratio of the assay, or increasing the sensitivity of the assay. In some embodiments, optimizing the nucleic acid assay comprises decreasing the noise of the assay. In some embodiments, optimizing the nucleic acid assay comprises increasing the signal to noise ratio of the assay. In some embodiments, optimizing the nucleic acid assay comprises increasing the sensitivity of the assay.

[0061] In an aspect, provided herein is a method for calibrating a nucleic acid assay comprising analyzing a plurality of mixtures comprising contrived sample matrices to determine a simulated presence or a quantitative measure of human nucleic acids in the plurality of mixtures . In some embodiments, a method for calibrating a nucleic acid assay provided herein comprises determining an assay characteristic of the nucleic acid assay based at least in part on the analyzing the plurality of mixtures. In some embodiments, a method for calibrating a nucleic acid assay provided herein comprises calibrating the nucleic acid assay based at least in parton a determined assay characteristic.

[0062] In some embodiments, provided herein is a method for calibrating a nucleic acid assay comprising: (a) analyzing a plurality of mixtures comprising contrived sample matrices to determine a simulated presence or a quantitative measure of human nucleic acids in the plurality of mixtures; (b) determining an assay characteristic of the nucleic acid assay based at least in part on the analyzing the plurality of mixtures; and (c) calibrating the nucleic acid assay based at least in part on the assay characteristic determined in (b).WSGR Docket No. 63228-706.601

[0063] Provided herein in some embodiments is a method for detecting microbial nucleic acids in a mixture comprising human nucleic acids comprising performing a nucleic acid assay (e.g. , a nucleic acid assay provided herein) on a biological sample (e.g., a biological sample provided herein). The nucleic acid assay can be configured to process nucleic acids obtained or derived form a biological sample (e.g., a biological sample provided herein). In some embodiments, the biological sample comprises whole blood, blood serum, blood plasma, bile, breath, urine, feces, sebum, tissue, breast milk, hair, saliva, sputum, sweat, tears, lymphatic fluid, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, cerebrospinal fluid, seminal fluid, vaginal secretion, amniotic fluid, nasal fluid, otic fluid, interstitial fluid, gastric fluid, intestinal fluid, transudates, exudates, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs, or biological washes. In certain embodiments, the biological sample comprises cerebrospinal fluid (CSF), blood plasma, peritoneal fluid, pleural fluid, synovial fluid, or pericardial fluid. In certain specific embodiments, the biological sample comprises CSF. In certain specific embodiments, the biological sample comprises blood plasma. In certain specific embodiments, the biological sample comprises peritoneal fluid. In certain specific embodiments, the biological sample comprises pleural fluid. In certain specific embodiments, the biological sample comprises synovial fluid. In certain specific embodiments, the biological sample comprises pericardial fluid. In certain specific embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs).

[0064] Provided herein in some embodiments is a method for detecting microbial nucleic acids in a mixture comprising human nucleic acids comprising performing a nucleic acid assay. In some embodiments, performing the nucleic acid assay comprises sequencing. For example, the nucleic acid assay can comprise a metagenomic next generation sequencing (mNGS) assay. In some embodiments, the sequencing comprises nucleic acid extraction, nucleic acid isolation, nucleic acid fragmentation, reverse transcription (e.g., of RNA), transcript fragmentation, adapter ligation, amplification, targeted enrichment, nucleic acid library preparation, bisulfite conversion, or methylation conversion. In some embodiments, sequencing comprises one or more of targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy -based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, duplex sequencing, cycle sequencing, single-base extension sequencing, solid phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, coamplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencingWSGR Docket No. 63228-706.601by reversible dye terminator, paired -end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, long-read sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, and MS-PET sequencing. In some embodiments, sequencing comprises a polymerase chain reaction (PCR) or isothermal amplification. In specific embodiments, the amplification comprises amplification reagents, the amplification reagents comprising reagents for one or more of polymerase chain reaction (PCR), transcription mediated amplification (TMA), helicase dependent amplification (HD A), circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA).

[0065] In some embodiments, a test for diagnosing an infection or detecting a microbe provided herein comprises a wet lab operation, e.g., a nucleic acid assay, such as a nucleic acid assay provided herein (FIG. 4). A wet lab operation can comprise an automated protocol focused on precision, sterility, speed, and / or throughput. A wet lab operation, for example, a nucleic acid assay, can comprise one or more of: providing a sample, e.g., a cerebrospinal fluid (CSF) sample; lysis of all organisms in the sample; purification of DNA and RNA; enrichment for RNA viruses; enrichment for DNA viruses, bacteria, fungi, and / or parasites; conversion to DNA libraries and RNA libraries, amplification, and pooling; and sequencing. In some embodiments, a wet lab operation comprises the addition of a stabilization buffer to a sample, e.g., a cerebrospinal fluid (CSF) sample; a physical extraction operation; an automation operation; the addition of internal controls to a sample, e.g., a cerebrospinal fluid (CSF) sample; and / or the use of external controls.

[0066] In some embodiments, performing a nucleic acid assay, e.g., a nucleic acid assay of a wet lab operation provided herein, comprises extracting microbial nucleic acids or human nucleic acid in a mixture, e.g., a mixture provided herein. In some embodiments, performing a nucleic acid assay comprises preparing a nucleic acid library . In some embodiments, performing the nucleic acid assay comprises (i) extracting the microbial nucleic acids or the human nucleic acids and (ii) preparing the nucleic acid library. In some embodiments, performing the nucleicWSGR Docket No. 63228-706.601acid assay comprises adding two or more internal controls to a mixture, e.g., a mixture provided herein.

[0067] Provided herein, in some embodiments, are methods for detecting microbial nucleic acids in a mixture comprising human nucleic acids, comprising reducing the amount of human or microbial in a mixture or a biological sample. In some embodiments, performing a nucleic acid assay, e.g., a nucleic acid assay of a wet lab operation provided herein, comprises binding and blocking human rRNA sequences (see, e.g., FIG. 17B). The binding and blocking of human rRNA sequences can reduce the biomass of the sequences or the number of reads of the sequences in a nucleic acid assay provided herein. The binding and blocking of human rRNA sequences may be achieved through use of a commercially available kit. A non -limiting example of a kit for binding and blocking human rRNA sequences is the FastSelect kit (Qiagen). In some embodiments of a nucleic acid assay provided herein, hemoglobin transcripts or bacterial rRNA are bound and blocked. In some specific embodiments, a commercially available kit, e.g., FastSelect, is used in a nucleic acid assay to reduce hemoglobin transcripts or bacterial rRNA.

[0068] In some embodiments, performing a nucleic acid assay, e.g., a nucleic acid assay of a wet lab operation provided herein, comprises lysing a microbial or a human cell. The microbial or human cell can be lysed using a chemical reagent or by bead bashing. The mixture or biological sample comprising the microbial or human cell can then be spun down to form a pellet comprising microbial nucleic acids. A portion of the supernatant can then be removed from the mixture or biological sample. The volume of the supernatant removed can be about 50%, 60%, 70%, 80%, 85%, 90%, or 95%, of an initial volume of the mixture. In some embodiments, the volume of the supernatant removed is about 50% -99%, 75% -95%, or 85-95% of an initial volume of the mixture. For example, if the starting volume of a mixture is 1 mL, the mixture can be spun down to form a pellet, and then 900 uL of the supernatant can be removed (see, e.g., FIG. 17C and FIG. 19).

[0069] In some embodiments, a wet lab operation provided herein comprises a stabilization buffer, e.g., a nucleic acid stabilization buffer. For example, a nucleic acid stabilization buffer stabilization buffer can be added to a sample, e.g., a cerebrospinal fluid (CSF) sample from a subject. A stabilization buffer can preserve microbial nucleic acids, lyse microbial cells, and / or inactive pathogens. In some embodiments, a stabilization buffer is added to a sample immediately after collection of the sample. In some embodiments, a stabilization buffer can protect DNA, RNA, or both from degradation, and can eliminate the need for cold chain during shipping and handling while also improving biosafety. The use of a stabilization buffer can make handling of a sample safer for laboratory staff. In some embodiments, a stabilization buffer is provided in a pre-filled tube to clinicians collecting the sample (FIG. 3). In someWSGR Docket No. 63228-706.601embodiments, a stabilization buffer is provided in a pre-filled tube with an autometered transfer pipet and instructions for use to clinician. The pre-filled stabilization buffer tube can be barcoded, e.g., to be compatible with high-throughput automation.

[0070] In some embodiments, a stabilization buffer provided herein comprises a detergent. In some embodiments, a stabilization buffer provided herein comprises a guanidinium salt. Example stabilization buffers include but are not limited to: DNA / RNA Shield (Zymo Research), Primestore MTM Media (EKF Diagnostics), RNA Later (Invitrogen), Monarch StabiLyse DNA / RNA Buffer (New England Biolabs), and PrepProtect Buffer (Miltenyi Biotech).

[0071] In some embodiments, a wet lab operation provided herein comprises a physical extraction operation. The physical extraction operation can be optimized for hard-to-lyse species. Examples of species that a physical extraction operation can be optimized for, e.g., hard-to-lyse species, include but are not limited to fungi, yeasts, and gram positive bacteria. The physical extraction operation can be optimized for hard-to-lyse-species, while not reducing the integrity of DNA or RNA from easier-to-lyse organisms, e.g., DNA viruses, RNA viruses, gram negative bacteria, or parasites. In some embodiments, DNA and RNA extraction occurs in parallel, e.g., simultaneously, on a single liquid handling instrument, e.g., to reduce processing time and complexity.

[0072] In some embodiments, a physical sample extraction operation comprises physical sample agitation, e.g., bead bashing. In some embodiments a physical extraction operation comprises bead bashing. Examples of beads, e.g, for bead bashing, include but are not limited to: MP Bio Matrix B beads, MagMax Microbiome Beads, Zymo Research Bashing Beads, Omni Beads, MP Bio Matrix F Beads, Bio Matrix A Beads.

[0073] In some embodiments, the duration of the physical sample extraction operation is less than about 30 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 25 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 20 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 15 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 10 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 9 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 8 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 7 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 6 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 5 minutes. In some embodiments, theWSGR Docket No. 63228-706.601duration of the physical sample extraction operation is less than about 4 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 2 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 1 minutes. In some embodiments, the duration of the physical sample extraction operation is less than about 30 seconds. In some embodiments, the duration of the physical sample extraction operation is less than about 15 seconds. In some embodiments, the duration of the physical sample extraction operation is less than about 5 seconds.

[0074] In some embodiments, the physical sample extraction operation comprises combining beads and a clinical sample. In some embodiments, the physical sample extraction operation comprises centrifuging a composition comprising beads and a physical samples. In some embodiments, the beads and clinical sample are centrifuged at 10 m / s. In some embodiments, the beads and clinical sample are centrifuged at 9 m / s. In some embodiments, the beads and clinical sample are centrifuged at 8 m / s. In some embodiments, the beads and clinical sample are centrifuged at 7.5 m / s. In some embodiments, the beads and clinical sample are centrifuged at 7 m / s. In some embodiments, the beads and clinical sample are centrifuged at 6.5 m / s. In some embodiments, the beads and clinical sample are centrifuged at 6 m / s. In some embodiments, the beads and clinical sample are centrifuged at 5.5 m / s. In some embodiments, the beads and clinical sample are centrifuged at 5 m / s. In some embodiments, the beads and clinical sample are centrifuged at 4.5 m / s. In some embodiments, the beads and clinical sample are centrifuged at 4 m / s. In some embodiments, the beads and clinical sample are centrifuged at 3 m / s. In some embodiments, the beads and clinical sample are centrifuged at 2 m / s. In some embodiments, the beads and clinical sample are centrifuged at 1 m / s.

[0075] In some embodiments, a wet lab operation provided herein comprises automation. Automation can increase assay consistency; decrease exogenous contamination and cross -contamination; and / or improve assay speed (e.g., through parallel extraction of DNA and RNA).

[0076] Adapter concentration can be varied to optimize library quality and adapter dimer content. Number of PCR cycles can be varied to optimize library quality and diversity. Buffer composition of the fragmentation incubation step can be varied to optimize fragment length. Extraction bead concentration can be varied to optimize library yield. Bead bashing conditions (bead size, bead material, bashing speed, bashing time, bashing temperature, and speed and length of centrifugation step prior to bead bashing) can be varied to improve extraction efficiency for different microbial organisms, such as fungus and gram positive bacteria. Stabilization buffer composition can be varied to improve extraction efficiency for different microbial organisms such as fungus and gram positive bacteria.WSGR Docket No. 63228-706.601

[0077] In some embodiments, a wet lab operation provided herein comprisesan internal control. In some embodiments, an internal control is added to a sample, e.g., a cerebrospinal fluid (CSF) sample from a subject. The use of one or more internal controls can ensure low assay failure rates; allow relative quantification of human material (e.g., for research purposes); and / or limit the detection of background microorganisms. In some embodiments, the use of one or more internal controls limits the detection of background microorganisms in low input samples. In some embodiments, two or more internal controls are added to a sample, e.g., a cerebrospinal fluid (CSF) sample from a subject. In some embodiments, a contrived sample matrix (e.g., a contrived sample matrix added to a mixture provided herein) comprises two or more internal controls. In some embodiments, three or more internal controls are added to a sample, e.g., a cerebrospinal fluid (CSF) sample from a subject. In some embodiments, a contrived sample matrix (e.g., a contrived sample matrix added to a mixture provided herein) comprises three or more internal controls. In some embodiments, four or more internal controls are added to a sample, e.g., a cerebrospinal fluid (CSF) sample from a subject. In some embodiments, a contrived sample matrix (e.g., a contrived sample matrix added to a mixture provided herein) comprises four or more internal controls.

[0078] Internal controls can be used singly or in combination to evaluate and monitor extraction efficiency. An internal control can be an extraction control (e.g., an internal control added to a sample prior to DNA or RNA extraction) or a calibration control (e.g. , an internal control added to a sample after DNA or RNA extraction and prior to DNA or RNA library preparation) In some embodiments, one or more internal controls are added to a sample prior to extraction. In some embodiments, one or more internal controls are added to a sample after DNA extraction and prior to DNA library preparation. In some embodiments, one or more internal controls are added to a sample after RNA extraction and prior to RNA library preparation. In some embodiments, two internal controls are added to a sample prior to extraction. In some embodiments, internal controls are added to a sample after DNA extraction and prior to DNA library preparation. In some embodiments, internal controls are added to a sample after RNA extraction and prior to RNA library preparation. A ratio of the number of reads of two internal controls can be used to measure and monitor the efficiency of DNA or RNA extraction. For example, a first internal control can be added to a sample prior DNA extraction and a second internal control can be added to a sample after DNA extraction, and the ratio of a first amount of DNA from the first control to a second amount of DNA from the second control present in the sample can indicate DNA extraction efficiency. In some embodiments, an extraction control can be added to a sample prior DNA extraction and a calibration control can be added to a sample after DNA extraction, and the ratio of a first amount of DNA from the extraction control to aWSGR Docket No. 63228-706.601second amount of DNA from the calibration control present in the sample can indicate DNA extraction efficiency. In some embodiments, an extraction control can be added to a sample prior RNA extraction and a calibration control can be added to a sample after RNA extraction, and the ratio of a first amount of RNA from the extraction control to a second amount of RNA from the calibration control present in the sample can indicate RNA extraction efficiency.

[0079] In some embodiments, an internal control comprises an internal deoxyribonucleic acid (DNA) control. In some embodiments, two or more internal controls comprise two or more internal DNA controls. In some embodiments, an internal control comprises an internal ribonucleic acid (RNA) control. In some embodiments, two or more internal controls comprise two or more internal RNA controls. In some embodiments, a mixture (e.g., a mixture provided herein) comprises (i) two or more internal DNA controls or (ii) two or more internal RNA controls. In some embodiments, a mixture (e.g., a mixture provided herein) comprises (i) two or more internal DNA controls and (ii) two or more internal RNA controls. In some embodiments, the mixture (e.g. a mixture provided herein) comprises (i) a first internal DNA control and a second internal DNA control and (ii) a first internal RNA control and a second internal RNA control. Examples of internal controls include but are not limited to viruses, phages, bacteria, eukaryotic cells, plasmids, bacterial artificial chromosomes (BACs), polymerase chain reaction (PCR) products, synthetic DNA, synthetic RNA, or DNA or RNA purified from an organism. The internal controls can comprise two or more of T1 DNA phage, MS2 RNA phage, Lambda DNA phage, and an RNA standard. A non-limiting example of an RNA standard is the External RNA Controls Consortium (ERCC) standard.

[0080] In some embodiments, a method provided herein comprises adding two or more internal controls to a mixture prior to extracting microbial nucleic acids or human nucleic acids. In some embodiments, a method provided herein comprises adding two or more internal controls to a mixture after extracting microbial nucleic acids or human nucleic acids. The two or more internal controls can be added to the mixture after the extracting the microbial nucleic acids or the human nucleic acid and prior to the preparing the nucleic acid library. In some embodiments, the first internal DNA control and the first internal RNA control are added to the mixture prior to the extracting the microbial nucleic acids or the human nucleic acid. In some embodiments, the second internal DNA control and the second internal RNA control are added to the mixture after the extracting the microbial nucleic acids or the human nucleic acid. In some embodiments, the first internal DNA control and the first internal RNA control are added to the mixture prior to the extracting the microbial nucleic acids or the human nucleic acid, and the second internal DNA control and the second internal RNA control are added to the mixture after the extracting the microbial nucleic acids or the human nucleic acid.WSGR Docket No. 63228-706.601

[0081] In some embodiments, one or more internal controls can be used as a calibration control. A calibration internal control can be used to (1) determine the amount of nucleic acid from a target organism present in a sample; (2) determine the total amount of microbial nucleic acid present in sample (comprising negative controls, positive controls, and clinical sample); (3) determine whether a sample is contaminated; (4) determine the amount of human nucleic acid present in a sample; and (5) estimate the sensitivity of the mNGS assay to microbial organisms in a sample.

[0082] In some embodiments, a wet lab operation provided herein comprises an external control. An external control can ensure consistent extraction for hard -to-lyse species. Examples of species that are hard to lyse include but are not limited to filamentous fungi, yeasts, and gram positive bacteria. An external control can improve understanding of background organism content, e.g., human content and can be used to assess the extraction efficiency of the mNGS assay for different microorganism types. In some embodiments, a microorganism used as an external control is a fungus (e.g., a filamentous fungus), a gram negative bacteria, a yeast, a parasite, a gram positive bacteria, a DNA virus, or an RNA virus. Non-limiting examples of microorganisms that can be used as external controls are provided in Table 1. Examples of external controls include but are not limited to viruses, phages, bacteria, eukaryotic cells, plasmids, BACs, PCR products, synthetic DNA, synthetic RNA, or DNA or RNA purified from an organism.

[0083] Provided herein in some embodiments are systems and methods for detection of microbial DNA or RNA, the systems and methods comprising interpretation and reporting of clinical results. For example, a system or methods for detection of microbial DNA and RNA provided herein can aid in the interpretation and reporting of metagenomic next-generation sequencing (mNGS) results.

[0084] Provided herein in some embodiments, is method for detecting microbial nucleic acids in a mixture comprising human nucleic acids, the human nucleic acids comprising human DNA or human RNA. The mixture can comprise at least 5 human cells per microliter, at least 250 human cells per microliter, or at least 500 human cells per microliter. In some embodiments, the mixture comprises 5 human cells per microliter. In some embodiments, the mixture comprises 250 human cells per microliter. In some embodiments, the mixture comprises 500 human cells per microliter. In some embodiments, the human nucleic acids comprise human DNA. In some embodiments, the contrived sample matrix has about 0.01 picograms (pg) to about 1 microgram (pg) or about 1,000 pg to about 100,000 pg of human DNA. In some embodiments, the contrived sample matrix has about 0.01 picograms (pg) to about 1 microgram (pg) of human DNA. In some embodiments, the contrived sample matrix has about 1,000 pg to about 100,000WSGR Docket No. 63228-706.601pg of human DNA. In some specific embodiments, the contrived sample matrix comprises about 1,200, about 1,900 pg, or about 76,000 pg of human DNA. In some even more specific embodiments, the contrived sample matrix has about 1,200 pg of human DNA. In some even more specific embodiments, the contrived sample matrix has about 1 ,900 pg of human DNA. In some even more specific embodiments, the contrived sample matrix has about 76,000 pg of human DNA. In some embodiments, the human nucleic acids comprise human RNA. In some embodiments, the human nucleic acids comprise human ribosomal RNA. The contrived sample matrix can have about 0.01 pg to about 100 pg of human RNA. In some embodiments, the contrived sample matrix has about 1 pg to about 10,000 pg of human RNA. In some specific embodiments, the contrived sample matrix has about 7.9 pg, about 121 pg, or about 7,400 pg of human RNA. In some specific embodiments, the contrived sample matrix has about 7.9 pg of human RNA. In some specific embodiments, the contrived sample matrix has about 121 pg of human RNA. In some specific embodiments, the contrived sample matrix has about 7,400 pg of human RNA.

[0085] In some embodiments, a system or method for detection of microbial DNA or RNA provided herein is designed to aid in the diagnosis of microbial infections, such as CNS infections. For example, a system or method provided herein can comprise data analysis and results reporting tailored to metagenomic sequencing of a subject sample, such as a CSF sample.

[0086] In some embodiments, a system or method for detection of microbial DNA or RNA provided herein comprises a dry lab operation (FIG. 5). A dry lab operation provided herein can have improved speed, accuracy, scalability, and / or auditability. A dry lab operation provided herein can comprise one or more of: preprocessing; background extraction (e.g., human host background extraction); microbial identification; identification of viral, bacterial, fungal, and parasitic pathogens in the sample; clinical interpretation; and automated results report.

[0087] Provided herein in some embodiments is a system or method for detection of microbial DNA or RNA comprising a modular and scalable mNGS platform. A system or method provided herein can comprise a curated database of potential microbial pathogens, e.g., CNS pathogens. A curated database can comprise a subset of a larger database, e.g., an NCBI NT database, on taxa implicated (or potentially implicated) in microbial infection (FIG. 7).Redundant entries in the data can be collapsed by sequence clustering, low-quality submissions can be removed, and high-quality reference sequences (RefSeqs) can be prioritized when available.

[0088] In some embodiments of a system or method for detection of microbial nucleic acids in a mixture provided herein, the microbial nucleic acids comprise nucleic acids from one or more microbes. In some embodiments, the microbial nucleic acids comprise microbial DNA. someWSGR Docket No. 63228-706.601embodiments, the microbial nucleic acids comprise microbial RNA. The one or more microbes can comprise one or more of a filamentous fungus, a gram negative bacteria, a gram positive bacteria, a yeast, a parasite, a DNA virus, or an RNA virus. The one or more microbes can comprise a strain or a subtype of a species of one or more of a filamentous fungus, a gram negative bacteria, a gram positive bacteria, a yeast, a parasite, a DNA virus, and an RNA virus.

[0089] In certain embodiments, the filamentous fungus comprises one or more of a fungus of the genus Acre monium. Alternaria, Aspergillus, Cladosporium, Cryptococcus, Curvularia, Fusarium, Histoplasma, Lichtheimia, I.omenlospora, Mucor, Paecilomyces, Penicillium, Rhizomucor , Rhizopus, Scedosporium, Schizophyllum, and Trichoderma. In some embodiments, the filamentous fungus comprises one or more of Acremonium spp., Alternaria spp., Alternaria alternata, Alternaria infectoria, Aspergillus spp., Aspergillus carneus, Aspergillus clavatus, Aspergillus flavus, Aspergillus jumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor, Aspergillus parasiticus, Cladosporium spp., Cryptococcus spp., Cryptococcus neoformans, Curvularia spp., Fusarium spp., Fusarium avenaceum, Fusarium culmorum, Fusarium equiseti, Fusarium fujikuroi, Fusarium graminearum, Fusarium nivale, Fusarium proliferatum, Fusarium oxysporum, Fusarium roseum, Fusarium solani, Fusarium verticillioides, Histoplasma spp., Histoplasma capsulatum, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramose, Lomentospora spp., I.omenlospor prolificans, Mucor spp., Mucor circinelloides, Paecilomyces spp., Paecilomyces variotii, Penicillium spp., Penicillium aurantiogriseum, Penicillium brunneum, Penicillium citreoviride , Penicillium citrinin, Penicillium claviforme, Penicillium crustosum, Penicillium expansum, Penicillium griseojulvum, Penicillium hirsutum, Penicillium islandicum, Penicillium kloeckeri, Penicillium roqueforti, Penicillium rubrum, Penicillium rugulosum, Penicillium verrucossum, Penicillium viridicatum, Rhizomucor spp., Rhizomucor pusillus, Rhizopus spp., Rhizopus arrhizus, Rhizopus microspores, Scedosporium spp., Scedosporium apiospermum, Schizophyllum spp., Schizophyllum commune, and Trichoderma spp.

[0090] In certain embodiments, the gram negative gram negative bacteria comprises one or more of a bacterium of the genus Acinetobacter, Bacteroides, Cereibacter , Chlamydia, Citrobacter , Enterobacter , Escherichia, Haemophilus, Klebsiella, Moraxella, Morganella, Neisseria, Pantoea, Proteus, Pseudomonas, Shigella, Salmonella, and Yersinia. In some embodiments, the gram negative bacteria comprises one or more of Acinetobacter spp., Acinetobacter baumannii, Acinetobacter johnsonii, Bacteroides sp., Bacteroides fragilis, Bacteroides thetaiotaomicron, Chlamydia spp., Chlamydia trachomatis, Citrobacter spp., Citrobacter freundii, Citrobacter koseri, Enterobacter spp., Enterobacter aerogenes,WSGR Docket No. 63228-706.601Enterococcus avium, Enterobacter cloacae complex, Escherichia coli, Escherichia coli Extended-Spectrum Beta-Lactamase (ESBL), Haemophilus spp., Haemophilus influenzae, Klebsiella spp., Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella pneumoniae Extended- Spectrum Beta-Lactamase (ESBL), Klebsiella variicola, Moraxella spp., Moraxella sp. K1664, Moraxella bovis, Moraxella canis, Moraxella catarrhalis, Moraxella lacunata, Moraxella nonliquefaciens, Moraxella osloensis, Moraxella phenylpyruvica, Morganella spp., Morganella morganii, Neisseria spp ., Neisseria gonorrhoeae, Pantoea spp ., Pantoea agglomerans, Proteus spp., Proteus mirabilis, Pseudomonas spp., Pseudomonas aeruginosa, Salmonella spp., Salmonella enterica, Salmonella bongori, Shigella spp., Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Yersinia spp., and Yersinia pestis.

[0091] In certain embodiments, the gram positive bacteria comprises one or more of a bacterium of the genus Bacillus, Brevibacterium, Clostridium, Corynebacterium, Eggerthella, Enterococcus, Granulicatella, Micrococcus, Mycobacterium, Staphylococcus, and Streptococcus. In some embodiments, the gram positive bacteria comprises one or more of Bacillus sp., Brevibacterium sp., Clostridium spp., Clostridium difficile, Corynebacterium sp., Eggerthella sp., Eggerthella lenta, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Vancomycin resistant Enterococcus faecium (VRE), Enterococcus faecium, Vancomycin resistant Enterococcus faecium (VRE), Enterococcus gallinarum, Granulicatella spp., Granulicatella adiace ns, Micrococcus spp., Micrococcus lute us, Mycobacterium spp., and Mycobacterium tuberculosis Staphylococcus spp., Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA), Methicillin / Oxacillin Resistant Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus simulans, Staphylococcus warneri, Streptococcus spp., Streptococcus agalactiae, Streptococcus aginosus, Streptococcus canis, Streptococcus constellatus, Streptococcus intermedins, Streptococcus mitis, Streptococcus pneumoniae , Streptococcus pyogenes, Streptococcus salivarius, Streptococcus uberis, and Streptococcus warneri.

[0092] In certain embodiments, the yeast comprises one or more of a yeast of the genus Candida or Cryptococcus . In some embodiments, the yeast comprises one or more of Candida auris, Candida albicans, Candida glabr ata, Candida parap silo sis, Candida tropicalis, Candida krusei, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, and Cryptococcus uniguttulatus .

[0093] In certain embodiments, the parasite comprises one or more of a protozoa, a helminth, or an ectoparasite. In some specific embodiments, the protozoa is a species of the genus Neospora or the genus Plasmodium.WSGR Docket No. 63228-706.601

[0094] In certain embodiments, the DNA virus comprises one or more of herpesvirus, cytomegalovirus (CMV), muromegalovirus, human papillomavirus (HPV), adenovirus, hepatitis B virus (HBV), poxvirus, and polyomavirus. In some embodiments, the herpesvirus comprises one or more of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Kaposi sarcoma-associated herpesvirus (gamma herpesvirus), varicella-zoster virus (VZV), Roseolovirus, and Epstein-Barr virus (EBV). In some embodiments, the polyomavirus comprises Human polyomavirus 1 (BK Virus).

[0095] In some embodiments, the RNA virus comprises one or more of influenza virus, respiratory syncytial virus (RSV), coronavirus (e.g., SARS-CoV-2), enterovirus, norovirus, rotavirus, human immune deficiency virus (HIV), hepatitis C virus (HCV), Rift Valley fever virus, morbillivirus (e.g. , Morbilivirus canis), Tick-borne encephalitis virus, Zika virus, Dengue virus, West Nile virus, Ebola virus, yellow fever virus, Saint Louis encephalitis virus (SLEV), Eastern Equine encephalitis virus (EEEV), La Crosse encephalitis virus (LCEV), and Japanese encephalitis virus.

[0096] In some embodiments, a system or methods provided herein can comprise a bioinformatics pipeline. A bioinformatics pipeline can comprise self -contained, interchangeable components (modules) with standardized inputs and outputs (SeqSamples) (FIG. 6). The SeqSample object can be a custom dataclass that can serve as a standardized input and output of all operation / modules in the pipeline. A single file, e.g., a FastQ file, can initialize a SeqSample. In some embodiments, the pipeline is organized as a series of tasks, e.g., tasks managed by Flyte. Each task can execute in its own containerized (docker) environment and can accept (as input) and outputs a SeqSample. In some embodiments, a bioinformatics pipeline provided herein facilitates rapid prototyping and independent testing of each module.

[0097] Provided herein in some embodiments is a kit for use in detection of microbial RNA and DNA (FIG.3). In some embodiments, a kit provided herein comprises a sample collection tube thatis pre-filled with a stabilization buffer, e.g., a stabilization buffer disclosed herein. In some embodiments, a kit provided herein comprises a sample collection tube, e.g., a tube pre-filled with a stabilization buffer, that is barcoded, e.g., with a sticker comprising a barcode. A barcoded tube can be compatible with high-throughput automation, such as in a system or method for detection of microbial RNA or DNA provided herein. A kit provided herein can comprise a box, and the box can comprise a slot to hold a collection tube erect without the use of hands. A kit provided herein can comprise an autometered transfer pipet for transferring a sample, e.g., a patient sample, into a collection tube, e.g., a collection tube pre-filled with a stabilization buffer. In some embodiments, a kit provided herein comprises a safety bag. AWSGR Docket No. 63228-706.601collection tube comprising stabilization buffer and a patient sample can be placed inside the safety bag for transportation. In some embodiments, a kit comprises pre-paid postage.EXAMPLESExample 1: Development of a matrix for optimizing and assessing clinical metagenomic Next-Generation Sequencing (mNGS) for meningitis and encephalitis

[0098] Since human cerebrospinal fluid (CSF) is not readily available in large amounts for research and development purposes, the development of a commutable matrix to be used for development and validation purposed in accordance with CLSI guidelines EP30-A was pursued. The amount of human nucleic acid typically present in CSF samples sent for mNGS testing was investigated. The assay methodology was performed on 48 previously characterized, de-identified clinical remnant CSF samples, of which half contained a pathogen detected by metagenomic Next-Generation Sequencing (mNGS). Human DNA biomass detected in the CSF samples is provided in FIG. 9A (see also Table 1) and human RNA biomass detected in the CSF samples is provided in FIG. 9B (see also Table 2). Three synthetic samples were tested using similar techniques for comparison (see Table 3, for comparison). Sequencing data were analyzed with a pipeline provided herein, and human DNA and RNA content were calculated by normalizing reads per Million (rpM) mapping to human rpM mapping to internal controls of known concentration.

[0099] Table 1: Individual human DNA sample metricsWSGR Docket No. 63228-706.601

[0100] Table 2: Individual human RNA sample metrics

[0101] In 48 clinical remnant CSF samples, a median of 1,500 pg human DNA background (Table 2 mean 1,600 pg, range 12 pg - 960 ng) and a median of 1.0 pg human RNA background (Table 3 mean 1.3 pg, range 0.34-57 pg) was found. Different matrices were assayed to mimic human CSF (Table 1). A pooled human CSF sample generated by mixing 64 individual remnant CSF samples had 1,900 pg human DNA and 7.9 pg human RNA. Synthetic CSF contained no nucleic acid material. Synthetic CSF with human donor PBMCs added at 10 cells / uL and 500 cells / uL mimicked median and high human content human CSF samples. The levels of human RNA in human CSF were lower than in synthetic CSF with human donor PBMCs. Degradation of RNA in individual clinical remnant samples, such as samples stored frozen for 1-8 years prior to addition of stabilization buffer, may provide lower levels of human RNA in human CSF. The frozen storage times of the 64 remnant CSF samples comprising the pooled human sample are unknown.

[0102] Table 3: Human and synthetic sample typesWSGR Docket No. 63228-706.601

[0103] To investigate how human nucleic acid content affects pathogen detection sensitivity, clinically -relevant concentrations of microorganisms were spiked into each contrived sample matrix. Signal from spiked organisms was highly dependent on the matrix (Table 4 and Table 5; FIGs. 10A-10G)

[0104] Each of FIGS. 10A-10G shows microbial signal (rpM) for one of the seven organisms (filamentous fungus (FIG. 10A), gram negative bacteria (FIG. 10B), yeast (FIG. 10C), DNA virus (FIG. 10D), parasite (FIG. 10E), gram positive bacteria (FIG. 10F), and RNA virus (FIG.10G)) at high and low levels of microbial material. Squares on the left and dotted horizontal lines show signal in the pooled human CSF sample. Diamonds on the right show signal in samples with (a) no human content added, (b) 10 human cells / uL added, and (c) 500 human cells / uL added (high microbe only). The sample with microbes added but no human genetic material added contained residual human signal equivalent to approximately 0.1 cells / uL due to the methods used for microbial culture. All datapoints represent mean and standard deviation of at least three replicates.

[0105] Table 4: Signal with high microbial contentWSGR Docket No. 63228-706.601

[0106] Table 5: Signal with low microbial content

[0107] Since microbial and human nucleic acid compete for the same flow cell space in mNGS, the amountof human background in aclinical sample will impact test sensitivity. In the absence of added human matrix, microbial rpMs are artificially high, surpassing the signal in the pooled human sample by more than 10 fold for each organism. With 10 human cells / uL added, signals are close to those observed in the pooled human sample, indicating this to be a better matrix for assay development and testing. Adding 500 human cell / uL resulted in a high background sample that is a useful mimic of a sample with unusually high cellular content.

[0108] Signal from spiked organisms was highly dependent on the matrix, demonstrating the importance of using an appropriate matrix for assay development and performance assessment. In addition, processing of previously confirmed positive and negative clinical remnant samples allowed evaluation of sensitivity and specificity for methods of microbial DNA and RNA detection provided herein and demonstrated that for methods of microbial DNA and RNAWSGR Docket No. 63228-706.601detection provided herein can identify a variety of viruses, bacteria, fungi, and parasites at clinically significant levels.

[0109] When human material is left out of a matrix, microbial signal can appear artificially high, masking the challenge of human background in clinical mNGS. Given the high variability of human content in clinical CSF samples, it is useful to consider the impact of different amounts of human content on assay sensitivity. A contrived sample matrix that mimics human background was developed to ensure that assay development and assessment are indicative of performance on real samples.Example 2: Use of Internal Controls to Evaluate and Monitor Extraction Efficiency

[0110] The efficacy of using internal controls (two each of extraction and calibration controls) to evaluate and monitor extraction efficiency was tested.

[0111] DNA extraction control in varying amounts and RNA extraction control in varying amounts were added to a composition comprising a microbial community standard and synthetic CSF (sCSF) prior to DNA and RNA extraction. 25 pg of purified DNA calibration control DNA were added to the sample after DNA extraction prior to DNA library preparation, and 25 pg of RNA calibration control were added to the sample after RNA extraction prior to RNA library preparation. The amount of DNA extraction control and RNA extraction control added prior to extraction were varied to produce dilutions of 1 : 10, 1 :50, and 1 :250 of DNA extraction control and RNA extraction control relative to the undiluted starting concentration. The efficiency of DNA extraction was measured and monitored by calculating the ratio of DNA extraction control: DNA calibration control after amplification. The efficiency of RNA extraction was measured and monitored by calculating the ration of the amount of and of RNA extraction control: RNA calibration control after amplification.

[0112] Reducing the extraction control concentrations increased the relative read capacity for pathogen organism detection and enabled improved assay sensitivity (Table 6). The target microorganism’s rpM ratio was calculated by dividing the target’s rpM in each sample of different extraction control inputs by the average target rpM in sCSF with the same extraction control input. When the sCSF rpM was less than 1, the rpM ratio was calculated by dividing the microorganism rpM by 1.WSGR Docket No. 63228-706.601

[0113] Table 6: Mean target organism rpM in sCSF with different extraction control dilutions

[0114] When the sample had undetectable or low human background, the rpM ratios for the target organisms generally increased with the lowering of the extraction control input. In samples containing high human background (FIG. 11C), the PC organism rpM ratios were lower than in samples with no (FIG. 11 A) or low (FIG. 11B) human background and the rpM ratios remained the same regardless of extraction control dilution . Further, reducing the extraction control input increased the target organisms’ rpM in samples with no (FIG. 12A) or low (5 human cells / uL, FIG. 12B) human background. In samples with high human background (500 human cells / uL, FIG. 12C), there was no significant observed impact of the concentration of extraction control used on the rpM and rpM ratio of the target microorganisms.Example 3: Evaluation of Nucleic Acid Assay on Various Biological Samples Nucleic acid assays for detecting microbial nucleic acids in biological samples provided herein (see, e.g., FIG. 17A, FIG. 17B, and FIG. 17C) were evaluated on various biological sample types. The performance of the assay with regard to human content (Table 7, Table 8, and Table 9; FIG. 13A, FIG. 13B, and FIG. 13C), microbial content (FIG. 14A and FIG. 14B), and assay interference (FIG. 15Aand 15B) were considered. Based on the evaluation, Limit of Detection (LOD) predictions and Loss of Sensitivity estimates (Table 10) were made for various sample types.WSGR Docket No. 63228-706.601

[0115] Table 7: Human Biomass DNAWSGR Docket No. 63228-706.601

[0116] Table 8: Human Biomass RNA

[0117] Table 9: Percent of human biomass that maps to rRNAWSGR Docket No. 63228-706.601

[0118] Table 10: Estimated Reductions in SensitivityWSGR Docket No. 63228-706.601

[0119] Biological samples known to be positive for microbial nucleic acids (Table 11) were tested to evaluate the efficacy of the nucleic acid assay in detecting microbial DNA. A summary of positive samples of plasm, peritoneal fluid, pleural fluid, synovial fluid, ocular fluid, and pericardial fluid that were available for testing is provided in Table 12.

[0120] Table 11: Examples of PositivesWSGR Docket No. 63228-706.601WSGR Docket No. 63228-706.601

[0121] Table 12: Available Positive and Negative Samples

[0122] 125 positive control plasma samples were assayed.29 bacterial, fiveDNA virus and one RNA virus taxa were detected in this assay (Table 13).

[0123] Table 13: Summary of plasma samplesWSGR Docket No. 63228-706.601& &WSGR Docket No. 63228-706.601

[0124] 23 positive control peritoneal fluid samples were assayed.20 bacterial taxa were detected in this assay (Table 14).

[0125] Table 14: Summary of peritoneal fluid samples& & & & &&& &&WSGR Docket No. 63228-706.601

[0126] 30 positive control pleural fluid samples were assayed. 21 bacterial and 1 fungal taxa were detected in this assay (Table 15).

[0127] Table 15: Summary of pleural fluid samples& & & & & &WSGR Docket No. 63228-706.601&

[0128] 28 positive control synovial fluid samples were assayed. 13 bacterial taxa were detected in this assay (Table 16).

[0129] Table 16: Summary of synovial fluid taxa&WSGR Docket No. 63228-706.601

[0130] 2 positive control ocular fluid samples were assayed. 2 bacterial taxa were detected in this assay (Table 17).

[0131] Table 17: Ocular

[0132] 9 positive control pericardial fluid samples were assayed. 5 bacterial, 1 DNA virus, and 1 RNA virus taxa were detected in this assay (Table 18).

[0133] Table 18: PericardialWSGR Docket No. 63228-706.601

[0134] These results demonstrate that nucleic acid assays provided herein can detect microbial nucleic acids in a wide range of biological samples types, including, but not limited to CSF, plasma, pleural fluid, peritoneal fluid, synovial fluid, ocular fluid, and pericardial fluid.

[0135] Considerations for collection of biological samples, exemplified by plasma, pleural, and synovial samples, are provided in Table 19.

[0136] Table 19: Sample Collection Method at the Clinical SitesExample 4: Evaluation of Methods of Depletion of Human Nucleic Acids in Biological Samples

[0137] Eight methods were evaluated for their efficacy in depletion of human nucleic acids in biological samples. The feasibility of the use of these methods in a nucleic acid assay for the detection of microbial nucleic acids provided herein was tested. The methods evaluated were: (l)FastSelect (RNA); (2) cellular separation by pre-Shield spin; (3) partial lysis by post-Shield spin; (4) partial lysis b post-Shield extraction bead binding; (5) removal of proteinase K; (6) size selection after extraction; (7) duplexDNAse digestion; and (8) methyl pulldown (FIG. 16). The effects of these eight methods on human biomass, microbial biomass, and microbial reads per million, as well as the financial cost, increase in turnaround time (TAT), and additional steps involved with the methods is provided in Table 20.WSGR Docket No. 63228-706.601

[0138] Table 20: Methods Evaluated for Human DepletionWSGR Docket No. 63228-706.601WSGR Docket No. 63228-706.601

[0139] Based on the initial evaluation, the (a) FastSelect and (b) partial lysis by post-Shield spin, remove 900 uL (“supernatant removal”) methods were evaluated more closely.

[0140] FastSelect is an off-the-shelf reagent from Qiagen. FastSelect binds and blocks human ribosomal RNA (rRNA; see, e.g., FIG. 13C) sequences during random priming, prior to first strand synthesis. FastSelect may also reduce hemoglobin transcripts and bacterial rRNA. A summary of the efficacy of FastSelect in reducing human biomass and the change microbial nucleic acid biomass and microbial reads per Million in CSF, plasma, peritoneal, pleural, synovial, pericardial, and peripheral blood mononuclear cell (PBMC) biological samples is provided in Table 21.WSGR Docket No. 63228-706.601

[0141] Table 21: Evaluation of FastSelectWSGR Docket No. 63228-706.601

[0142] The evaluation of the FastSelect method (FIG. 17B) indicated that biological samples in which a high percentage of human RNA is rRNA benefit most from the use of FastSelect.

[0143] The percent of human nucleic acid reads that map to hemoglobin in plasma, serum, whole blood, PBMC, and synthetic CSF (sCSF) samples was measured to evaluate the feasibility of using FastSelect to remove hemoglobin reads from biological samples (FIG. 18).The results of this study indicate that less than 20% of reads in plasma were from hemoglobin.

[0144] Table 22: Evaluation of 900 uL spinWSGR Docket No. 63228-706.601

[0145] In the supernatant removal method, Shield (Zymo Research) lyses human and some microbial nucleic acids immediately after addition to a mixture (FIG. 17C). However, some microbes are not lysed until a bead bashing step. The microbes that are not lysed by Shield can be spun down into a pellet. It was found that the microbes in the pellet were often fungi and gram positive bacteria, and the microbial nucleic acids in the supernatant were often from gram negative bacteria, parasites, and viruses. Summaries of the efficacy of the supernatant removal method in reducing human biomass and the change microbial reads per Million in CSF, plasma, pleural, and peripheral blood mononuclear cell (PBMC) biological samples are provided in Table 22 and Table 23, respectively.

[0146] The evaluation of the supernatant removal method indicated that pelleting a 1 mL sample, and removing 900 uL of the supernatant, leaving 100 uL of supernatant (FIG. 19) was an automation-friendly way to enrich for the microbes in the pellet in a sample with high human biomass with minimal impact on the microbes in the supernatant.WSGR Docket No. 63228-706.601

[0147] Table 23: Evaluation of 900 removal method

[0148] The supernatant removal method was evaluated on the production positive control (PC).It was found that Cereibacter and Neospora reads decreased by 8-10X using the supernatant removal method, with 106 and 61 rpM, respectively. Increasing the concentration of Cereibacter and Neospora by 10X in the PC restored the rpMs (FIG. 20), making it less likely that the positive control would fail.

[0149] A summary of the effect of the FastSelect and the supernatant removal methods on the Turn Around Time (TAT) and Hands on Time (HOT) of methods provided herein is provided in Table 24WSGR Docket No. 63228-706.601

[0150] Table 24: Turn Around Time (TAT) and Hands on Time (HOT) of Evaluated Methods

[0151] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variationsWSGR Docket No. 63228-706.601or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WSGR Docket No. 63228-706.601CLAIMS1. A method for detecting microbial nucleic acids in a mixture comprising human nucleic acids, comprising:(a) adding a contrived sample matrix to the mixture, and(b) performing a nucleic acid assay on the mixture to detect a presence or quantitative measure of microbial nucleic acids in the mixture, wherein the nucleic acid assay has an assay sensitivity that is calibrated based at least in part on analyzing a plurality of mixtures comprising contrived sample matricesto determine a simulated presence or quantitative measure of human nucleic acids in the plurality of mixtures.

2. The method of claim 1, further comprising performing the nucleic acid assay on a biological sample.

3. The method of claim 1 or claim 2, wherein the nucleic acid assay is configured to process nucleic acids obtained or derived from the biological sample.

4. The method of claim 2 or claim 3, wherein the biological sample comprises whole blood, blood serum, blood plasma, bile, breath, urine, feces, sebum, tissue, breast milk, hair, saliva, sputum, sweat, tears, lymphatic fluid, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, cerebrospinal fluid, seminal fluid, vaginal secretion, amniotic fluid, nasal fluid, otic fluid, interstitial fluid, gastric fluid, intestinal fluid, transudates, exudates, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi -celled organisms, biological swabs, or biological washes.

5. The method of any one of claims 2-4, wherein the biological sample comprises cerebrospinal fluid (CSF), blood plasma, peritoneal fluid, pleural fluid, synovial fluid, or pericardial fluid.

6. The method of any one of claims 1 -5, further comprising determining the nucleic acid assay sensitivity to optimize the nucleic acid assay.

7. The method of claim 6, wherein optimizing the nucleic acid assay comprises one or more of decreasing the noise of the assay, increasing the signal to noise ratio of the assay, and increasing the sensitivity of the assay.

8. The method of any one of claims 1-7, wherein performing the nucleic acid assay comprises sequencing.WSGR Docket No. 63228-706.6019. The method of any one of claims 1-8, wherein the nucleic acid assay comprises a metagenomic next generation sequencing (mNGS) assay.

10. The method of claim 8 or claim 9, wherein the sequencing comprises one or more of nucleic acid extraction, nucleic acid isolation, nucleic acid fragmentation, reverse transcription (e.g., of RNA), transcript fragmentation, adapter ligation, amplification, targeted enrichment, nucleic acid library preparation, bisulfite conversion, and methylation conversion.

11. The method of any one of claims 8-10, wherein the sequencing comprises one or more of targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy -based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, duplex sequencing, cycle sequencing, single-base extension sequencing, solid phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, coamplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired -end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, long-read sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, and MS-PET sequencing.

12. The method of any one of claims 8-11, wherein the sequencing comprises a polymerase chain reaction (PCR) or isothermal amplification.

13. The method of any one of claims 10-12, wherein the amplification comprises use of amplification reagents, the amplification reagents comprising reagents for one or more of polymerase chain reaction (PCR), transcription mediated amplification (TMA), helicase dependent amplification (HD A), circular helicase dependent amplification (cHDA), strand displacement amplification (SDA), loop mediated amplification (LAMP), exponential amplification reaction (EXPAR), rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA).WSGR Docket No. 63228-706.60114. The method of any one of claims 1-13, wherein performing the nucleic acid assay comprises extracting the microbial nucleic acids or the human nucleic acid.

15. The method of any one of claims 1-14, wherein performing the nucleic acid assay comprises preparing a nucleic acid library.

16. The method of any one of claims 1-15, wherein performing the nucleic acid assay comprises (i) extracting the microbial nucleic acids or the human nucleic acids and (ii) preparing the nucleic acid library.

17. The method of any one of claims 1-16, wherein performing the nucleic acid assay comprises adding two or more internal controls to the mixture.

18. The method of any one of claims 1-17, wherein performing the nucleic acid assay comprises binding and blocking human rRNA sequences.

19. The method of any one of claims 1-18, wherein performing the nucleic acid assay comprises lysing a microbial or a human cell.

20. The method of claim 19, wherein the lysing comprises adding a chemical reagent.

21. The method of claim 19, wherein the lysing comprises bead bashing.

22. The method of any one of claims 1-21, wherein performing the nucleic acid assay comprises spinning down the mixture or the biological sample to form a pellet comprising microbial nucleic acids.

23. The method of any one of claims 1-22, wherein performing the nucleic acid assay comprises removing a supernatant.

24. The method of claim 23, wherein a volume of the supernatant removed is about 50%, 60%, 70%, 80%, 85%, 90%, or 95%, of an initial volume of the mixture.

25. The method of claim 23, wherein a volume of the supernatant removed is about 50% -99%, 75% -95%, or 85-95% of an initial volume of the mixture.

26. The method of any one of claims 1 to 25, wherein the contrived sample matrix comprises two or more internal controls.

27. The method of any one of claims 1 to 26, wherein the contrived sample matrix comprises three or more internal controls.

28. The method of any one of claims 1 to 27, wherein the contrived sample matrix comprises four or more internal controls.WSGR Docket No. 63228-706.60129. The method of any one of claims 26 to 28, wherein the two or more internal controls comprise two or more internal deoxyribonucleic acid (DNA) controls.

30. The method of any one of claims 26 to 29, wherein the two or more internal controls comprise two or more internal ribonucleic acid (RNA) controls.

31. The method of claim 30, wherein the mixture comprises (i) the two or more internal DNA controls or (ii) the two or more internal RNA controls.

32. The method of claim 31, wherein the mixture comprises (i) a first internal DNA control and a second internal DNA control and (ii) a first internal RNA control and a second internal RNA control.

33. The method of any one of claims 26-32, further comprising adding the two or more internal controls to the mixture prior to the extracting the microbial nucleic acids or the human nucleic acid.

34. The method of claim 26-32, further comprising adding the two or more internal controls to the mixture after the extracting the microbial nucleic acids or the human nucleic acid.

35. The method of any one of claims 26-32, wherein the two or more internal controls are added to the mixture after the extracting the microbial nucleic acids or the human nucleic acid and prior to the preparing the nucleic acid library.

36. The method of claim 35, wherein the first internal DNA control and the first internal RNA control are added to the mixture prior to the extracting the microbial nucleic acids or the human nucleic acid, and wherein the second internal DNA control and the second internal RNA control are added to the mixture after the extracting the microbial nucleic acids or the human nucleic acid.

37. The method of any one of claims 26-36, wherein the two or more internal controls comprise two or more of T1 DNA phage, MS2 RNA phage, Lambda DNA phage, and an RNA standard (e.g., the External RNA Controls Consortium (ERCC) standard).

38. The method of any one of claims 1-37, wherein the microbial nucleic acids comprise microbial DNA.

39. The method of any one of claims 1-38, wherein the microbial nucleic acids comprise microbial RNA.

40. The method of any one of claims 1-39, wherein the human nucleic acids comprise human DNA.WSGR Docket No. 63228-706.60141. The method of claim 40, wherein the contrived sample matrix has about 0.01 picograms (pg) to about 1 microgram (pg) of human DNA.

42. The method of claim 41, wherein the contrived sample matrix has about 1,000 pg to about 100,000 pg of human DNA.

43. The method of claim 42, wherein the contrived sample matrix has about 1,200, about 1,900 pg, or about 76,000 pg of human DNA.

44. The method of any one of claims 1-43, wherein the human nucleic acids comprise human RNA.

45. The method of claim 44, wherein the human nucleic acids comprise human ribosomal (rRNA).

46. The method of claim 44 or claim 45, wherein the contrived sample matrix has about 0.01 pg to about 100 pg of human RNA.

47. The method of claim 46, wherein the contrived sample matrix has about 1 pg to about 10,000 pg of human RNA.

48. The method of claim 47, wherein the contrived sample matrix has about 7.9 pg, about 121 pg, or about 7,400 pg of human RNA.

49. The method of any one of claims 1-48, wherein the mixture comprises at least 5 human cells per microliter, at least 250 human cells per microliter, or at least 500 human cells per microliter.

50. The method of any one of claims 1-49, wherein the microbial nucleic acids comprise nucleic acids from one or more microbes.

51. The method of claim 50, wherein the one or more microbes comprise one or more of a filamentous fungus, a gram negative bacteria, a gram positive bacteria, a yeast, a parasite, a DNA virus, and an RNA virus.

52. The method of claim 51, wherein the one or more microbes comprise a strain or a subtype of a species of one or more of a filamentous fungus, a gram negative bacteria, a gram positive bacteria, a yeast, a parasite, a DNA virus, and an RNA virus.

53. The method of claim 51 or claim 52, wherein the filamentous fungus comprises one or more of a fungus of the genus Acremonium, Ahernaria. Aspergillus, Cladosporium, Cryptococcus, Curvularia, Fusarium, Histoplasma, Lichtheimia, Lomentospora, Mucor,WSGR Docket No. 63228-706.601Paecilomyces, Penicillium, Rhizomucor, Rhizopus, Scedosporium, Schizophyllum, and Trichoderma .

54. The method of any one of claims 51-53, wherein the filamentous fungus comprises one or more of Acremonium spp., Alternaria spp., Alternaria a! tern ala. Alternaria infectoria, Aspergillus spp., Aspergillus came us, Aspergillus davalus. Aspergillus flavus. Aspergillus fumigatus, Aspergillus nidulans. Aspergillus niger. Aspergillus ochraceus. Aspergillus lerreus. Aspergillus uslus. Aspergillus versicolor, Aspergillus parasiticus, Cladosporium spp., Cryptococcus spp., Cryptococcus neoformans, Curvularia spp., Fusarium spp., Fusarium avenaceum, Fusarium culmorum, Fusarium equiseti, Fusarium fujikuroi, Fusarium graminearum, Fusarium nivale, Fusarium proliferatum, Fusarium oxysporum, Fusarium roseum, Fusarium solani, Fusarium verticillioides, Histoplasma spp., Histoplasma capsulatum, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramose, Lomentospora spp., Lomentospora prolificans, Mucor spp., Mucor circinelloides, Paecilomyces spp., Paecilomyces variotii, Penicillium spp., Penicillium aurantiogriseum, Penicillium brunneum, Penicillium citreoviride, Penicillium citrinin, Penicillium claviforme , Penicillium crustosum, Penicillium expansum, Penicillium griseofulvum, Penicillium hirsutum, Penicillium islandicum, Penicillium kloeckeri, Penicillium roqueforti, Penicillium rubrum, Penicillium rugulosum, Penicillium verrucossum, Penicillium viridicatum, Rhizomucor spp., Rhizomucor pusillus, Rhizopus spp., Rhizopus arrhizus, Rhizopus microspores, Scedosporium spp., Scedosporium apiospermum, Schizophyllum spp., Schizophyllum commune, and Trichoderma spp.

55. The method of claim 51 or claim 52, wherein the gram negative bacteria comprises one or more of a bacterium of the genus Acinetobacter , Bacteroides, Cereibacter, Chlamydia, Citrobacter , Enterobacter , Escherichia, Haemophilus, Klebsiella, Moraxella, Morganella, Neisseria, Pantoea, Proteus, Pseudomonas, Shigella, Salmonella, and Yersinia.

56. The method of claim 51 or claim 52, wherein the gram negative bacteria comprises one or more of Acinetobacter spp ., Acinetobacter baumannii, Acinetobacter johnsonii, Bacteroides sp., Bacteroides j gilis, Bacteroides thetaiotaomicron , Chlamydia spp., Chlamydia trachomatis, Citrobacter spp., Citrobacter freundii, Citrobacter koseri, Enterobacter spp., Enterobacter aerogenes, Enterococcus avium, Enterobacter cloacae complex, Escherichia coli, Escherichia coli Extended-Spectrum Beta-Lactamase (ESBL), Haemophilus spp., Haemophilus influenzae, Klebsiella spp., Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella pneumoniae Extended-Spectrum Beta-Lactamase (ESBL), Klebsiella variicola, Moraxella spp., Moraxella sp. KI 664, Moraxella bovis, Moraxella canis, Moraxella catarrhalis, Moraxella lacunata, Moraxella nonliquefaciens, Moraxella osloensis, Moraxella phenylpyruvica,WSGR Docket No. 63228-706.601Morganella spp., Morganellamorganii, Neisseria spp., Neisseria gonorrhoeae, Pantoea spp., Pantoea agglomerans, Proteus spp., Proteus mirabilis, Pseudomonas spp., Pseudomonas aeruginosa, Salmonella r ., Salmonella enterica, Salmonella bongori, Shigella spp., Shigella dysenteriae , Shigella flexneri, Shigella boydii, Shigella sonnei, Yersinia spp., and Yersinia pestis.

57. The method of claim 51 or claim 52, wherein the gram positive bacteria comprises one or more of a bacterium of the genus Bacillus, Brevibacterium, Clostridium, Corynebacterium, Eggerthella, Enterococcus, Granulicatella, Micrococcus, Mycobacterium, Staphylococcus, and Streptococcus.

58. The method of claim 51 or claim 52, wherein the gram positive bacteria comprises one or more of Bacillus sp., Brevibacterium sp., Clostridium spp., Clostridium difficile, Corynebacterium sp., Eggerthella sp., Eggerthella lenta, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Vancomycin resistant Enterococcus faecium (VRE), Enterococcus faecium, Vancomycin resistant Enterococcus faecium (VRE), Enterococcus gallinarum, Granulicatella spp., Granulicatella adiacens, Micrococcus spp., Micrococcus luteus, Mycobacterium spp., and Mycobacterium tuberculosis Staphylococcus spp., Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA), Methicillin / Oxacillin Resistant Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus simulans, Staphylococcus warneri, Streptococcus spp., Streptococcus agalactiae , Streptococcus aginosus, Streptococcus canis, Streptococcus constellatus, Streptococcus intermedins , Streptococcus mitis, Streptococcus pneumoniae , Streptococcus pyogenes, Streptococcus salivarius, Streptococcus uberis, and Streptococcus warneri.

59. The method of claim 51 or claim 52, wherein the yeast comprises one or more of a yeast of the genus Candida or Cryptococcus .

60. The method of claim 51 or claim 52, wherein the yeast comprises one or more of Candida auris, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, and Cryptococcus uniguttulatus .

61. The method of claim 51 or claim 52, wherein the parasite comprises one or more of a protozoa, a helminth, or an ectoparasite.WSGR Docket No. 63228-706.60162. The method of claim 51 or claim 52, wherein the DNA virus comprises one or more of herpesvirus, cytomegalovirus (CMV), muromegalovirus, human papillomavirus (HPV), adenovirus, hepatitis B virus (HBV), poxvirus, and polyomavirus.

63. The method of claim 62, wherein the herpesvirus comprises one or more of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Kaposi sarcoma-associated herpesvirus (gamma herpesvirus), varicella-zoster virus (VZV), Roseolovirus, and Epstein-Barr virus (EBV).

64. The method of claim 62, wherein the polyomavirus comprises Human polyomavirus 1 (BK Virus).

65. The method of claim 51 or claim 52, wherein the RNA virus comprises one or more of influenza virus, respiratory syncytial virus (RSV), coronavirus (e.g., SARS-CoV-2), enterovirus, norovirus, rotavirus, human immune deficiency virus (HIV), hepatitis C virus (HCV), Rift Valley fever virus, morbilivirus, Tick-borne encephalitis virus, Zika virus, Dengue virus, West Nile virus, Ebola virus, yellow fever virus, Saint Louis encephalitis virus (SLEV), Eastern Equine encephalitis virus (EEEV), La Crosse encephalitis virus (LCEV), and Japanese encephalitis virus.

66. A method for calibrating a nucleic acid assay, comprising:(a) analyzing a plurality of mixtures comprising contrived sample matrices to determine a simulated presence or a quantitative measure of human nucleic acids in the plurality of mixtures;(b) determining an assay characteristic of the nucleic acid assay based at least in part on the analyzing the plurality of mixtures; and(c) calibrating the nucleic acid assay based at least in part on the assay characteristic determined in (b).

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