Multi-step methods for detecting nucleic acids

Abstract

Provided herein are improved methods and systems for detecting AMR genetic markers using multi-step sequencing assays. Also disclosed are methods and systems for preparing samples for use in high-throughput sequencing assays to detect AMR genetic markers.

Description

MULTI-STEP METHODS FOR DETECTING NUCLEIC ACIDSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 554,922, filedFebruary 16, 2024, which application is incorporated herein by reference in its entirety.BACKGROUND

[0002] Massively parallel sequencing (MPS), as the name implies, is a high-throughput technology that can generate an enormous amount of information about the genetic makeup of an organism. MPS is particularly useful for genomic studies that analyze sequences across a genome such as whole genome sequencing. MPS can be used to study cell-associated DNA, as well as cell-free DNA shed into a variety of samples including blood. MPS can also be used for metagenomic sequencing applications that detect microbial nucleic acids in a sample.

[0003] Detecting genetic markers is essential for diagnosing and treating diseases. In infectious diseases, identifying antimicrobial resistance (AMR) is particularly important for effective management. Early detection of AMR genes in microbes enables targeted antibiotic therapy, reducing the misuse of broad-spectrum antibiotics that may drive drug resistance. Rapid AMR identification can also aid in controlling outbreaks, especially in hospital settings where multidrug-resistant organisms pose serious risks. Additionally, AMR surveillance informs global health policies, guiding antibiotic stewardship programs and the development of new treatments. Without reliable AMR detection, infections become increasingly difficult to treat, leading to higher morbidity, mortality, and healthcare costs.

[0004] Current AMR detection methods often rely on traditional bacterial culture techniques, which are low throughput, time-consuming and labor-intensive. While sequencing-based approaches offer potential alternatives, they have limitations in sensitivity and accuracy. For example, molecular assays like PCR can identify known resistance genes but may not sufficiently provide information on phenotypic resistance of the organism. Therefore, there is a need for improved diagnostic tools that can rapidly and accurately detect a broad range of AMR determinants to inform effective treatment strategies. Provided herein are novel approaches for detecting AMR markers or carrier organisms harboring said AMR markers, determining antimicrobial resistance of the carrier organisms, and preparing samples useful for AMR detection.SUMMARY

[0005] There remains a need to develop improved methods for detecting genetic markers in the context of disease diagnosis and treatment. The present disclosure provides novel approaches for detecting genetic markers using high-throughput sequencing.

[0006] This Summary introduces a selection of concepts that are described further below in the Detailed Description. This Summary is not intended to limit the scope of the claimed subject matter.

[0007] Disclosed herein are methods and compositions for performing a multi-step assay for detecting nucleic acids in a sample from a subject, the method comprising: providing a first aliquot of the sample from the subject, wherein the sample comprises nucleic acids comprising a first target nucleic acid and a second target nucleic acid that is different from the first target nucleic acid; performing a sequencing assay on the nucleic acids to produce sequence reads comprising sequence reads associated with the first target nucleic acid; analyzing the sequence reads, thereby obtaining an identification of the first target nucleic acid; after the identification of the first target nucleic acid is obtained, providing a second aliquot of the sample comprising nucleic acids comprising the first target nucleic acid and the second target nucleic acid; introducing primers into the second aliquot or to nucleic acids derived from the second aliquot wherein the primers specifically target the second target nucleic acid and do not target the first target nucleic acid; and conducting an amplification reaction on the second target nucleic acid with the primers, thereby amplifying the second target nucleic acid and producing amplicons associated with the second target nucleic acid. In some embodiments, the methods disclosed herein further comprise performing a high-throughput sequencing assay on the amplicons associated with the second target nucleic acid. In some embodiments, the second target nucleic acid is not detected by a sequencing assay. In some embodiments, the first nucleic acid is associated with a genome of an organism but not a phenotype of interest of the organism and the second nucleic acid is associated with the phenotype of interest of the organism. In some embodiments, the first target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 first target nucleic acids; or the second target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 second target nucleic acids. In some embodiments, the first target nucleic acid or the second target nucleic acid are not negative or positive controls for any step of the multi-step assay. In some embodiments, the primers comprise multiple primers targeting multiple target nucleic acids. In some embodiments, the first target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises nucleic acids from an animal, a virus, abacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises a cancer marker. In some embodiments, the first target nucleic acid is associated with a carrier microbe harboring a target genetic marker and the second target nucleic acid comprises a sequence associated with the target genetic marker. In some embodiments, the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the target genetic marker comprises an antimicrobial resistance (AMR) genetic marker. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the AMR genetic marker to a threshold value. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the gene cassette to a threshold value. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the AMR genetic marker is below the threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the gene cassette is below the threshold. In some embodiments, the methods disclosed herein further comprise quantifying mcfNA sequencing reads from the one or more microbes. In some embodiments, the methods disclosed herein further comprise comparing the abundance of the mcfNA sequencing reads from the one or more microbes to a threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the mcfNA sequencing reads from the one or more microbes is below the threshold. In some embodiments, the methods disclosed herein further comprise performing high-throughput sequencing on the amplicons associated with the AMR genetic marker. In some embodiments, the sequencing assay on the first nucleic acids comprises a high-throughput sequencing assay. In some embodiments, the methods disclosed herein further comprise conducting a polymerase chain reaction (PCR) to amplify the AMR genetic marker, thereby producing amplicons associated with the AMR genetic marker. In some embodiments, the PCR comprises multiplex PCR, random PCR (rPCR), non-biased PCR, Nested PCR, Hot Start PCR, or Assembly PCR. In some embodiments, the methods disclosed herein further comprise attaching an adapter sequence to the second nucleic acids. In some embodiments, the primers comprise an adapter sequence. In some embodiments, the methods disclosed herein further comprise physically manipulating the sample to produce a fraction of cfNA enriched for degraded cfNA, wherein the fraction of cfNA comprises the AMR genetic marker. In some embodiments, the degraded cfNA comprises ultra short cfNA, single stranded cfNA, or nicked double stranded cfNA. In some embodiments, the ultra short cfNA comprises cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the ultra short cfNA comprisescfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, the methods disclosed herein further comprise performing size selection of the nucleic acids in the sample. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, performing size selection using a method selected from the group consisting of: chromatography, size-exclusion chromatography, electrophoresis, gel electrophoresis, automated electrophoresis, capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation, filtration, membrane ultrafiltration, magnetic beads, and affinity-based beads. In some embodiments, the second aliquot comprises at least 500 pl of plasma. In some embodiments, detecting the carrier microbe comprises determining an abundance of the carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per microliter of sample (MPM). In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number from the amplification or sequencing of the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to the carrier microbe using the abundance of the mcfNA from the carrier microbe and the AMR gene copy number. In some embodiments, the AMR gene copy number is an episomal gene copy number. In some embodiments, the methods disclosed herein comprise introducing at least 200 primers targeting the plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of the microbe infecting the subject. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample is not subjected to a process that primarily causes cell lysis. In some embodiments, the methods disclosed herein further comprise preparing a library from the amplicons associated with the AMR genetic marker. In some embodiments, the primers are added directly to the second aliquot. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, the second nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, intact microbes are not actively lysed prior to performing the sequencing assay. In some embodiments, the microbial nucleic acids comprise microbial cell-free nucleic acids (mcfNA) from the microbe. In some embodiments, the microbial nucleic acids comprise nucleic acids associated with a microbial cell. In some embodiments, the methods disclosed herein comprise enriching for at least at least 75%, at least 80%, at least 85%, or at least 90% ofthe mcfNA in the sample. In some embodiments, the degraded cfNA comprise ultra short cfNA, single stranded cfNA, nicked double stranded cfNA, or any combination thereof. In some embodiments, the degraded cfNA comprise cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the degraded cfNA comprise cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, the methods disclosed herein further comprise generating sequence reads from the cfNA from the subject, wherein the sequence reads comprise microbial sequencing reads derived from the microbe infecting the subject. In some embodiments, the sequence reads further comprise microbial sequencing reads from one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise calculating an abundance of the mcfNA from the microbe in the sample and an abundance of the mcfNA from the one or more carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample. In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number for each of the one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise detecting one or more carrier microbe from the sequencing assay. In some embodiments, the methods disclosed herein further comprise identifying the one or more carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise obtaining an abundance of the one or more carrier microbes based on an abundance of carrier microbe sequences in the sample. In some embodiments, conducting a statistical analysis comprises using the abundance of the microbial nucleic acids of the one or more potential carrier microbes. In some embodiments, conducting a statistical analysis comprises using the AMR gene copy number. In some embodiments, the methods disclosed herein further comprise calculating a probability of each of the one or more potential carrier microbes being the microbe harboring the AMR genetic marker, thereby identifying the potential carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining the antimicrobial resistance of the microbe infecting the subject based on the calculated probability. In some embodiments, the detecting comprises performing a sequencing assay. In some embodiments, the sequencing assay comprises a high-throughput sequencing assay. In some embodiments, the amplification reaction comprises introducing primers targeting a plurality of AMR genetic markers. In some embodiments, the amplification reaction produces amplicons associated with the AMR genetic marker. In some embodiments, the sample comprises plasma. In someembodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample does not undergo cell lysis. In some embodiments, intact microbes are not actively lysed. In some embodiments, the amplicons associated with the AMR genetic marker undergoes direct library preparation. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise cell-free nucleic acids. In some embodiments, the second nucleic acids comprise cell-free nucleic acids. In some embodiments, the methods disclosed herein comprise adding primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein comprise adding at least 200 primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the second aliquot of the sample comprises at least 500 pl of plasma. In some embodiments, detecting the pathogen comprises detecting the abundance of mcfNA from the pathogen in the sample over a threshold. In some embodiments, the methods disclosed herein further comprise calculating a positive percent agreement (PPA), negative percent agreement (NPA), diagnostic yield (DY), or any combination thereof. In some embodiments, the methods disclosed herein further comprise detecting cfDNA from one or more housekeeping genes. In some embodiments, the methods disclosed herein further comprise spiking one or more control molecules into the sample at a known concentration. In some embodiments, the one or more control molecules are synthetic oligonucleotides. In some embodiments, the control molecules comprise whole assay internal control (WINC) molecules. In some embodiments, the methods disclosed herein further comprise spiking at least 25,000 unique WINC molecules at known concentrations. In some embodiments, the methods disclosed herein further comprise generating a report listing the carrier microbes or pathogen detected in the subject. In some embodiments, the report further comprises the abundance of microbial cell-free DNA (mcfDNA) from microbes detected in the sample or the antimicrobial resistance of the microbes infecting the subject. In some embodiments, the sample comprises a biological sample obtained from the subject. In some embodiments, the biological sample is a whole blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a synovial fluid sample, a urine sample, a stool sample, a bronchoalveolar lavage sample, or any combination thereof. In some embodiments, the sample is a plasma sample. In some embodiments, the AMR genetic marker comprises a gene, a genetic element, a genetic cassette, an allele, a mutation, or any combination thereof. In some embodiments, the AMR genetic marker is associated with one or more genes selected from the group consisting of SCCmec, mecA, mecC, vanA, vanB, blacrx-M, blctKPC, OXA-48-Uke, OXA-23,NDM, VIM, IMP, or mcr-1. In some embodiments, the AMR genetic marker provides resistance to an anti-microbial agent selected from the group consisting of: methicillin, vancomycin, cephalosporin, carbapenem, and oxyimino-cephalosporin / aztreonam resistance. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a gram-positive bacterium or a fungus. In some embodiments, the microbe infecting the subject or the carrier microbe is a microbe listed in Table 1. In some embodiments, the microbe infecting the subject or the carrier microbe harbors at last two AMR genetic markers. In some embodiments, the antimicrobial resistance is phenotypic antimicrobial resistance. In some embodiments, the sequencing comprises nextgeneration sequencing or a sequencing method beyond next generation sequencing. In some embodiments, the sequencing comprises sequencing by synthesis. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, the subject has an infection by the microbe infecting the subject harboring the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise administering an anti- infective agent to the subject. In some embodiments, the subject has been treated with an anti- infective agent for an infection. In some embodiments, the methods disclosed herein further comprise adjusting the anti -microbial agent received by the subject at least in part based on the antimicrobial resistance of the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of a microbe. In some embodiments, the microbe comprises a microbe infecting the subject. In some embodiments, the microbe comprises a carrier microbe. In some embodiments, the methods disclosed herein comprise determining whether an AMR gene is carried by a microbe infecting the subject or by a carrier microbe. In some embodiments, the determining comprises linking an AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the determining comprises determining a copy number of the AMR genetic marker in the microbe. In some embodiments, the linking comprises performing a statistical analysis on the sequence reads. In some embodiments, the statistical analysis comprises a generalized linear model, a maximum-likelihood estimation, a probit model, a logistic regression, a linear probability, a linear regression, a complimentary log-log, a Poisson regression, a support vector machine, a decision tree, a random forest, a neural network, a gradient boosted model, a Bayesian model, a hidden Markov model, or any combination thereof. In some embodiments, the method further comprises comparing the sequence read data to the amplification data. In some embodiments, the method further comprises calculating estimated deduplicated templates (EDT) for an AMR gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads froma microorganism present in a sequenced library. In some embodiments, the method further comprises calculating an EDT for a housekeeping gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the methods disclosed herein further comprise determining a presence of an organism in a sample by calculating a ratio of an AMR estimated deduplicated templates (EDT) to a housekeeping gene EDT.

[0008] Disclosed herein are methods and compositions for performing a multi-step sample processing procedure for detecting nucleic acids in a sample from a subject: providing a first aliquot of the sample from the subject, wherein the sample comprises one or more first nucleic acids, wherein the one or more first nucleic acids comprise one or more microbial nucleic acids derived from one or more carrier microbes harboring an antimicrobial resistance (AMR) genetic marker; performing a sequencing assay on the one or more first nucleic acids to produce sequence reads comprising microbial nucleic acid sequence reads from the one or more carrier microbes; detecting the one or more carrier microbes by analyzing the microbial nucleic acid sequence reads; after at least one carrier microbe is detected, providing a second aliquot of the sample from the subject, wherein the second aliquot comprises second nucleic acids from at least one AMR genetic marker; annealing primers targeting at least one AMR genetic marker to the second nucleic acids; and amplifying the AMR genetic marker in an amplification reaction to produce amplicons associated with the at least one AMR genetic marker. In some embodiments, the methods disclosed herein further comprise performing a high-throughput sequencing assay on the amplicons associated with the second target nucleic acid. In some embodiments, the second target nucleic acid is not detected by a sequencing assay. In some embodiments, the first nucleic acid is associated with a genome of an organism but not a phenotype of interest of the organism and the second nucleic acid is associated with the phenotype of interest of the organism. In some embodiments, the first target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 first target nucleic acids; or the second target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 second target nucleic acids. In some embodiments, the first target nucleic acid or the second target nucleic acid are not negative or positive controls for any step of the multi-step assay. In some embodiments, the primers comprise multiple primers targeting multiple target nucleic acids. In some embodiments, the first target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises a cancer marker. In some embodiments, the first target nucleic acid isassociated with a carrier microbe harboring a target genetic marker and the second target nucleic acid comprises a sequence associated with the target genetic marker. In some embodiments, the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the target genetic marker comprises an antimicrobial resistance (AMR) genetic marker. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the AMR genetic marker to a threshold value. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the gene cassette to a threshold value. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the AMR genetic marker is below the threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the gene cassette is below the threshold. In some embodiments, the methods disclosed herein further comprise quantifying mcfNA sequencing reads from the one or more microbes. In some embodiments, the methods disclosed herein further comprise comparing the abundance of the mcfNA sequencing reads from the one or more microbes to a threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the mcfNA sequencing reads from the one or more microbes is below the threshold. In some embodiments, the methods disclosed herein further comprise performing high-throughput sequencing on the amplicons associated with the AMR genetic marker. In some embodiments, the sequencing assay on the first nucleic acids comprises a high-throughput sequencing assay. In some embodiments, the methods disclosed herein further comprise conducting a polymerase chain reaction (PCR) to amplify the AMR genetic marker, thereby producing amplicons associated with the AMR genetic marker. In some embodiments, the PCR comprises multiplex PCR, random PCR (rPCR), non-biased PCR, Nested PCR, Hot Start PCR, or Assembly PCR. In some embodiments, the methods disclosed herein further comprise attaching an adapter sequence to the second nucleic acids. In some embodiments, the primers comprise an adapter sequence. In some embodiments, the methods disclosed herein further comprise physically manipulating the sample to produce a fraction of cfNA enriched for degraded cfNA, wherein the fraction of cfNA comprises the AMR genetic marker. In some embodiments, the degraded cfNA comprises ultra short cfNA, single stranded cfNA, or nicked double stranded cfNA. In some embodiments, the ultra short cfNA comprises cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the ultra short cfNA comprises cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, the methods disclosed herein further comprise performing size selection of the nucleic acids inthe sample. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, performing size selection using a method selected from the group consisting of chromatography, size-exclusion chromatography, electrophoresis, gel electrophoresis, automated electrophoresis, capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation, filtration, membrane ultrafiltration, magnetic beads, and affinity-based beads. In some embodiments, the second aliquot comprises at least 500 pl of plasma. In some embodiments, detecting the carrier microbe comprises determining an abundance of the carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per microliter of sample (MPM). In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number from the amplification or sequencing of the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to the carrier microbe using the abundance of the mcfNA from the carrier microbe and the AMR gene copy number. In some embodiments, the AMR gene copy number is an episomal gene copy number. In some embodiments, the methods disclosed herein comprise introducing at least 200 primers targeting the plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of the microbe infecting the subject. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample is not subjected to a process that primarily causes cell lysis. In some embodiments, the methods disclosed herein further comprise preparing a library from the amplicons associated with the AMR genetic marker. In some embodiments, the primers are added directly to the second aliquot. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, the second nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, intact microbes are not actively lysed prior to performing the sequencing assay. In some embodiments, the microbial nucleic acids comprise microbial cell-free nucleic acids (mcfNA) from the microbe. In some embodiments, the microbial nucleic acids comprise nucleic acids associated with a microbial cell. In some embodiments, the methods disclosed herein comprise enriching for at least at least 75%, at least 80%, at least 85%, or at least 90% of the mcfNA in the sample. In some embodiments, the degraded cfNA comprise ultra short cfNA, single stranded cfNA, nicked double stranded cfNA, or any combination thereof. In someembodiments, the degraded cfNA comprise cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the degraded cfNA comprise cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, the methods disclosed herein further comprise generating sequence reads from the cfNA from the subject, wherein the sequence reads comprise microbial sequencing reads derived from the microbe infecting the subject. In some embodiments, the sequence reads further comprise microbial sequencing reads from one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise calculating an abundance of the mcfNA from the microbe in the sample and an abundance of the mcfNA from the one or more carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample. In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number for each of the one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise detecting one or more carrier microbe from the sequencing assay. In some embodiments, the methods disclosed herein further comprise identifying the one or more carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise obtaining an abundance of the one or more carrier microbes based on an abundance of carrier microbe sequences in the sample. In some embodiments, conducting a statistical analysis comprises using the abundance of the microbial nucleic acids of the one or more potential carrier microbes. In some embodiments, conducting a statistical analysis comprises using the AMR gene copy number. In some embodiments, the methods disclosed herein further comprise calculating a probability of each of the one or more potential carrier microbes being the microbe harboring the AMR genetic marker, thereby identifying the potential carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining the antimicrobial resistance of the microbe infecting the subject based on the calculated probability. In some embodiments, the detecting comprises performing a sequencing assay. In some embodiments, the sequencing assay comprises a high-throughput sequencing assay. In some embodiments, the amplification reaction comprises introducing primers targeting a plurality of AMR genetic markers. In some embodiments, the amplification reaction produces amplicons associated with the AMR genetic marker. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample does not undergo cell lysis. In some embodiments,intact microbes are not actively lysed. In some embodiments, the amplicons associated with the AMR genetic marker undergoes direct library preparation. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise cell-free nucleic acids. In some embodiments, the second nucleic acids comprise cell-free nucleic acids. In some embodiments, the methods disclosed herein comprise adding primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein comprise adding at least 200 primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the second aliquot of the sample comprises at least 500 pl of plasma. In some embodiments, detecting the pathogen comprises detecting the abundance of mcfNA from the pathogen in the sample over a threshold. In some embodiments, the methods disclosed herein further comprise calculating a positive percent agreement (PPA), negative percent agreement (NPA), diagnostic yield (DY), or any combination thereof. In some embodiments, the methods disclosed herein further comprise detecting cfDNA from one or more housekeeping genes. In some embodiments, the methods disclosed herein further comprise spiking one or more control molecules into the sample at a known concentration. In some embodiments, the one or more control molecules are synthetic oligonucleotides. In some embodiments, the control molecules comprise whole assay internal control (WINC) molecules. In some embodiments, the methods disclosed herein further comprise spiking at least 25,000 unique WINC molecules at known concentrations. In some embodiments, the methods disclosed herein further comprise generating a report listing the carrier microbes or pathogen detected in the subject. In some embodiments, the report further comprises the abundance of microbial cell-free DNA (mcfDNA) from microbes detected in the sample or the antimicrobial resistance of the microbes infecting the subject. In some embodiments, the sample comprises a biological sample obtained from the subject. In some embodiments, the biological sample is a whole blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a synovial fluid sample, a urine sample, a stool sample, a bronchoalveolar lavage sample, or any combination thereof. In some embodiments, the sample is a plasma sample. In some embodiments, the AMR genetic marker comprises a gene, a genetic element, a genetic cassette, an allele, a mutation, or any combination thereof. In some embodiments, the AMR genetic marker is associated with one or more genes selected from the group consisting of: SCCmec, mecA, mecC, vanA, vanB, blacrx-M, blctKPC, OXA-48-like, OXA-23, NDM, VIM, IMP, or mcr-1. In some embodiments, the AMR genetic marker provides resistance to an anti-microbial agent selected from the group consisting of: methicillin, vancomycin,cephalosporin, carbapenem, and oxyimino-cephalosporin / aztreonam resistance. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a gram-positive bacterium or a fungus. In some embodiments, the microbe infecting the subject or the carrier microbe is a microbe listed in Table 1. In some embodiments, the microbe infecting the subject or the carrier microbe harbors at last two AMR genetic markers. In some embodiments, the antimicrobial resistance is phenotypic antimicrobial resistance. In some embodiments, the sequencing comprises nextgeneration sequencing or a sequencing method beyond next generation sequencing. In some embodiments, the sequencing comprises sequencing by synthesis. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, the subject has an infection by the microbe infecting the subject harboring the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise administering an anti- infective agent to the subject. In some embodiments, the subject has been treated with an anti- infective agent for an infection. In some embodiments, the methods disclosed herein further comprise adjusting the anti -microbial agent received by the subject at least in part based on the antimicrobial resistance of the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of a microbe. In some embodiments, the microbe comprises a microbe infecting the subject. In some embodiments, the microbe comprises a carrier microbe. In some embodiments, the methods disclosed herein comprise determining whether an AMR gene is carried by a microbe infecting the subject or by a carrier microbe. In some embodiments, the determining comprises linking an AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the determining comprises determining a copy number of the AMR genetic marker in the microbe. In some embodiments, the linking comprises performing a statistical analysis on the sequence reads. In some embodiments, the statistical analysis comprises a generalized linear model, a maximum-likelihood estimation, a probit model, a logistic regression, a linear probability, a linear regression, a complimentary log-log, a Poisson regression, a support vector machine, a decision tree, a random forest, a neural network, a gradient boosted model, a Bayesian model, a hidden Markov model, or any combination thereof. In some embodiments, the method further comprises comparing the sequence read data to the amplification data. In some embodiments, the method further comprises calculating estimated deduplicated templates (EDT) for an AMR gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the method further comprises calculating an EDT for a housekeeping gene, wherein calculating EDT comprisescalculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the methods disclosed herein further comprise determining a presence of an organism in a sample by calculating a ratio of an AMR estimated deduplicated templates (EDT) to a housekeeping gene EDT.

[0009] Disclosed herein are methods and compositions for assaying nucleic acids in a sample from a subject comprising: providing a first aliquot of the sample from the subject, wherein the sample comprises one or more first nucleic acids, wherein the one or more first nucleic acids comprise one or more microbial nucleic acids derived from one or more carrier microbes harboring an antimicrobial resistance (AMR) genetic marker; performing a sequencing assay on the one or more first nucleic acids to produce sequence reads comprising microbial nucleic acid sequence reads from the one or more carrier microbes; detecting the one or more carrier microbes by analyzing the microbial sequence reads; providing a second aliquot of the sample from the subject, wherein the second aliquot comprises second nucleic acids, wherein the second aliquot comprises second nucleic acids from at least one AMR genetic marker; annealing primers to the second nucleic acids that target the at least one AMR genetic marker, wherein the primers that target the at least one AMR genetic marker are annealed prior to amplification of nucleic acids in the sample; and conducting an amplification reaction on the second nucleic acids using the primers targeting the at least one AMR genetic marker, thereby amplifying at least one AMR genetic marker and producing amplicons associated with the at least one AMR genetic marker. In some embodiments, the methods disclosed herein further comprise performing a high- throughput sequencing assay on the amplicons associated with the second target nucleic acid. In some embodiments, the second target nucleic acid is not detected by a sequencing assay. In some embodiments, the first nucleic acid is associated with a genome of an organism but not a phenotype of interest of the organism and the second nucleic acid is associated with the phenotype of interest of the organism. In some embodiments, the first target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 first target nucleic acids; or the second target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 second target nucleic acids. In some embodiments, the first target nucleic acid or the second target nucleic acid are not negative or positive controls for any step of the multi-step assay. In some embodiments, the primers comprise multiple primers targeting multiple target nucleic acids. In some embodiments, the first target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises a cancer marker. In someembodiments, the first target nucleic acid is associated with a carrier microbe harboring a target genetic marker and the second target nucleic acid comprises a sequence associated with the target genetic marker. In some embodiments, the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the target genetic marker comprises an antimicrobial resistance (AMR) genetic marker. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the AMR genetic marker to a threshold value. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the gene cassette to a threshold value. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the AMR genetic marker is below the threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the gene cassette is below the threshold. In some embodiments, the methods disclosed herein further comprise quantifying mcfNA sequencing reads from the one or more microbes. In some embodiments, the methods disclosed herein further comprise comparing the abundance of the mcfNA sequencing reads from the one or more microbes to a threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the mcfNA sequencing reads from the one or more microbes is below the threshold. In some embodiments, the methods disclosed herein further comprise performing high-throughput sequencing on the amplicons associated with the AMR genetic marker. In some embodiments, the sequencing assay on the first nucleic acids comprises a high- throughput sequencing assay. In some embodiments, the methods disclosed herein further comprise conducting a polymerase chain reaction (PCR) to amplify the AMR genetic marker, thereby producing amplicons associated with the AMR genetic marker. In some embodiments, the PCR comprises multiplex PCR, random PCR (rPCR), non-biased PCR, Nested PCR, Hot Start PCR, or Assembly PCR. In some embodiments, the methods disclosed herein further comprise attaching an adapter sequence to the second nucleic acids. In some embodiments, the primers comprise an adapter sequence. In some embodiments, the methods disclosed herein further comprise physically manipulating the sample to produce a fraction of cfNA enriched for degraded cfNA, wherein the fraction of cfNA comprises the AMR genetic marker. In some embodiments, the degraded cfNA comprises ultra short cfNA, single stranded cfNA, or nicked double stranded cfNA. In some embodiments, the ultra short cfNA comprises cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the ultra short cfNA comprises cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments,the methods disclosed herein further comprise performing size selection of the nucleic acids in the sample. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, performing size selection using a method selected from the group consisting of: chromatography, size-exclusion chromatography, electrophoresis, gel electrophoresis, automated electrophoresis, capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation, filtration, membrane ultrafiltration, magnetic beads, and affinity-based beads. In some embodiments, the second aliquot comprises at least 500 pl of plasma. In some embodiments, detecting the carrier microbe comprises determining an abundance of the carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per microliter of sample (MPM). In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number from the amplification or sequencing of the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to the carrier microbe using the abundance of the mcfNA from the carrier microbe and the AMR gene copy number. In some embodiments, the AMR gene copy number is an episomal gene copy number. In some embodiments, the methods disclosed herein comprise introducing at least 200 primers targeting the plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of the microbe infecting the subject. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample is not subjected to a process that primarily causes cell lysis. In some embodiments, the methods disclosed herein further comprise preparing a library from the amplicons associated with the AMR genetic marker. In some embodiments, the primers are added directly to the second aliquot. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, the second nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, intact microbes are not actively lysed prior to performing the sequencing assay. In some embodiments, the microbial nucleic acids comprise microbial cell-free nucleic acids (mcfNA) from the microbe. In some embodiments, the microbial nucleic acids comprise nucleic acids associated with a microbial cell. In some embodiments, the methods disclosed herein comprise enriching for at least at least 75%, at least 80%, at least 85%, or at least 90% of the mcfNA in the sample. In some embodiments, the degraded cfNA comprise ultra short cfNA,single stranded cfNA, nicked double stranded cfNA, or any combination thereof. In some embodiments, the degraded cfNA comprise cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the degraded cfNA comprise cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, the methods disclosed herein further comprise generating sequence reads from the cfNA from the subject, wherein the sequence reads comprise microbial sequencing reads derived from the microbe infecting the subject. In some embodiments, the sequence reads further comprise microbial sequencing reads from one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise calculating an abundance of the mcfNA from the microbe in the sample and an abundance of the mcfNA from the one or more carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample. In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number for each of the one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise detecting one or more carrier microbe from the sequencing assay. In some embodiments, the methods disclosed herein further comprise identifying the one or more carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise obtaining an abundance of the one or more carrier microbes based on an abundance of carrier microbe sequences in the sample. In some embodiments, conducting a statistical analysis comprises using the abundance of the microbial nucleic acids of the one or more potential carrier microbes. In some embodiments, conducting a statistical analysis comprises using the AMR gene copy number. In some embodiments, the methods disclosed herein further comprise calculating a probability of each of the one or more potential carrier microbes being the microbe harboring the AMR genetic marker, thereby identifying the potential carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining the antimicrobial resistance of the microbe infecting the subject based on the calculated probability. In some embodiments, the detecting comprises performing a sequencing assay. In some embodiments, the sequencing assay comprises a high-throughput sequencing assay. In some embodiments, the amplification reaction comprises introducing primers targeting a plurality of AMR genetic markers. In some embodiments, the amplification reaction produces amplicons associated with the AMR genetic marker. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acidsare DNA. In some embodiments, the sample does not undergo cell lysis. In some embodiments, intact microbes are not actively lysed. In some embodiments, the amplicons associated with the AMR genetic marker undergoes direct library preparation. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise cell-free nucleic acids. In some embodiments, the second nucleic acids comprise cell-free nucleic acids. In some embodiments, the methods disclosed herein comprise adding primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein comprise adding at least 200 primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the second aliquot of the sample comprises at least 500 pl of plasma. In some embodiments, detecting the pathogen comprises detecting the abundance of mcfNA from the pathogen in the sample over a threshold. In some embodiments, the methods disclosed herein further comprise calculating a positive percent agreement (PPA), negative percent agreement (NPA), diagnostic yield (DY), or any combination thereof. In some embodiments, the methods disclosed herein further comprise detecting cfDNA from one or more housekeeping genes. In some embodiments, the methods disclosed herein further comprise spiking one or more control molecules into the sample at a known concentration. In some embodiments, the one or more control molecules are synthetic oligonucleotides. In some embodiments, the control molecules comprise whole assay internal control (WINC) molecules. In some embodiments, the methods disclosed herein further comprise spiking at least 25,000 unique WINC molecules at known concentrations. In some embodiments, the methods disclosed herein further comprise generating a report listing the carrier microbes or pathogen detected in the subject. In some embodiments, the report further comprises the abundance of microbial cell-free DNA (mcfDNA) from microbes detected in the sample or the antimicrobial resistance of the microbes infecting the subject. In some embodiments, the sample comprises a biological sample obtained from the subject. In some embodiments, the biological sample is a whole blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a synovial fluid sample, a urine sample, a stool sample, a bronchoalveolar lavage sample, or any combination thereof. In some embodiments, the sample is a plasma sample. In some embodiments, the AMR genetic marker comprises a gene, a genetic element, a genetic cassette, an allele, a mutation, or any combination thereof. In some embodiments, the AMR genetic marker is associated with one or more genes selected from the group consisting of: SCCmec, mecA, mecC, vanA, vanB, blacrx-M, blctKPC, OXA-48-like, OXA-23, NDM, VIM, IMP, or mcr-1. In some embodiments, the AMR genetic marker provides resistanceto an anti-microbial agent selected from the group consisting of: methicillin, vancomycin, cephalosporin, carbapenem, and oxyimino-cephalosporin / aztreonam resistance. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a gram-positive bacterium or a fungus. In some embodiments, the microbe infecting the subject or the carrier microbe is a microbe listed in Table 1. In some embodiments, the microbe infecting the subject or the carrier microbe harbors at last two AMR genetic markers. In some embodiments, the antimicrobial resistance is phenotypic antimicrobial resistance. In some embodiments, the sequencing comprises nextgeneration sequencing or a sequencing method beyond next generation sequencing. In some embodiments, the sequencing comprises sequencing by synthesis. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, the subject has an infection by the microbe infecting the subject harboring the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise administering an anti- infective agent to the subject. In some embodiments, the subject has been treated with an anti- infective agent for an infection. In some embodiments, the methods disclosed herein further comprise adjusting the anti -microbial agent received by the subject at least in part based on the antimicrobial resistance of the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of a microbe. In some embodiments, the microbe comprises a microbe infecting the subject. In some embodiments, the microbe comprises a carrier microbe. In some embodiments, the methods disclosed herein comprise determining whether an AMR gene is carried by a microbe infecting the subject or by a carrier microbe. In some embodiments, the determining comprises linking an AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the determining comprises determining a copy number of the AMR genetic marker in the microbe. In some embodiments, the linking comprises performing a statistical analysis on the sequence reads. In some embodiments, the statistical analysis comprises a generalized linear model, a maximum-likelihood estimation, a probit model, a logistic regression, a linear probability, a linear regression, a complimentary log-log, a Poisson regression, a support vector machine, a decision tree, a random forest, a neural network, a gradient boosted model, a Bayesian model, a hidden Markov model, or any combination thereof. In some embodiments, the method further comprises comparing the sequence read data to the amplification data. In some embodiments, the method further comprises calculating estimated deduplicated templates (EDT) for an AMR gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the method furthercomprises calculating an EDT for a housekeeping gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the methods disclosed herein further comprise determining a presence of an organism in a sample by calculating a ratio of an AMR estimated deduplicated templates (EDT) to a housekeeping gene EDT.

[0010] Disclosed herein are methods and compositions for performing a multi-step sample processing procedure for detecting nucleic acids in a sample from a subject: providing a first aliquot of the sample from the subject, wherein the sample comprises one or more first nucleic acids, wherein the one or more first nucleic acids comprise one or more microbial nucleic acids derived from one or more carrier microbes harboring an antimicrobial resistance (AMR) genetic marker; performing a sequencing assay on the one or more first nucleic acids to produce sequence reads comprising microbial nucleic acid sequence reads from the one or more carrier microbes; detecting the one or more carrier microbes by analyzing the microbial nucleic acid sequence reads; after at least one carrier microbe is detected, providing a second aliquot of the sample from the subject, wherein the second aliquot comprises second nucleic acids from at least one AMR genetic marker; annealing primers to the second nucleic acids that target at least one AMR genetic marker; and amplifying the AMR genetic marker and producing amplicons associated with the at least one AMR genetic marker. In some embodiments, the methods disclosed herein further comprise performing a high-throughput sequencing assay on the amplicons associated with the second target nucleic acid. In some embodiments, the second target nucleic acid is not detected by a sequencing assay. In some embodiments, the first nucleic acid is associated with a genome of an organism but not a phenotype of interest of the organism and the second nucleic acid is associated with the phenotype of interest of the organism. In some embodiments, the first target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 first target nucleic acids; or the second target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 second target nucleic acids. In some embodiments, the first target nucleic acid or the second target nucleic acid are not negative or positive controls for any step of the multi-step assay. In some embodiments, the primers comprise multiple primers targeting multiple target nucleic acids. In some embodiments, the first target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises a cancer marker. In some embodiments, the first target nucleic acid is associated with a carrier microbe harboring a target genetic marker and the second target nucleic acid comprises asequence associated with the target genetic marker. In some embodiments, the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the target genetic marker comprises an antimicrobial resistance (AMR) genetic marker. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the AMR genetic marker to a threshold value. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the gene cassette to a threshold value. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the AMR genetic marker is below the threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the gene cassette is below the threshold. In some embodiments, the methods disclosed herein further comprise quantifying mcfNA sequencing reads from the one or more microbes. In some embodiments, the methods disclosed herein further comprise comparing the abundance of the mcfNA sequencing reads from the one or more microbes to a threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the mcfNA sequencing reads from the one or more microbes is below the threshold. In some embodiments, the methods disclosed herein further comprise performing high-throughput sequencing on the amplicons associated with the AMR genetic marker. In some embodiments, the sequencing assay on the first nucleic acids comprises a high-throughput sequencing assay. In some embodiments, the methods disclosed herein further comprise conducting a polymerase chain reaction (PCR) to amplify the AMR genetic marker, thereby producing amplicons associated with the AMR genetic marker. In some embodiments, the PCR comprises multiplex PCR, random PCR (rPCR), non-biased PCR, Nested PCR, Hot Start PCR, or Assembly PCR. In some embodiments, the methods disclosed herein further comprise attaching an adapter sequence to the second nucleic acids. In some embodiments, the primers comprise an adapter sequence. In some embodiments, the methods disclosed herein further comprise physically manipulating the sample to produce a fraction of cfNA enriched for degraded cfNA, wherein the fraction of cfNA comprises the AMR genetic marker. In some embodiments, the degraded cfNA comprises ultra short cfNA, single stranded cfNA, or nicked double stranded cfNA. In some embodiments, the ultra short cfNA comprises cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the ultra short cfNA comprises cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, the methods disclosed herein further comprise performing size selection of the nucleic acids in the sample. In some embodiments, physically manipulating the sample comprises performingsize selection of the nucleic acids in the sample. In some embodiments, performing size selection using a method selected from the group consisting of: chromatography, size-exclusion chromatography, electrophoresis, gel electrophoresis, automated electrophoresis, capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation, filtration, membrane ultrafiltration, magnetic beads, and affinity-based beads. In some embodiments, the second aliquot comprises at least 500 pl of plasma. In some embodiments, detecting the carrier microbe comprises determining an abundance of the carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per microliter of sample (MPM). In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number from the amplification or sequencing of the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to the carrier microbe using the abundance of the mcfNA from the carrier microbe and the AMR gene copy number. In some embodiments, the AMR gene copy number is an episomal gene copy number. In some embodiments, the methods disclosed herein comprise introducing at least 200 primers targeting the plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of the microbe infecting the subject. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample is not subjected to a process that primarily causes cell lysis. In some embodiments, the methods disclosed herein further comprise preparing a library from the amplicons associated with the AMR genetic marker. In some embodiments, the primers are added directly to the second aliquot. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, the second nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, intact microbes are not actively lysed prior to performing the sequencing assay. In some embodiments, the microbial nucleic acids comprise microbial cell-free nucleic acids (mcfNA) from the microbe. In some embodiments, the microbial nucleic acids comprise nucleic acids associated with a microbial cell. In some embodiments, the methods disclosed herein comprise enriching for at least at least 75%, at least 80%, at least 85%, or at least 90% of the mcfNA in the sample. In some embodiments, the degraded cfNA comprise ultra short cfNA, single stranded cfNA, nicked double stranded cfNA, or any combination thereof. In some embodiments, the degraded cfNA comprise cfNA fragments that are less than 100 nucleotides inlength. In some embodiments, the degraded cfNA comprise cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, the methods disclosed herein further comprise generating sequence reads from the cfNA from the subject, wherein the sequence reads comprise microbial sequencing reads derived from the microbe infecting the subject. In some embodiments, the sequence reads further comprise microbial sequencing reads from one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise calculating an abundance of the mcfNA from the microbe in the sample and an abundance of the mcfNA from the one or more carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample. In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number for each of the one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise detecting one or more carrier microbe from the sequencing assay. In some embodiments, the methods disclosed herein further comprise identifying the one or more carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise obtaining an abundance of the one or more carrier microbes based on an abundance of carrier microbe sequences in the sample. In some embodiments, conducting a statistical analysis comprises using the abundance of the microbial nucleic acids of the one or more potential carrier microbes. In some embodiments, conducting a statistical analysis comprises using the AMR gene copy number. In some embodiments, the methods disclosed herein further comprise calculating a probability of each of the one or more potential carrier microbes being the microbe harboring the AMR genetic marker, thereby identifying the potential carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining the antimicrobial resistance of the microbe infecting the subject based on the calculated probability. In some embodiments, the detecting comprises performing a sequencing assay. In some embodiments, the sequencing assay comprises a high-throughput sequencing assay. In some embodiments, the amplification reaction comprises introducing primers targeting a plurality of AMR genetic markers. In some embodiments, the amplification reaction produces amplicons associated with the AMR genetic marker. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample does not undergo cell lysis. In some embodiments, intact microbes are not actively lysed. In some embodiments, the amplicons associated with theAMR genetic marker undergoes direct library preparation. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise cell-free nucleic acids. In some embodiments, the second nucleic acids comprise cell-free nucleic acids. In some embodiments, the methods disclosed herein comprise adding primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein comprise adding at least 200 primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the second aliquot of the sample comprises at least 500 pl of plasma. In some embodiments, detecting the pathogen comprises detecting the abundance of mcfNA from the pathogen in the sample over a threshold. In some embodiments, the methods disclosed herein further comprise calculating a positive percent agreement (PPA), negative percent agreement (NPA), diagnostic yield (DY), or any combination thereof. In some embodiments, the methods disclosed herein further comprise detecting cfDNA from one or more housekeeping genes. In some embodiments, the methods disclosed herein further comprise spiking one or more control molecules into the sample at a known concentration. In some embodiments, the one or more control molecules are synthetic oligonucleotides. In some embodiments, the control molecules comprise whole assay internal control (WINC) molecules. In some embodiments, the methods disclosed herein further comprise spiking at least 25,000 unique WINC molecules at known concentrations. In some embodiments, the methods disclosed herein further comprise generating a report listing the carrier microbes or pathogen detected in the subject. In some embodiments, the report further comprises the abundance of microbial cell-free DNA (mcfDNA) from microbes detected in the sample or the antimicrobial resistance of the microbes infecting the subject. In some embodiments, the sample comprises a biological sample obtained from the subject. In some embodiments, the biological sample is a whole blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a synovial fluid sample, a urine sample, a stool sample, a bronchoalveolar lavage sample, or any combination thereof. In some embodiments, the sample is a plasma sample. In some embodiments, the AMR genetic marker comprises a gene, a genetic element, a genetic cassette, an allele, a mutation, or any combination thereof. In some embodiments, the AMR genetic marker is associated with one or more genes selected from the group consisting of: SCCmec, mecA, mecC, vanA, vanB, blacrx-M, blctKPC, OXA-48-like, OXA-23, NDM, VIM, IMP, or mcr-1. In some embodiments, the AMR genetic marker provides resistance to an anti-microbial agent selected from the group consisting of: methicillin, vancomycin, cephalosporin, carbapenem, and oxyimino-cephalosporin / aztreonam resistance. In someembodiments, the microbe infecting the subject or the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a gram-positive bacterium or a fungus. In some embodiments, the microbe infecting the subject or the carrier microbe is a microbe listed in Table 1. In some embodiments, the microbe infecting the subject or the carrier microbe harbors at last two AMR genetic markers. In some embodiments, the antimicrobial resistance is phenotypic antimicrobial resistance. In some embodiments, the sequencing comprises nextgeneration sequencing or a sequencing method beyond next generation sequencing. In some embodiments, the sequencing comprises sequencing by synthesis. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, the subject has an infection by the microbe infecting the subject harboring the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise administering an anti- infective agent to the subject. In some embodiments, the subject has been treated with an anti- infective agent for an infection. In some embodiments, the methods disclosed herein further comprise adjusting the anti -microbial agent received by the subject at least in part based on the antimicrobial resistance of the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of a microbe. In some embodiments, the microbe comprises a microbe infecting the subject. In some embodiments, the microbe comprises a carrier microbe. In some embodiments, the methods disclosed herein comprise determining whether an AMR gene is carried by a microbe infecting the subject or by a carrier microbe. In some embodiments, the determining comprises linking an AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the determining comprises determining a copy number of the AMR genetic marker in the microbe. In some embodiments, the linking comprises performing a statistical analysis on the sequence reads. In some embodiments, the statistical analysis comprises a generalized linear model, a maximum-likelihood estimation, a probit model, a logistic regression, a linear probability, a linear regression, a complimentary log-log, a Poisson regression, a support vector machine, a decision tree, a random forest, a neural network, a gradient boosted model, a Bayesian model, a hidden Markov model, or any combination thereof. In some embodiments, the method further comprises comparing the sequence read data to the amplification data. In some embodiments, the method further comprises calculating estimated deduplicated templates (EDT) for an AMR gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the method further comprises calculating an EDT for a housekeeping gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in asequenced library. In some embodiments, the methods disclosed herein further comprise determining a presence of an organism in a sample by calculating a ratio of an AMR estimated deduplicated templates (EDT) to a housekeeping gene EDT.

[0011] Disclosed herein are methods and compositions for assaying nucleic acids in a sample from a subject comprising: providing a first aliquot of the sample from the subject, wherein the first aliquot comprises first nucleic acids, and the first nucleic acids comprise an AMR genetic marker and a gene cassette comprising the AMR genetic marker derived from one or more carrier microbes harboring the AMR genetic marker; performing a sequencing assay on the first nucleic acids to produce sequence reads comprising microbial nucleic acid sequence reads from the one or more carrier microbes; detecting the AMR genetic marker or the gene cassette comprising the AMR genetic marker in the sample and quantifying microbial sequence reads from the AMR genetic marker and / or microbial sequence reads from the gene cassette; after and based on the quantification, providing a second aliquot of the sample from the subject, wherein the second aliquot comprises second nucleic acids, wherein the second nucleic acids comprise the AMR genetic marker; introducing primers targeting the AMR genetic marker to the second nucleic acids, wherein the primers targeting the AMR genetic marker are introduced prior to amplification of nucleic acids in the second aliquot; and conducting an amplification reaction on the second nucleic acids using the primers targeting the AMR genetic marker, thereby amplifying the AMR genetic marker and producing amplicons associated with the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise performing a high-throughput sequencing assay on the amplicons associated with the second target nucleic acid. In some embodiments, the second target nucleic acid is not detected by a sequencing assay. In some embodiments, the first nucleic acid is associated with a genome of an organism but not a phenotype of interest of the organism and the second nucleic acid is associated with the phenotype of interest of the organism. In some embodiments, the first target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 first target nucleic acids; or the second target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 second target nucleic acids. In some embodiments, the first target nucleic acid or the second target nucleic acid are not negative or positive controls for any step of the multi-step assay. In some embodiments, the primers comprise multiple primers targeting multiple target nucleic acids. In some embodiments, the first target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises a cancer marker. In someembodiments, the first target nucleic acid is associated with a carrier microbe harboring a target genetic marker and the second target nucleic acid comprises a sequence associated with the target genetic marker. In some embodiments, the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the target genetic marker comprises an antimicrobial resistance (AMR) genetic marker. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the AMR genetic marker to a threshold value. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the gene cassette to a threshold value. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the AMR genetic marker is below the threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the gene cassette is below the threshold. In some embodiments, the methods disclosed herein further comprise quantifying mcfNA sequencing reads from the one or more microbes. In some embodiments, the methods disclosed herein further comprise comparing the abundance of the mcfNA sequencing reads from the one or more microbes to a threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the mcfNA sequencing reads from the one or more microbes is below the threshold. In some embodiments, the methods disclosed herein further comprise performing high-throughput sequencing on the amplicons associated with the AMR genetic marker. In some embodiments, the sequencing assay on the first nucleic acids comprises a high- throughput sequencing assay. In some embodiments, the methods disclosed herein further comprise conducting a polymerase chain reaction (PCR) to amplify the AMR genetic marker, thereby producing amplicons associated with the AMR genetic marker. In some embodiments, the PCR comprises multiplex PCR, random PCR (rPCR), non-biased PCR, Nested PCR, Hot Start PCR, or Assembly PCR. In some embodiments, the methods disclosed herein further comprise attaching an adapter sequence to the second nucleic acids. In some embodiments, the primers comprise an adapter sequence. In some embodiments, the methods disclosed herein further comprise physically manipulating the sample to produce a fraction of cfNA enriched for degraded cfNA, wherein the fraction of cfNA comprises the AMR genetic marker. In some embodiments, the degraded cfNA comprises ultra short cfNA, single stranded cfNA, or nicked double stranded cfNA. In some embodiments, the ultra short cfNA comprises cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the ultra short cfNA comprises cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments,the methods disclosed herein further comprise performing size selection of the nucleic acids in the sample. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, performing size selection using a method selected from the group consisting of: chromatography, size-exclusion chromatography, electrophoresis, gel electrophoresis, automated electrophoresis, capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation, filtration, membrane ultrafiltration, magnetic beads, and affinity-based beads. In some embodiments, the second aliquot comprises at least 500 pl of plasma. In some embodiments, detecting the carrier microbe comprises determining an abundance of the carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per microliter of sample (MPM). In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number from the amplification or sequencing of the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to the carrier microbe using the abundance of the mcfNA from the carrier microbe and the AMR gene copy number. In some embodiments, the AMR gene copy number is an episomal gene copy number. In some embodiments, the methods disclosed herein comprise introducing at least 200 primers targeting the plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of the microbe infecting the subject. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample is not subjected to a process that primarily causes cell lysis. In some embodiments, the methods disclosed herein further comprise preparing a library from the amplicons associated with the AMR genetic marker. In some embodiments, the primers are added directly to the second aliquot. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, the second nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, intact microbes are not actively lysed prior to performing the sequencing assay. In some embodiments, the microbial nucleic acids comprise microbial cell-free nucleic acids (mcfNA) from the microbe. In some embodiments, the microbial nucleic acids comprise nucleic acids associated with a microbial cell. In some embodiments, the methods disclosed herein comprise enriching for at least at least 75%, at least 80%, at least 85%, or at least 90% of the mcfNA in the sample. In some embodiments, the degraded cfNA comprise ultra short cfNA,single stranded cfNA, nicked double stranded cfNA, or any combination thereof. In some embodiments, the degraded cfNA comprise cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the degraded cfNA comprise cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, the methods disclosed herein further comprise generating sequence reads from the cfNA from the subject, wherein the sequence reads comprise microbial sequencing reads derived from the microbe infecting the subject. In some embodiments, the sequence reads further comprise microbial sequencing reads from one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise calculating an abundance of the mcfNA from the microbe in the sample and an abundance of the mcfNA from the one or more carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample. In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number for each of the one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise detecting one or more carrier microbe from the sequencing assay. In some embodiments, the methods disclosed herein further comprise identifying the one or more carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise obtaining an abundance of the one or more carrier microbes based on an abundance of carrier microbe sequences in the sample. In some embodiments, conducting a statistical analysis comprises using the abundance of the microbial nucleic acids of the one or more potential carrier microbes. In some embodiments, conducting a statistical analysis comprises using the AMR gene copy number. In some embodiments, the methods disclosed herein further comprise calculating a probability of each of the one or more potential carrier microbes being the microbe harboring the AMR genetic marker, thereby identifying the potential carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining the antimicrobial resistance of the microbe infecting the subject based on the calculated probability. In some embodiments, the detecting comprises performing a sequencing assay. In some embodiments, the sequencing assay comprises a high-throughput sequencing assay. In some embodiments, the amplification reaction comprises introducing primers targeting a plurality of AMR genetic markers. In some embodiments, the amplification reaction produces amplicons associated with the AMR genetic marker. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acidsare DNA. In some embodiments, the sample does not undergo cell lysis. In some embodiments, intact microbes are not actively lysed. In some embodiments, the amplicons associated with the AMR genetic marker undergoes direct library preparation. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise cell-free nucleic acids. In some embodiments, the second nucleic acids comprise cell-free nucleic acids. In some embodiments, the methods disclosed herein comprise adding primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein comprise adding at least 200 primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the second aliquot of the sample comprises at least 500 pl of plasma. In some embodiments, detecting the pathogen comprises detecting the abundance of mcfNA from the pathogen in the sample over a threshold. In some embodiments, the methods disclosed herein further comprise calculating a positive percent agreement (PPA), negative percent agreement (NPA), diagnostic yield (DY), or any combination thereof. In some embodiments, the methods disclosed herein further comprise detecting cfDNA from one or more housekeeping genes. In some embodiments, the methods disclosed herein further comprise spiking one or more control molecules into the sample at a known concentration. In some embodiments, the one or more control molecules are synthetic oligonucleotides. In some embodiments, the control molecules comprise whole assay internal control (WINC) molecules. In some embodiments, the methods disclosed herein further comprise spiking at least 25,000 unique WINC molecules at known concentrations. In some embodiments, the methods disclosed herein further comprise generating a report listing the carrier microbes or pathogen detected in the subject. In some embodiments, the report further comprises the abundance of microbial cell-free DNA (mcfDNA) from microbes detected in the sample or the antimicrobial resistance of the microbes infecting the subject. In some embodiments, the sample comprises a biological sample obtained from the subject. In some embodiments, the biological sample is a whole blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a synovial fluid sample, a urine sample, a stool sample, a bronchoalveolar lavage sample, or any combination thereof. In some embodiments, the sample is a plasma sample. In some embodiments, the AMR genetic marker comprises a gene, a genetic element, a genetic cassette, an allele, a mutation, or any combination thereof. In some embodiments, the AMR genetic marker is associated with one or more genes selected from the group consisting of: SCCmec, mecA, mecC, vanA, vanB, blacrx-M, blctKPC, OXA-48-like, OXA-23, NDM, VIM, IMP, or mcr-1. In some embodiments, the AMR genetic marker provides resistanceto an anti-microbial agent selected from the group consisting of: methicillin, vancomycin, cephalosporin, carbapenem, and oxyimino-cephalosporin / aztreonam resistance. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a gram-positive bacterium or a fungus. In some embodiments, the microbe infecting the subject or the carrier microbe is a microbe listed in Table 1. In some embodiments, the microbe infecting the subject or the carrier microbe harbors at last two AMR genetic markers. In some embodiments, the antimicrobial resistance is phenotypic antimicrobial resistance. In some embodiments, the sequencing comprises nextgeneration sequencing or a sequencing method beyond next generation sequencing. In some embodiments, the sequencing comprises sequencing by synthesis. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, the subject has an infection by the microbe infecting the subject harboring the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise administering an anti- infective agent to the subject. In some embodiments, the subject has been treated with an anti- infective agent for an infection. In some embodiments, the methods disclosed herein further comprise adjusting the anti -microbial agent received by the subject at least in part based on the antimicrobial resistance of the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of a microbe. In some embodiments, the microbe comprises a microbe infecting the subject. In some embodiments, the microbe comprises a carrier microbe. In some embodiments, the methods disclosed herein comprise determining whether an AMR gene is carried by a microbe infecting the subject or by a carrier microbe. In some embodiments, the determining comprises linking an AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the determining comprises determining a copy number of the AMR genetic marker in the microbe. In some embodiments, the linking comprises performing a statistical analysis on the sequence reads. In some embodiments, the statistical analysis comprises a generalized linear model, a maximum-likelihood estimation, a probit model, a logistic regression, a linear probability, a linear regression, a complimentary log-log, a Poisson regression, a support vector machine, a decision tree, a random forest, a neural network, a gradient boosted model, a Bayesian model, a hidden Markov model, or any combination thereof. In some embodiments, the method further comprises comparing the sequence read data to the amplification data. In some embodiments, the method further comprises calculating estimated deduplicated templates (EDT) for an AMR gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the method furthercomprises calculating an EDT for a housekeeping gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the methods disclosed herein further comprise determining a presence of an organism in a sample by calculating a ratio of an AMR estimated deduplicated templates (EDT) to a housekeeping gene EDT.

[0012] Disclosed herein are methods and compositions for preparing a cell-free DNA fraction from a subject infected with a microbe useful for analyzing an antimicrobial resistance (AMR) genetic marker, comprising: providing a sample from the subject, wherein: the sample comprises cell-free nucleic acids (cfNA) from the subject and microbial nucleic acids from the microbe infecting the subject; the cfNA comprises degraded cfNA, single-stranded cfNA and intact cfNA; and the microbe harbors an AMR genetic marker; physically manipulating the sample in order to produce a fraction of cfNA enriched for degraded cfNA and single-stranded cfNA, wherein the fraction of cfNA comprises microbial nucleic acids comprising a genetic locus associated with the AMR genetic marker; performing a sequencing assay on the fraction of cfNA enriched for the degraded cfNA and single-stranded cfNA; and analyzing the genetic locus associated with the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise performing a high-throughput sequencing assay on the amplicons associated with the second target nucleic acid. In some embodiments, the second target nucleic acid is not detected by a sequencing assay. In some embodiments, the first nucleic acid is associated with a genome of an organism but not a phenotype of interest of the organism and the second nucleic acid is associated with the phenotype of interest of the organism. In some embodiments, the first target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 first target nucleic acids; or the second target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 second target nucleic acids. In some embodiments, the first target nucleic acid or the second target nucleic acid are not negative or positive controls for any step of the multi-step assay. In some embodiments, the primers comprise multiple primers targeting multiple target nucleic acids. In some embodiments, the first target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises a cancer marker. In some embodiments, the first target nucleic acid is associated with a carrier microbe harboring a target genetic marker and the second target nucleic acid comprises a sequence associated with the target genetic marker. In some embodiments, the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the target genetic markercomprises an antimicrobial resistance (AMR) genetic marker. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the AMR genetic marker to a threshold value. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the gene cassette to a threshold value. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the AMR genetic marker is below the threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the gene cassette is below the threshold. In some embodiments, the methods disclosed herein further comprise quantifying mcfNA sequencing reads from the one or more microbes. In some embodiments, the methods disclosed herein further comprise comparing the abundance of the mcfNA sequencing reads from the one or more microbes to a threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the mcfNA sequencing reads from the one or more microbes is below the threshold. In some embodiments, the methods disclosed herein further comprise performing high-throughput sequencing on the amplicons associated with the AMR genetic marker. In some embodiments, the sequencing assay on the first nucleic acids comprises a high- throughput sequencing assay. In some embodiments, the methods disclosed herein further comprise conducting a polymerase chain reaction (PCR) to amplify the AMR genetic marker, thereby producing amplicons associated with the AMR genetic marker. In some embodiments, the PCR comprises multiplex PCR, random PCR (rPCR), non-biased PCR, Nested PCR, Hot Start PCR, or Assembly PCR. In some embodiments, the methods disclosed herein further comprise attaching an adapter sequence to the second nucleic acids. In some embodiments, the primers comprise an adapter sequence. In some embodiments, the methods disclosed herein further comprise physically manipulating the sample to produce a fraction of cfNA enriched for degraded cfNA, wherein the fraction of cfNA comprises the AMR genetic marker. In some embodiments, the degraded cfNA comprises ultra short cfNA, single stranded cfNA, or nicked double stranded cfNA. In some embodiments, the ultra short cfNA comprises cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the ultra short cfNA comprises cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, the methods disclosed herein further comprise performing size selection of the nucleic acids in the sample. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, performing size selection using a method selected from the group consisting of: chromatography, size-exclusionchromatography, electrophoresis, gel electrophoresis, automated electrophoresis, capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation, filtration, membrane ultrafiltration, magnetic beads, and affinity-based beads. In some embodiments, the second aliquot comprises at least 500 pl of plasma. In some embodiments, detecting the carrier microbe comprises determining an abundance of the carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per microliter of sample (MPM). In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number from the amplification or sequencing of the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to the carrier microbe using the abundance of the mcfNA from the carrier microbe and the AMR gene copy number. In some embodiments, the AMR gene copy number is an episomal gene copy number. In some embodiments, the methods disclosed herein comprise introducing at least 200 primers targeting the plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of the microbe infecting the subject. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample is not subjected to a process that primarily causes cell lysis. In some embodiments, the methods disclosed herein further comprise preparing a library from the amplicons associated with the AMR genetic marker. In some embodiments, the primers are added directly to the second aliquot. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, the second nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, intact microbes are not actively lysed prior to performing the sequencing assay. In some embodiments, the microbial nucleic acids comprise microbial cell-free nucleic acids (mcfNA) from the microbe. In some embodiments, the microbial nucleic acids comprise nucleic acids associated with a microbial cell. In some embodiments, the methods disclosed herein comprise enriching for at least at least 75%, at least 80%, at least 85%, or at least 90% of the mcfNA in the sample. In some embodiments, the degraded cfNA comprise ultra short cfNA, single stranded cfNA, nicked double stranded cfNA, or any combination thereof. In some embodiments, the degraded cfNA comprise cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the degraded cfNA comprise cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, physically manipulating thesample comprises performing size selection of the nucleic acids in the sample. In some embodiments, the methods disclosed herein further comprise generating sequence reads from the cfNA from the subject, wherein the sequence reads comprise microbial sequencing reads derived from the microbe infecting the subject. In some embodiments, the sequence reads further comprise microbial sequencing reads from one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise calculating an abundance of the mcfNA from the microbe in the sample and an abundance of the mcfNA from the one or more carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample. In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number for each of the one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise detecting one or more carrier microbe from the sequencing assay. In some embodiments, the methods disclosed herein further comprise identifying the one or more carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise obtaining an abundance of the one or more carrier microbes based on an abundance of carrier microbe sequences in the sample. In some embodiments, conducting a statistical analysis comprises using the abundance of the microbial nucleic acids of the one or more potential carrier microbes. In some embodiments, conducting a statistical analysis comprises using the AMR gene copy number. In some embodiments, the methods disclosed herein further comprise calculating a probability of each of the one or more potential carrier microbes being the microbe harboring the AMR genetic marker, thereby identifying the potential carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining the antimicrobial resistance of the microbe infecting the subject based on the calculated probability. In some embodiments, the detecting comprises performing a sequencing assay. In some embodiments, the sequencing assay comprises a high-throughput sequencing assay. In some embodiments, the amplification reaction comprises introducing primers targeting a plurality of AMR genetic markers. In some embodiments, the amplification reaction produces amplicons associated with the AMR genetic marker. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample does not undergo cell lysis. In some embodiments, intact microbes are not actively lysed. In some embodiments, the amplicons associated with the AMR genetic marker undergoes direct library preparation. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA.In some embodiments, the first nucleic acids comprise cell-free nucleic acids. In some embodiments, the second nucleic acids comprise cell-free nucleic acids. In some embodiments, the methods disclosed herein comprise adding primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein comprise adding at least 200 primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the second aliquot of the sample comprises at least 500 pl of plasma. In some embodiments, detecting the pathogen comprises detecting the abundance of mcfNA from the pathogen in the sample over a threshold. In some embodiments, the methods disclosed herein further comprise calculating a positive percent agreement (PPA), negative percent agreement (NPA), diagnostic yield (DY), or any combination thereof. In some embodiments, the methods disclosed herein further comprise detecting cfDNA from one or more housekeeping genes. In some embodiments, the methods disclosed herein further comprise spiking one or more control molecules into the sample at a known concentration. In some embodiments, the one or more control molecules are synthetic oligonucleotides. In some embodiments, the control molecules comprise whole assay internal control (WINC) molecules. In some embodiments, the methods disclosed herein further comprise spiking at least 25,000 unique WINC molecules at known concentrations. In some embodiments, the methods disclosed herein further comprise generating a report listing the carrier microbes or pathogen detected in the subject. In some embodiments, the report further comprises the abundance of microbial cell-free DNA (mcfDNA) from microbes detected in the sample or the antimicrobial resistance of the microbes infecting the subject. In some embodiments, the sample comprises a biological sample obtained from the subject. In some embodiments, the biological sample is a whole blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a synovial fluid sample, a urine sample, a stool sample, a bronchoalveolar lavage sample, or any combination thereof. In some embodiments, the sample is a plasma sample. In some embodiments, the AMR genetic marker comprises a gene, a genetic element, a genetic cassette, an allele, a mutation, or any combination thereof. In some embodiments, the AMR genetic marker is associated with one or more genes selected from the group consisting of SCCmec, mecA, mecC, vanA, vanB, blacrx-M, blctKPC, OXA-48-like, OXA-23, NDM, VIM, IMP, or mcr-1. In some embodiments, the AMR genetic marker provides resistance to an anti-microbial agent selected from the group consisting of methicillin, vancomycin, cephalosporin, carbapenem, and oxyimino-cephalosporin / aztreonam resistance. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the microbeinfecting the subject or the carrier microbe comprises a gram-positive bacterium or a fungus. In some embodiments, the microbe infecting the subject or the carrier microbe is a microbe listed in Table 1. In some embodiments, the microbe infecting the subject or the carrier microbe harbors at last two AMR genetic markers. In some embodiments, the antimicrobial resistance is phenotypic antimicrobial resistance. In some embodiments, the sequencing comprises nextgeneration sequencing or a sequencing method beyond next generation sequencing. In some embodiments, the sequencing comprises sequencing by synthesis. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, the subject has an infection by the microbe infecting the subject harboring the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise administering an anti- infective agent to the subject. In some embodiments, the subject has been treated with an anti- infective agent for an infection. In some embodiments, the methods disclosed herein further comprise adjusting the anti -microbial agent received by the subject at least in part based on the antimicrobial resistance of the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of a microbe. In some embodiments, the microbe comprises a microbe infecting the subject. In some embodiments, the microbe comprises a carrier microbe. In some embodiments, the methods disclosed herein comprise determining whether an AMR gene is carried by a microbe infecting the subject or by a carrier microbe. In some embodiments, the determining comprises linking an AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the determining comprises determining a copy number of the AMR genetic marker in the microbe. In some embodiments, the linking comprises performing a statistical analysis on the sequence reads. In some embodiments, the statistical analysis comprises a generalized linear model, a maximum-likelihood estimation, a probit model, a logistic regression, a linear probability, a linear regression, a complimentary log-log, a Poisson regression, a support vector machine, a decision tree, a random forest, a neural network, a gradient boosted model, a Bayesian model, a hidden Markov model, or any combination thereof. In some embodiments, the method further comprises comparing the sequence read data to the amplification data. In some embodiments, the method further comprises calculating estimated deduplicated templates (EDT) for an AMR gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the method further comprises calculating an EDT for a housekeeping gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the methods disclosed herein further comprisedetermining a presence of an organism in a sample by calculating a ratio of an AMR estimated deduplicated templates (EDT) to a housekeeping gene EDT.

[0013] Disclosed herein are methods and compositions for determining antimicrobial resistance of a microbe infecting a subject, comprising: providing a sample comprising nucleic acids from the subject, wherein: the nucleic acids comprise microbial nucleic acids from one or more potential carrier microbes harboring an antimicrobial resistance (AMR) genetic marker; performing a sequencing assay on the nucleic acids from the subject to generate sequence reads and detecting the one or more potential carrier microbe based on the sequence reads; if a carrier microbe is detected in (c), performing an amplification reaction on an aliquot of the sample, wherein the amplification reaction targets one or more AMR genetic markers in order to provide a copy number for the one or more AMR genetic markers; conducting a statistical analysis to link one or more of the AMR genetic markers to one of the one or more potential carrier microbe in the subject, thereby determining that a potential carrier microbe comprises an AMR genetic marker. In some embodiments, the methods disclosed herein further comprise performing a high- throughput sequencing assay on the amplicons associated with the second target nucleic acid. In some embodiments, the second target nucleic acid is not detected by a sequencing assay. In some embodiments, the first nucleic acid is associated with a genome of an organism but not a phenotype of interest of the organism and the second nucleic acid is associated with the phenotype of interest of the organism. In some embodiments, the first target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 first target nucleic acids; or the second target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 second target nucleic acids. In some embodiments, the first target nucleic acid or the second target nucleic acid are not negative or positive controls for any step of the multi-step assay. In some embodiments, the primers comprise multiple primers targeting multiple target nucleic acids. In some embodiments, the first target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises a cancer marker. In some embodiments, the first target nucleic acid is associated with a carrier microbe harboring a target genetic marker and the second target nucleic acid comprises a sequence associated with the target genetic marker. In some embodiments, the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the target genetic marker comprises an antimicrobial resistance (AMR) genetic marker. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequencereads from the AMR genetic marker to a threshold value. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the gene cassette to a threshold value. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the AMR genetic marker is below the threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the gene cassette is below the threshold. In some embodiments, the methods disclosed herein further comprise quantifying mcfNA sequencing reads from the one or more microbes. In some embodiments, the methods disclosed herein further comprise comparing the abundance of the mcfNA sequencing reads from the one or more microbes to a threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the mcfNA sequencing reads from the one or more microbes is below the threshold. In some embodiments, the methods disclosed herein further comprise performing high-throughput sequencing on the amplicons associated with the AMR genetic marker. In some embodiments, the sequencing assay on the first nucleic acids comprises a high- throughput sequencing assay. In some embodiments, the methods disclosed herein further comprise conducting a polymerase chain reaction (PCR) to amplify the AMR genetic marker, thereby producing amplicons associated with the AMR genetic marker. In some embodiments, the PCR comprises multiplex PCR, random PCR (rPCR), non-biased PCR, Nested PCR, Hot Start PCR, or Assembly PCR. In some embodiments, the methods disclosed herein further comprise attaching an adapter sequence to the second nucleic acids. In some embodiments, the primers comprise an adapter sequence. In some embodiments, the methods disclosed herein further comprise physically manipulating the sample to produce a fraction of cfNA enriched for degraded cfNA, wherein the fraction of cfNA comprises the AMR genetic marker. In some embodiments, the degraded cfNA comprises ultra short cfNA, single stranded cfNA, or nicked double stranded cfNA. In some embodiments, the ultra short cfNA comprises cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the ultra short cfNA comprises cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, the methods disclosed herein further comprise performing size selection of the nucleic acids in the sample. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, performing size selection using a method selected from the group consisting of: chromatography, size-exclusion chromatography, electrophoresis, gel electrophoresis, automated electrophoresis, capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation,filtration, membrane ultrafiltration, magnetic beads, and affinity-based beads. In some embodiments, the second aliquot comprises at least 500 pl of plasma. In some embodiments, detecting the carrier microbe comprises determining an abundance of the carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per microliter of sample (MPM). In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number from the amplification or sequencing of the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to the carrier microbe using the abundance of the mcfNA from the carrier microbe and the AMR gene copy number. In some embodiments, the AMR gene copy number is an episomal gene copy number. In some embodiments, the methods disclosed herein comprise introducing at least 200 primers targeting the plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of the microbe infecting the subject. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample is not subjected to a process that primarily causes cell lysis. In some embodiments, the methods disclosed herein further comprise preparing a library from the amplicons associated with the AMR genetic marker. In some embodiments, the primers are added directly to the second aliquot. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, the second nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, intact microbes are not actively lysed prior to performing the sequencing assay. In some embodiments, the microbial nucleic acids comprise microbial cell-free nucleic acids (mcfNA) from the microbe. In some embodiments, the microbial nucleic acids comprise nucleic acids associated with a microbial cell. In some embodiments, the methods disclosed herein comprise enriching for at least at least 75%, at least 80%, at least 85%, or at least 90% of the mcfNA in the sample. In some embodiments, the degraded cfNA comprise ultra short cfNA, single stranded cfNA, nicked double stranded cfNA, or any combination thereof. In some embodiments, the degraded cfNA comprise cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the degraded cfNA comprise cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, the methods disclosed herein further comprise generating sequence reads from thecfNA from the subject, wherein the sequence reads comprise microbial sequencing reads derived from the microbe infecting the subject. In some embodiments, the sequence reads further comprise microbial sequencing reads from one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise calculating an abundance of the mcfNA from the microbe in the sample and an abundance of the mcfNA from the one or more carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample. In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number for each of the one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise detecting one or more carrier microbe from the sequencing assay. In some embodiments, the methods disclosed herein further comprise identifying the one or more carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise obtaining an abundance of the one or more carrier microbes based on an abundance of carrier microbe sequences in the sample. In some embodiments, conducting a statistical analysis comprises using the abundance of the microbial nucleic acids of the one or more potential carrier microbes. In some embodiments, conducting a statistical analysis comprises using the AMR gene copy number. In some embodiments, the methods disclosed herein further comprise calculating a probability of each of the one or more potential carrier microbes being the microbe harboring the AMR genetic marker, thereby identifying the potential carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining the antimicrobial resistance of the microbe infecting the subject based on the calculated probability. In some embodiments, the detecting comprises performing a sequencing assay. In some embodiments, the sequencing assay comprises a high-throughput sequencing assay. In some embodiments, the amplification reaction comprises introducing primers targeting a plurality of AMR genetic markers. In some embodiments, the amplification reaction produces amplicons associated with the AMR genetic marker. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample does not undergo cell lysis. In some embodiments, intact microbes are not actively lysed. In some embodiments, the amplicons associated with the AMR genetic marker undergoes direct library preparation. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise cell-free nucleic acids. In some embodiments, the second nucleic acids comprise cell-free nucleic acids. In some embodiments,the methods disclosed herein comprise adding primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein comprise adding at least 200 primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the second aliquot of the sample comprises at least 500 pl of plasma. In some embodiments, detecting the pathogen comprises detecting the abundance of mcfNA from the pathogen in the sample over a threshold. In some embodiments, the methods disclosed herein further comprise calculating a positive percent agreement (PPA), negative percent agreement (NPA), diagnostic yield (DY), or any combination thereof. In some embodiments, the methods disclosed herein further comprise detecting cfDNA from one or more housekeeping genes. In some embodiments, the methods disclosed herein further comprise spiking one or more control molecules into the sample at a known concentration. In some embodiments, the one or more control molecules are synthetic oligonucleotides. In some embodiments, the control molecules comprise whole assay internal control (WINC) molecules. In some embodiments, the methods disclosed herein further comprise spiking at least 25,000 unique WINC molecules at known concentrations. In some embodiments, the methods disclosed herein further comprise generating a report listing the carrier microbes or pathogen detected in the subject. In some embodiments, the report further comprises the abundance of microbial cell-free DNA (mcfDNA) from microbes detected in the sample or the antimicrobial resistance of the microbes infecting the subject. In some embodiments, the sample comprises a biological sample obtained from the subject. In some embodiments, the biological sample is a whole blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a synovial fluid sample, a urine sample, a stool sample, a bronchoalveolar lavage sample, or any combination thereof. In some embodiments, the sample is a plasma sample. In some embodiments, the AMR genetic marker comprises a gene, a genetic element, a genetic cassette, an allele, a mutation, or any combination thereof. In some embodiments, the AMR genetic marker is associated with one or more genes selected from the group consisting of: SCCmec, mecA, mecC, vanA, vanB, blacrx-M, blctKPC, OXA-48-like, OXA-23, NDM, VIM, IMP, or mcr-1. In some embodiments, the AMR genetic marker provides resistance to an anti-microbial agent selected from the group consisting of: methicillin, vancomycin, cephalosporin, carbapenem, and oxyimino-cephalosporin / aztreonam resistance. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a gram-positive bacterium or a fungus. In some embodiments, the microbe infecting the subject or the carrier microbe is a microbe listedin Table 1. In some embodiments, the microbe infecting the subject or the carrier microbe harbors at last two AMR genetic markers. In some embodiments, the antimicrobial resistance is phenotypic antimicrobial resistance. In some embodiments, the sequencing comprises nextgeneration sequencing or a sequencing method beyond next generation sequencing. In some embodiments, the sequencing comprises sequencing by synthesis. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, the subject has an infection by the microbe infecting the subject harboring the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise administering an anti- infective agent to the subject. In some embodiments, the subject has been treated with an anti- infective agent for an infection. In some embodiments, the methods disclosed herein further comprise adjusting the anti -microbial agent received by the subject at least in part based on the antimicrobial resistance of the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of a microbe. In some embodiments, the microbe comprises a microbe infecting the subject. In some embodiments, the microbe comprises a carrier microbe. In some embodiments, the methods disclosed herein comprise determining whether an AMR gene is carried by a microbe infecting the subject or by a carrier microbe. In some embodiments, the determining comprises linking an AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the determining comprises determining a copy number of the AMR genetic marker in the microbe. In some embodiments, the linking comprises performing a statistical analysis on the sequence reads. In some embodiments, the statistical analysis comprises a generalized linear model, a maximum-likelihood estimation, a probit model, a logistic regression, a linear probability, a linear regression, a complimentary log-log, a Poisson regression, a support vector machine, a decision tree, a random forest, a neural network, a gradient boosted model, a Bayesian model, a hidden Markov model, or any combination thereof. In some embodiments, the method further comprises comparing the sequence read data to the amplification data. In some embodiments, the method further comprises calculating estimated deduplicated templates (EDT) for an AMR gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the method further comprises calculating an EDT for a housekeeping gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the methods disclosed herein further comprise determining a presence of an organism in a sample by calculating a ratio of an AMR estimated deduplicated templates (EDT) to a housekeeping gene EDT.

[0014] Disclosed herein are methods and compositions for determining antimicrobial resistance of a microbe infecting a subject, comprising: providing a first aliquot of the sample comprising first nucleic acids, wherein the first nucleic acids comprise microbial nucleic acids derived from a potential carrier microbe harboring an antimicrobial resistance (AMR) genetic marker; detecting the potential carrier microbe by analyzing the first nucleic acids; after the potential carrier microbe is detected, providing a second aliquot of the sample comprising second nucleic acids, wherein the second nucleic acids comprise microbial nucleic acids from the potential carrier microbe harboring an AMR genetic marker; conducting an amplification reaction on the second nucleic acids to detect the AMR genetic marker; and determining the antimicrobial resistance of the microbe infecting the subject based on the AMR genetic marker detection. In some embodiments, the methods disclosed herein further comprise performing a high-throughput sequencing assay on the amplicons associated with the second target nucleic acid. In some embodiments, the second target nucleic acid is not detected by a sequencing assay. In some embodiments, the first nucleic acid is associated with a genome of an organism but not a phenotype of interest of the organism and the second nucleic acid is associated with the phenotype of interest of the organism. In some embodiments, the first target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 first target nucleic acids; or the second target nucleic acid comprises at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or at least 25 second target nucleic acids. In some embodiments, the first target nucleic acid or the second target nucleic acid are not negative or positive controls for any step of the multi-step assay. In some embodiments, the primers comprise multiple primers targeting multiple target nucleic acids. In some embodiments, the first target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the second target nucleic acid comprises a cancer marker. In some embodiments, the first target nucleic acid is associated with a carrier microbe harboring a target genetic marker and the second target nucleic acid comprises a sequence associated with the target genetic marker. In some embodiments, the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the target genetic marker comprises an antimicrobial resistance (AMR) genetic marker. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the AMR genetic marker to a threshold value. In some embodiments, the methods disclosed herein further comprise comparing an abundance of the microbial sequence reads from the gene cassette to a threshold value. In some embodiments, the methods disclosed herein furthercomprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the AMR genetic marker is below the threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the gene cassette is below the threshold. In some embodiments, the methods disclosed herein further comprise quantifying mcfNA sequencing reads from the one or more microbes. In some embodiments, the methods disclosed herein further comprise comparing the abundance of the mcfNA sequencing reads from the one or more microbes to a threshold. In some embodiments, the methods disclosed herein further comprise providing a second aliquot for the amplification reaction when the abundance of the mcfNA sequencing reads from the one or more microbes is below the threshold. In some embodiments, the methods disclosed herein further comprise performing high-throughput sequencing on the amplicons associated with the AMR genetic marker. In some embodiments, the sequencing assay on the first nucleic acids comprises a high- throughput sequencing assay. In some embodiments, the methods disclosed herein further comprise conducting a polymerase chain reaction (PCR) to amplify the AMR genetic marker, thereby producing amplicons associated with the AMR genetic marker. In some embodiments, the PCR comprises multiplex PCR, random PCR (rPCR), non-biased PCR, Nested PCR, Hot Start PCR, or Assembly PCR. In some embodiments, the methods disclosed herein further comprise attaching an adapter sequence to the second nucleic acids. In some embodiments, the primers comprise an adapter sequence. In some embodiments, the methods disclosed herein further comprise physically manipulating the sample to produce a fraction of cfNA enriched for degraded cfNA, wherein the fraction of cfNA comprises the AMR genetic marker. In some embodiments, the degraded cfNA comprises ultra short cfNA, single stranded cfNA, or nicked double stranded cfNA. In some embodiments, the ultra short cfNA comprises cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the ultra short cfNA comprises cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, the methods disclosed herein further comprise performing size selection of the nucleic acids in the sample. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, performing size selection using a method selected from the group consisting of: chromatography, size-exclusion chromatography, electrophoresis, gel electrophoresis, automated electrophoresis, capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation, filtration, membrane ultrafiltration, magnetic beads, and affinity-based beads. In some embodiments, the second aliquot comprises at least 500 pl of plasma. In some embodiments, detecting the carrier microbe comprises determining an abundance of the carrier microbe. Insome embodiments, the abundance is expressed as molecules of mcfNA per microliter of sample (MPM). In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number from the amplification or sequencing of the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to the carrier microbe using the abundance of the mcfNA from the carrier microbe and the AMR gene copy number. In some embodiments, the AMR gene copy number is an episomal gene copy number. In some embodiments, the methods disclosed herein comprise introducing at least 200 primers targeting the plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of the microbe infecting the subject. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample is not subjected to a process that primarily causes cell lysis. In some embodiments, the methods disclosed herein further comprise preparing a library from the amplicons associated with the AMR genetic marker. In some embodiments, the primers are added directly to the second aliquot. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, the second nucleic acids comprise microbial cell-free nucleic acids. In some embodiments, intact microbes are not actively lysed prior to performing the sequencing assay. In some embodiments, the microbial nucleic acids comprise microbial cell-free nucleic acids (mcfNA) from the microbe. In some embodiments, the microbial nucleic acids comprise nucleic acids associated with a microbial cell. In some embodiments, the methods disclosed herein comprise enriching for at least at least 75%, at least 80%, at least 85%, or at least 90% of the mcfNA in the sample. In some embodiments, the degraded cfNA comprise ultra short cfNA, single stranded cfNA, nicked double stranded cfNA, or any combination thereof. In some embodiments, the degraded cfNA comprise cfNA fragments that are less than 100 nucleotides in length. In some embodiments, the degraded cfNA comprise cfNA fragments that are from 30 nucleotides to 70 nucleotides in length. In some embodiments, physically manipulating the sample comprises performing size selection of the nucleic acids in the sample. In some embodiments, the methods disclosed herein further comprise generating sequence reads from the cfNA from the subject, wherein the sequence reads comprise microbial sequencing reads derived from the microbe infecting the subject. In some embodiments, the sequence reads further comprise microbial sequencing reads from one or more carrier microbe. In some embodiments,the methods disclosed herein further comprise calculating an abundance of the mcfNA from the microbe in the sample and an abundance of the mcfNA from the one or more carrier microbe. In some embodiments, the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample. In some embodiments, the methods disclosed herein further comprise calculating an AMR gene copy number for each of the one or more carrier microbe. In some embodiments, the methods disclosed herein further comprise detecting one or more carrier microbe from the sequencing assay. In some embodiments, the methods disclosed herein further comprise identifying the one or more carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise linking the AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise obtaining an abundance of the one or more carrier microbes based on an abundance of carrier microbe sequences in the sample. In some embodiments, conducting a statistical analysis comprises using the abundance of the microbial nucleic acids of the one or more potential carrier microbes. In some embodiments, conducting a statistical analysis comprises using the AMR gene copy number. In some embodiments, the methods disclosed herein further comprise calculating a probability of each of the one or more potential carrier microbes being the microbe harboring the AMR genetic marker, thereby identifying the potential carrier microbe as the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining the antimicrobial resistance of the microbe infecting the subject based on the calculated probability. In some embodiments, the detecting comprises performing a sequencing assay. In some embodiments, the sequencing assay comprises a high-throughput sequencing assay. In some embodiments, the amplification reaction comprises introducing primers targeting a plurality of AMR genetic markers. In some embodiments, the amplification reaction produces amplicons associated with the AMR genetic marker. In some embodiments, the sample comprises plasma. In some embodiments, the first nucleic acids are DNA. In some embodiments, the second nucleic acids are DNA. In some embodiments, the sample does not undergo cell lysis. In some embodiments, intact microbes are not actively lysed. In some embodiments, the amplicons associated with the AMR genetic marker undergoes direct library preparation. In some embodiments, the sample comprises cell-free nucleic acids. In some embodiments, the sample comprises cell-free DNA. In some embodiments, the first nucleic acids comprise cell-free nucleic acids. In some embodiments, the second nucleic acids comprise cell-free nucleic acids. In some embodiments, the methods disclosed herein comprise adding primers targeting a plurality of AMR genetic markers into the second aliquot of the sample. In some embodiments, the methods disclosed herein comprise adding at least 200 primers targeting a plurality of AMR genetic markers intothe second aliquot of the sample. In some embodiments, the methods disclosed herein further comprise introducing primers targeting housekeeping genes into the second aliquot of the sample. In some embodiments, the second aliquot of the sample comprises at least 500 pl of plasma. In some embodiments, detecting the pathogen comprises detecting the abundance of mcfNA from the pathogen in the sample over a threshold. In some embodiments, the methods disclosed herein further comprise calculating a positive percent agreement (PPA), negative percent agreement (NPA), diagnostic yield (DY), or any combination thereof. In some embodiments, the methods disclosed herein further comprise detecting cfDNA from one or more housekeeping genes. In some embodiments, the methods disclosed herein further comprise spiking one or more control molecules into the sample at a known concentration. In some embodiments, the one or more control molecules are synthetic oligonucleotides. In some embodiments, the control molecules comprise whole assay internal control (WINC) molecules. In some embodiments, the methods disclosed herein further comprise spiking at least 25,000 unique WINC molecules at known concentrations. In some embodiments, the methods disclosed herein further comprise generating a report listing the carrier microbes or pathogen detected in the subject. In some embodiments, the report further comprises the abundance of microbial cell-free DNA (mcfDNA) from microbes detected in the sample or the antimicrobial resistance of the microbes infecting the subject. In some embodiments, the sample comprises a biological sample obtained from the subject. In some embodiments, the biological sample is a whole blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a synovial fluid sample, a urine sample, a stool sample, a bronchoalveolar lavage sample, or any combination thereof. In some embodiments, the sample is a plasma sample. In some embodiments, the AMR genetic marker comprises a gene, a genetic element, a genetic cassette, an allele, a mutation, or any combination thereof. In some embodiments, the AMR genetic marker is associated with one or more genes selected from the group consisting of: SCCmec, mecA, mecC, vanA, vanB, blacrx-M, blctKPC, OXA-48-like, OXA-23, NDM, VIM, IMP, or mcr-1. In some embodiments, the AMR genetic marker provides resistance to an anti-microbial agent selected from the group consisting of: methicillin, vancomycin, cephalosporin, carbapenem, and oxyimino-cephalosporin / aztreonam resistance. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the microbe infecting the subject or the carrier microbe comprises a gram-positive bacterium or a fungus. In some embodiments, the microbe infecting the subject or the carrier microbe is a microbe listed in Table 1. In some embodiments, the microbe infecting the subject or the carrier microbe harbors at last two AMR genetic markers. In some embodiments, the antimicrobial resistance is phenotypic antimicrobial resistance. In some embodiments, the sequencing comprises next-generation sequencing or a sequencing method beyond next generation sequencing. In some embodiments, the sequencing comprises sequencing by synthesis. In some embodiments, the subject is an animal. In some embodiments, the subject is a human. In some embodiments, the subject has an infection by the microbe infecting the subject harboring the AMR genetic marker. In some embodiments, the methods disclosed herein further comprise administering an anti- infective agent to the subject. In some embodiments, the subject has been treated with an anti- infective agent for an infection. In some embodiments, the methods disclosed herein further comprise adjusting the anti -microbial agent received by the subject at least in part based on the antimicrobial resistance of the microbe infecting the subject. In some embodiments, the methods disclosed herein further comprise determining antimicrobial resistance of a microbe. In some embodiments, the microbe comprises a microbe infecting the subject. In some embodiments, the microbe comprises a carrier microbe. In some embodiments, the methods disclosed herein comprise determining whether an AMR gene is carried by a microbe infecting the subject or by a carrier microbe. In some embodiments, the determining comprises linking an AMR genetic marker to one of the carrier microbes or the microbe infecting the subject. In some embodiments, the determining comprises determining a copy number of the AMR genetic marker in the microbe. In some embodiments, the linking comprises performing a statistical analysis on the sequence reads. In some embodiments, the statistical analysis comprises a generalized linear model, a maximum-likelihood estimation, a probit model, a logistic regression, a linear probability, a linear regression, a complimentary log-log, a Poisson regression, a support vector machine, a decision tree, a random forest, a neural network, a gradient boosted model, a Bayesian model, a hidden Markov model, or any combination thereof. In some embodiments, the method further comprises comparing the sequence read data to the amplification data. In some embodiments, the method further comprises calculating estimated deduplicated templates (EDT) for an AMR gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the method further comprises calculating an EDT for a housekeeping gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library. In some embodiments, the methods disclosed herein further comprise determining a presence of an organism in a sample by calculating a ratio of an AMR estimated deduplicated templates (EDT) to a housekeeping gene EDT.

[0015] In some embodiments, the method further comprises administering an alternative therapy to the subject if an AMR marker is detected. In some embodiments, the method further comprises detecting methicillin-resistant Staph aureus by detecting SCCmec, mecA, or mecC. In some embodiments, the method further comprises detecting methicillin-resistantStaph aureus and administering ceftaroline, ceftobiprole, vancomycin, linezolid, daptomycin, ceftobipole or combination thereof to the subject in order to treat the methicillin-resistant staph aureus. In some embodiments, the method further comprises detecting vancomycin resistant enterococcus by detecting VanA or VanB. In some embodiments, the method further comprises detecting vancomycin resistant enterococcus and administering linezolid or daptomycin to the subject in order to treat the vancomycin resistant enterococcus.

[0016] Described herein in some embodiments is a method to detect phenotypic antimicrobial susceptibility of a human host comprising: a) providing a sample comprising cell-free nucleic acids from a pathogen harboring at least one antimicrobial resistance (AMR) marker, wherein the cell-free nucleic acids from the pathogen are obtained from a biological sample of the human host; b) performing high-throughput sequencing of said cell-free nucleic acids from the pathogen to provide cell-free sequence reads; c) using said cell-free sequence reads from the pathogen in the biological sample to identify said at least one AMR marker. In one embodiment, the disclosed method further comprises adding adapters to said cell-free nucleic acids and producing a sequencing library comprising said cell-free nucleic acids. In one embodiment, the disclosed method further comprises enriching for ultra short, ultra rare target fragments in the biological sample prior to sequencing. In one embodiment, the disclosed method further comprises spiking whole assay internal control (WINC) molecules into each sample at a known concentration as a control for sequencing depth or human background levels, wherein the control molecules are synthetic oligonucleotides. In one embodiment, the control molecules comprise a minimum of 25,000 unique WINC molecules. In one embodiment of the disclosed method, the AMR marker provides methicillin resistance. In one embodiment of the disclosed method, the AMR marker provides vancomycin resistance. In one embodiment of the disclosed method, the AMR marker provides cephalosporin resistance. In one embodiment of the disclosed method, the AMR marker provides oxyimino-cephalosporin and aztreonam resistance. In one embodiment of the disclosed method, the AMR marker provides carbapenem resistance. In one embodiment of the disclosed method, the AMR markers include SCCmec, mecA and mecC for methicillin resistance, vanA and vanB for vancomycin resistance, blaCTX-M for oxyimino-cephalosporin and aztreonam resistance, and blaKPC for carbapenem resistance. In one embodiment of the disclosed method, the human host is a patient having an infection by the pathogen harboring at least one AMR marker. In one embodiment, the disclosed method further comprises adjusting the antibiotic treatment of the patient to compensate of the presence of the AMR in the pathogen. In one embodiment of the disclosed method, the biological sample is selected from the group consisting of blood, serum, cerebrospinal fluid, synovial fluid, urine, and stool. In one embodiment of thedisclosed method, the sequence analysis comprises whole-genome sequencing (WGS). In one embodiment of the disclosed method, the pathogen comprises a target bacteria listed in Table 1.INCORPORATION BY REFERENCE

[0017] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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 will be 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 of which:

[0019] FIG. 1 depicts an overview of the Karius Test AMR workflow.

[0020] FIG. 2 shows blaCTX-M variant prevalence (%) taken from CARD Resistomes database. Dark grey represents CTX-M variants with >95% sequence identity to the reference CTX-M sequence used in primer design and light gray represents CTX-M variants with <75% sequence identity.

[0021] FIG. 3. Distribution of index correct read counts across 108 clinical samples.

[0022] FIG. 4. Distribution of EDTs in negative control samples for each AMR target gene.

[0023] FIGs. 5A-5B. Probit fits for estimating CTX-M presence LoD using the (FIG. 5A) original dilution series and (FIG. 5B) simulated dilution series assuming one CTX-M gene copy. Light grey lines reflect the probit model fit and dark grey lines reflect the observed data of fraction positive calls per concentration.

[0024] FIG. 6 shows a computer control system that is programmed or otherwise configured to implement the methods provided herein.

[0025] FIG. 7 shows a table of clinical validation performance summary.DETAILED DESCRIPTION

[0026] The following passages describe different aspects of the disclosure in greater detail. Each aspect, embodiment, or feature of the disclosure can be combined with any other aspect, embodiment, or feature of the disclosure unless clearly indicated to the contrary. There remains a need for developing improved methods for identifying genetic markers in the context of disease diagnosis and treatment.Overview

[0027] The present disclosure provides, in some embodiments, methods of performing a plurality of assays in order to detect whether a subject is infected with a microbe that contains an antimicrobial resistance (AMR) genetic marker. The methods provided herein are particularly useful for determining whether a detected microbe carries an AMR genetic marker. In some embodiments, the assays are performed sequentially. For example, in some embodiments the methods comprise performing unbiased high-throughput sequencing (HTS) to detect a potential carrier microbe, followed by performing targeted sequencing in order to detect an AMR genetic marker in a sample. In some cases, the method comprises performing a targeted sequencing assay to detect both a carrier microbe and its associated AMR genetic marker, if present. The methods provided herein, in some embodiments, can comprise performing a statistical analysis to link a carrier microbe to a particular AMR genetic marker. The statistical analysis can be particularly helpful when nucleic acids from multiple different carrier microbes are detected, which can make it difficult to determine which carrier microbe comprises a detected AMR genetic marker.

[0028] The methods provided herein, in some embodiments, comprise performing a multi-step assay for detecting an AMR genetic marker by analyzing at least two aliquots of the same sample in at least two sequential assays. In some embodiments, the first assay is a high throughput sequencing assay, and the second assay is a targeted PCR amplification assay, that can, in some cases, be followed by a second high throughput sequencing assay. In some embodiments, carrier microbes and AMR genetic markers are detected in the same assay. In some embodiments, the carrier microbes are detected using primers specific for housekeeping genes and the AMR markers are detected using primers specific for sets of AMR genes. In some embodiments, targeted amplification for multiple targets is performed, followed by, in some embodiments, performing a statistical analysis to link detected carrier microbes with AMR genetic markers.

[0029] The present disclosure provides, in some embodiments, methods of detecting an antimicrobial resistance (AMR) genetic marker by analyzing cell-free nucleic acids in a biological sample from a patient using high-throughput sequencing and / or targeted sequencing in order to quantify or detect an abundance of a carrier microbe and / or an AMR genetic marker. In some embodiments, the methods provided herein enable AMR marker detection by identifying one or more potential carrier microbes and one or more AMR genetic markers, and conducting a statistical analysis to link the AMR genetic marker to the carrier microbe harboring the AMR genetic marker. In some embodiments, the methods comprise calculating an abundance of mcfNA (e.g., expressed as molecules per microliter (MPM)) from the one or more potentialcarrier microbes and / or calculating an abundance or copy number of mcfNA associated with the AMR genetic marker. In some embodiments, the methods comprise calculating a copy number of the AMR genetic marker and / or calculating abundances of one or more microbial housekeeping genes. In some embodiments, the methods comprise linking the AMR genetic marker to its carrier microbe based on the abundance of the mcfNA associated with the carrier and the copy number of the AMR genetic marker. In some embodiments, the methods comprise linking the AMR genetic marker to its carrier microbe based on the copy number of both the AMR genetic marker and the one or more microbe-specific housekeeping genes. In some embodiments, the method comprises analyzing certain features or criteria to determine that a carrier microbe harbors the AMR genetic marker. In some embodiments, such criteria can comprise a copy number of the AMR marker, the size of the AMR marker, the size of a gene cassette comprising the AMR marker, an abundance of mcfNA associated with the AMR marker, an abundance of mcfNA associated with the carrier microbe, or an abundance of mcfNA associated with the AMR marker in comparison to an abundance of mcfNA associated with one or more microbial-specific housekeeping genes.

[0030] In some embodiments, the methods comprise performing an HTS assay such as a sequencing-by-synthesis assay in order to detect and / or quantify nucleic acids associated with the one or more potential carrier microbes. In some embodiments, a targeted assay is then performed to detect the AMR genetic marker, particularly if a carrier microbe was identified in the first high throughput sequencing assay. For example, in some embodiments, detection of a carrier microbe in the first high throughput sequencing assay triggers obtaining a second aliquot of the sample and performing a targeted assay to detect nucleic acids in the second aliquot associated with the AMR genetic marker. The targeted assay can, for example, comprise performing a PCR reaction with primers specific for one or more AMR genetic markers, optionally followed by high throughput sequencing to detect the AMR genetic markers. In some embodiments, the methods comprise calculating abundances of microbial nucleic acids detected in the targeted assay, such as an estimated deduplicated transcripts or templates (EDT) or a copy number. In some embodiments, the methods comprise calculating an EDT of one or more AMR genetic markers, a copy number of the one or more AMR genetic markers, or an EDT of one or more microbial-specific housekeeping genes. In some embodiments, the methods comprise linking an AMR genetic marker to its carrier microbe based on the results from the targeted assay. In some embodiments, the methods comprise linking an AMR genetic marker to its carrier microbe using the copy number of the AMR genetic marker and the abundances of the one or more microbial-specific housekeeping genes.

[0031] In some embodiments, criteria associated with an HTS assay that detected a carrier microbe or an AMR gene cassette are analyzed in order to determine whether a subsequent assay such as a targeted sequencing assay is performed. In some cases, the methods comprise comparing the amount or abundance of mcfNA (e.g., MPM) associated with a carrier microbe, an AMR gene cassette, and / or an AMR gene with a threshold value. In some embodiments, the methods comprise proceeding to the targeted assay for the AMR genetic marker based on the comparison with the threshold value. In some instances, the methods comprise the use of primers targeting the AMR genetic marker for the targeted amplification of the AMR genetic marker, which is optionally followed by high throughput sequencing to detect the AMR genetic marker.

[0032] In some embodiments, a nucleic acid fraction enriched for ultra-short, degraded, nicked double-stranded, and / or single- stranded cfNA is used in the assays provided herein. In some cases, the methods comprise an extraction step. In some cases, the cfNA are amplified within a sample prior to extraction. In some cases, adapters or primers for the cfNA are added directly to the sample prior to extraction.

[0033] The high-throughput nature of the methods provided herein generally enables comprehensive profiling of the subject’s microbiome, infectome, and / or resistome. For example, the methods provided herein can further detect co-infections in a patient with complex infections. The methods can also comprise treating patients based on the identification of the AMR genetic markers, which can reduce or inform the use of broad-spectrum antibiotics and allow for a more targeted approach to treatment of infectious diseases. For example, the methods can comprise treating the patients who have received one class of antimicrobial treatment with a different class of antimicrobial treatments to overcome AMR.Genetic markers

[0034] Disclosed herein in some embodiments are methods for detecting one or more genetic markers associated with a disease or a disorder using high-throughput sequencing. In some embodiments, the one or more genetic markers are associated with an infectious disease. In some embodiments, the one or more genetic markers are associated with a non-communicable disease. In some embodiments, the one or more genetic markers comprise a genetic marker from a prokaryotic organism. In some embodiments, the one or more genetic markers comprise a genetic marker from a eukaryotic organism.

[0035] In some embodiments, the one or more genetic markers comprises a genetic locus, a gene, an allele, a genetic variant, a gene element (e.g., a regulatory element), a gene cassette, an operon, an integron, a resistance island, a resistance supercluster, a chromosome, a plasmid, or any combination thereof. In some embodiments, the one or more genetic markers comprise amutation associated with a disease or disorder. In some embodiments, the mutation comprises a point mutation (e.g., missense, nonsense, silent, frameshift), a structural mutation (e.g., insertion, deletion, duplication, inversion, translocation), repeat expansions (e.g., trinucleotide repeats, MSI), a gain-of-function mutation, a loss-of-function mutation, a dominant negative mutation, or any combination thereof.

[0036] In some embodiments, the genetic markers comprise a microbial genetic marker. In some embodiments, the microbial genetic marker is from a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae. In some embodiments, the microbial genetic marker comprises an antimicrobial resistance (AMR) marker. In some embodiments, the genetic markers comprise a mammalian genetic marker. In some embodiments, the mammalian genetic marker is from a human. In some embodiments, the genetic marker is associated with a human disease. In some embodiments, the genetic marker comprises a cancer marker.Antimicrobial Resistance (AMR) Markers

[0037] Disclosed herein in some embodiments are methods for detecting one or more antimicrobial resistance (AMR) genetic markers. An AMR genetic marker can be associated with one or more AMR genes that enable microorganisms to survive exposure to anti-microbial agents, leading to treatment failure and persistent infections.

[0038] The AMR genetic marker can be located anywhere in a microbial genome. In some embodiments, the AMR genetic marker is integrated in a microbial genome (e.g., chromosome), an episomal genome (e.g., plasmid), a transposon, or any combination thereof. In some embodiments, the AMR genetic marker is located on an episomal genome.

[0039] The AMR genetic marker can be of any size within a microbial genome. In some embodiments, the AMR genetic marker comprises a genetic locus, a gene, an allele, a genetic variant, a gene element (e.g., a regulatory element), a gene cassette, an operon, an integron, a resistance island, a resistance supercluster, a chromosome, a plasmid, or any combination thereof.

[0040] In some embodiments, the AMR genetic marker is associated with one or more AMR genes. In some embodiments, the AMR genetic marker is associated with at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 AMR genes.

[0041] In some embodiments, the AMR genetic marker is originated from one or more carrier microbes harboring the AMR genetic marker. In some embodiments, the AMR genetic marker is originated from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 AMR carrier microbes harboring the AMR genetic marker. For example,the mecA gene can be carried by multiple Staphylococcus species, such as S. aureus and S. epidermidis.

[0042] In some embodiments, the AMR genetic marker is linked to the one or more carrier microbes harboring the AMR genetic marker. In some embodiments, the AMR genetic marker is linked to the one or more carrier microbes detected by first sequencing assay in the method. In some embodiments, the methods detect a carrier microbe and then detect a phenotypic characteristic of the carrier microbe. In some cases, the phenotypic characteristic is antimicrobial resistance.

[0043] In some embodiments, the AMR genetic marker is associated with resistance to one or more anti-microbial or anti-infective agents. The AMR genetic marker can be associated with resistance to any class of anti-microbial agents. In some embodiments, the AMR genetic marker is associated with resistance to a P-Lactam drug (e.g., carbapenems, methicillin), oxyimino- cephalosporins / aztreonam, an aminoglycoside drug (e.g., gentamicin), a Macrolide- Lincosamide-Streptogramin (MLS) drug (e.g., clindamycin), a tetracycline drug (e.g., doxycycline), a fluoroquinolone drug (e.g., ciprofloxacin), a glycopeptide drug (e.g., vancomycin), a polymyxin drug (e.g., colistin), rifamycin (e.g., rifampin), or a sulfonamide drug (e.g., trimethoprim-sulfamethoxazole). In some embodiments, the AMR genetic marker is associated with resistance to a broad-spectrum anti-microbial drug. In some embodiments, the AMR genetic marker is associated with resistance to a narrow-spectrum anti-microbial drug.

[0044] In some embodiments, the AMR genetic marker is associated with or comprises one or more AMR genes selected from the group consisting of mecA, mecC, bla TEM, bla SHV, bla_CTX-M, bla KPC, blaJNDM, bla_OXA-48, bla_OXA-23, VIM, IMP aac(6')-Ib, aph(3')- Illa, ant(2")-Ia, erm(A), erm(B), erm(C), mef(A), mef(E), tet(A), tet(B), tet(M), gyrA / parC, qnrA, qnrB, qnrS, aac(6')-Ib-cr, vanA, vanB, mcr-1, pmrA / pmrB, rpoB, suit, sul2, sul3, dfrA genes, acrAB-TolC, mexAB-oprM., katG, inhA, rpoB, pncA, embB, rrs, gyrA, and gyrB.

[0045] In some embodiments, the AMR genetic marker is associated with a gene cassette. A gene cassette is a mobile genetic element that can harbor one or more AMR genes. In some embodiments, the gene cassette can be “empty” and does not harbor any AMR gene. In some embodiments, the gene cassette can integrate into a genome. In some embodiments, the gene cassette comprises SCCmec. SCCmec are mobile genetic elements that can integrate into coagulase-negative staphylococci species (e.g., Staphylococcus aureus). In some embodiments, the SCCmec can harbor one or more AMR genes. For example, SCCmec can harbor mecA / mecC in Staphylococcus aureus and additional AMR genes such as blaZ or aminoglycoside resistance genes. In some embodiments, SCCmec can be “empty” and does not harbor one or more AMRgenes. In some embodiments, SCCmec comprises Type I, Type II, Type III, Type IV, Type V or Type VIII SCCmec.

[0046] In some embodiments, a gene cassette comprises an integron. In some embodiments, an integron comprises a class 1 integron, a class 2 integron, or a class 2 integron.

[0047] In some embodiments, the AMR genetic markers can be present in a carrier microbe at any copy number. As used herein, “copy number” can refer to the number of times a microbial gene or genomic region (e.g., the AMR genetic marker) is present in a microbe. In some embodiments, the copy number of the AMR genetic marker comprises a chromosomal copy number, an integron copy number, or an episomal (or plasmid) copy number. In some embodiments, the copy number of the AMR genetic marker is 1. In some embodiments, the copy number of the AMR genetic marker is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10. In some embodiments, the copy number of the AMR genetic marker is calculated by PCR.Methods and Assay Workflow

[0048] The methods described herein can comprise performing a multi-step assay on a sample from a subject. In some embodiments, the multi-step assay comprises performing a first high- throughput sequencing (HTS) assay on nucleic acids from the sample followed by performing a targeted sequencing assay. In some instances, the targeted sequencing assay can comprise performing a PCR reaction on the sample, optionally followed by a second high-throughput sequencing assay. In some cases, performing a targeted sequencing assay is predicated on the results of a first HTS assay. In some embodiments, results from a first HTS assay may meet certain criteria before a targeted sequencing assay is performed. In some embodiments, after the targeted sequencing assay is performed, an analysis is performed to link the nucleic acids detected in the targeted sequencing assay with nucleic acids detected in the first high-throughput sequencing assay. In some cases, linking results from the two assays can provide phenotypic characteristics related to a genome identified in the first high throughput sequencing assay.

[0049] In some embodiments, the methods provided herein comprise performing a multi-step assay on a sample in order to detect a microbe of interest. In some instances, a first HTS assay is performed in order to detect a carrier microbe; in some embodiments, the first HTS is an unbiased sequencing assay. In some embodiments, a targeted sequencing assay is performed if the results of the first HTS assay meet certain criteria. In some cases, the targeted sequencing assay is performed if one or more microbes known to carry an AMR marker is detected in the first HTS. In some embodiments, if a carrier microbe or AMR cassette is identified in the first HTS, a targeted sequencing assay is performed in order to detect the AMR marker. If an AMR markeris detected, then, in some embodiments, a linkage analysis is performed in order to link the AMR marker with the carrier microbe detected in the first HTS. In some embodiments, both a carrier microbe and an AMR marker are detected in a targeted assay. In such cases, the methods may comprise performing an analysis in order to link the AMR marker with the carrier microbe detected in the targeted assay.

[0050] In some embodiments, the at least two sequencing assays can be the same sequencing assay. In some embodiments, the at least two sequencing assays can be different sequencing assays. In some embodiments, the at least two sequencing assays are performed in parallel. In some embodiments, the at least two sequencing assays are performed sequentially. In some embodiments, methods comprise performing a first high throughput sequencing assay before performing a targeted amplification or a second high throughput sequencing assay. In some embodiments, the second high throughput assay is a targeted sequencing assay. In some embodiments, only a targeted amplification or sequencing assay is performed. In some cases, an unbiased sequencing method is performed prior to the targeted assay.Sample Aliquots

[0051] In some embodiments, the methods comprise performing a high throughput sequencing assay or amplification assay on an aliquot of a sample. Generally, as used herein, an aliquot is a portion of a sample; but in some cases, the term aliquot as used herein refers to the entire sample. In some embodiments, the methods comprise obtaining multiple aliquots from a single sample. In some cases, the methods comprise performing an assay on a first sample and then performing an assay on a second sample that does not derive from the same sample as the first sample. In some embodiments, an aliquot is a remaining portion of the sample (or a fraction of the remaining portion of the sample), after an initial aliquot has been taken out of the sample. For example, a first aliquot can be taken out of the subject’s sample and analyzed by a first sequencing assay in the methods described herein. After that, in some embodiments, a second aliquot can be taken out of the subject’s sample and analyzed by a second sequencing assay (e.g., a targeted sequencing assay). In some cases, after the first aliquot is removed from the sample, the entire remaining sample is analyzed as the second aliquot in an amplification or a second sequencing assay. In some cases, after the first aliquot is removed from the sample, a portion or fraction of the remaining sample is analyzed as the second aliquot in the amplification or the second sequencing assay.

[0052] In some embodiments, assays within workflow analyze a different aliquot of the sample. In some embodiments, an aliquot of the sample can be any measured portion of the sample. In some cases, an aliquot is a portion of an original sample, such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% percent of the sample. In some cases, an aliquot is a portion of anoriginal sample, such as less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% percent of the sample. In some embodiments, the aliquot is at most 50 pl, at most 100 pl, at most 150 pl, at most 200 pl, at most 250 pl, at most 300 pl, at most 350 pl, at most 400 pl, at most 450 pl, at most 500 pl, at most 550 pl, at most 600 pl, at most 650 pl, at most 700 pl, at most 750 pl, at most 800 pl, at most 850 pl, at most 900 pl, at most 950 pl, or at most 1 ml of the sample. In some embodiments, the aliquot is 500 pl of a plasma sample obtained from a human. In some embodiments, the aliquot is 200 pl of the plasma sample.

[0053] In some embodiments, the methods comprise performing a first sequencing assay to analyze a first aliquot of the sample from the subject in order to detect first target nucleic acids, wherein the first aliquot comprises the first target nucleic acids. The methods described herein further comprise, in some embodiments, after detecting the first target nucleic acids, performing an amplification or a second sequencing assay to analyze a second aliquot of the sample in order to detect second target nucleic acids, wherein the second aliquot comprises the second target nucleic acids. In some embodiments, the first target nucleic acids and the second target nucleic acids comprise any one of the genetic markers provided herein. In some cases, the first target nucleic acids are associated with a carrier microbe or an AMR cassette; in some cases, the second target nucleic acids are AMR markers. In some embodiments, the first target nucleic acids and the second target nucleic acids comprise a genetic marker associated with an infection or a non- communicable disease or disorder. In some embodiments, the first target nucleic acids and the second target nucleic acids comprise the same genetic marker. In some embodiments, the first target nucleic acids and the second target nucleic acids comprise different genetic markers.HTS to Detect Carrier Microbes

[0054] In some embodiments, the methods comprise detecting a microbe using an HTS assay followed by performing a targeted sequencing assay. In some embodiments, an unbiased HTS is not performed prior to performing the targeted sequencing assay. In some embodiments, the methods comprise using unbiased HTS to detect a carrier microbe potentially harboring an AMR genetic marker. In some embodiments, the carrier microbe, but not the AMR marker, is detected by the unbiased HTS. In some embodiments, the methods comprise detecting two or more carrier microbes harboring an AMR genetic marker. In some embodiments, the methods comprise detecting at least three carrier microbes. In some embodiments, the methods comprise detecting at least 5, least 6, least 7, least 8, least 9, at least 10, least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 carrier microbes.

[0055] In some embodiments, the methods comprise detecting nucleic acids in the sequencing assay. In some embodiments, the methods comprise detecting microbial nucleic acids. In someembodiments, the methods comprise detecting microbial cell-free nucleic acids (mcfNA). In some embodiments, the methods comprise detecting mcfNA from one or more carrier microbes harboring an AMR genetic marker. In some embodiments, the methods comprise detecting mcfNA associated with or comprising an AMR genetic marker. In some embodiments, the methods comprise performing unbiased HTS to detect an AMR cassette (e.g., SCCmec cassette).

[0056] In some embodiments, the methods comprise generating nucleic acids sequence reads using the sequencing assay. In some embodiments, the methods comprise generating microbial nucleic acids sequence reads. In some embodiments, the methods comprise generating mcfNA sequence reads. In some embodiments, the methods comprise generating mcfNA sequence reads associated with one or more carrier microbes harboring an AMR genetic marker. In some embodiments, the methods comprise generating mcfNA sequence reads associated with or comprising an AMR genetic marker. In some embodiments, the methods comprise generating sequence reads associated with one or more housekeeping genes. In some embodiments, the methods comprise generating microbial sequence reads associated with one or more microbial- specific housekeeping genes. In some embodiments, the methods further comprise mapping nucleic acid sequence reads from the sequencing assay to a reference genome. In some embodiments, the methods further comprise mapping microbial nucleic acid sequence reads to one or more microbial genomes. In some embodiments, the methods further comprise mapping microbial nucleic acid sequence reads to an AMR reference sequence or AMR cassette reference sequence.Abundance

[0057] In some embodiments, the methods further comprise quantifying the nucleic acids detected in a sequencing assay, e.g., in an unbiased HTS or in a targeted sequencing assay. In some embodiments, quantifying the nucleic acids comprises calculating an abundance of the mcfNA detected in the sequencing assay. In some embodiments, the abundance of the mcfNA is an absolute quantity expressed in molecules per microliter (MPM). In some embodiments, the abundance of the mcfNA is an absolute quantity expressed in molecules per 100 nanoliters. In some embodiments, the methods further comprise calculating a copy number. In some embodiments, quantifying the nucleic acids comprises calculating an abundance of unique sequencing reads. In some embodiments, calculating an abundance of unique sequencing reads comprises estimating a copy number. In some embodiments, the abundance of the unique sequencing reads is a relative quantity expressed in estimated deduplicated reads (EDR). As used herein “estimated deduplicated reads” or “EDR” generally refers to the number of sequence reads after removing duplicate reads and then multiplying the total deduplicated reads by the estimated fraction of reads generated from each microbe.

[0058] In some embodiments, EDR is calculated per microbe. In some embodiments, calculating an EDR comprises determining an estimated fraction of reads. An estimated fraction of reads may be the calculated proportion of total sequencing reads that fall within a region of interest. The region of interest may be a genomic region. In some embodiments, the EDR is calculated for one or more microbes. In some embodiments, an estimated fraction of reads may be calculated by dividing the number of reads aligned to the region of interest by the total number of reads in the sequence data. In some embodiments, the EDR comprises identifying duplicates. In some embodiments, the EDR per microbe is calculated by removing duplicate reads and then multiplying the total deduplicated reads by the estimated fraction of reads generated from a microbe.

[0059] In some embodiments, quantifying the nucleic acids comprises calculating an abundance of one or more spike-in nucleic acids. In some embodiments, the spike-in nucleic acids are synthetic oligonucleotides directly added to the initial sample. In some embodiments, the synthetic spike-in nucleic acids comprise whole assay internal control (WINC). In some embodiments, the read depth of the region of interest may be normalized. Examples of normalization includes GC content normalization, mappability normalization, control genome normalization, Poisson latent factor models, and tangent normalization. In some embodiments, the copy number calculation comprises comparing the read depth of a genomic region of interest to a standard. A genomic region may be a window of the genome within a given range of continuous nucleotides between a start point and an end point. The range may be 100 bp, 200 bp, 300, bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kbp,10 kbp, 20 kbp, 30 kbp, 40 kbp, 50 kbp, 60 kbp, 70 kbp, 80 kbp, 90 kbp, 100 kbp, 1 mbp, 2 mbp, 3 mbp, 4 mbp, 5 mbp, or any combination thereof. In some embodiments, the standard has a known copy number. In some embodiments, the standard is the synthetic spike-in nucleic acids. In some embodiments, the standard is a selected region of the genome. In some embodiments, the genomic region of interest may be compared to the standard. Examples of comparisons include read depth (coverage) analysis, ration of normalized read counts, b-allele frequency analysis, control-FREEC (free copy number estimation from coverage), read depth per probe, hidden Markov model analysis, or sequencing depth windowing.Detection of AMR Cassettes and Markers

[0060] In some embodiments, the methods comprise detecting a gene cassette (such as a gene cassette described herein) using a sequencing assay. In some cases, a gene cassette (e.g., AMR cassette) is detected by an unbiased HTS; in some cases, the gene cassette is detected by a targeted sequencing assay. In some embodiments, the methods comprise detecting at least one gene cassette. In some embodiments, the methods detect two or more gene cassettes. In someembodiments, the methods detect at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 gene cassettes. In some embodiments, the methods comprise detecting a gene cassette harboring at least one AMR genetic marker. In some embodiments, the methods comprise detecting an empty gene cassette that does not harbor any AMR genetic marker. In some embodiments, the methods comprise detecting gene cassettes from one or more carrier microbes harboring at least one AMR genetic marker. In some embodiments, the methods comprise detecting gene cassettes from the same carrier microbe. In some embodiments, the methods comprise detecting an integrated gene cassette. In some embodiments, the methods comprise detecting an episomal gene cassette.

[0061] In some embodiments, the methods described herein comprise detecting an AMR genetic marker using a sequencing assay. In some embodiments, the methods comprise detecting two or more AMR genetic markers. In some embodiments, the methods comprise detecting at least three AMR genetic markers. In some embodiments, the methods comprise detecting at least 5, at least 10, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, at least 2000, at least 2100, at least 2200, at least 2300, at least 2400, at least 2500, at least 2600, at least 2700, at least 2800, at least 2900, or at least 3000 AMR genetic markers. In some embodiments, the methods comprise detecting AMR genetic markers from one or more carrier microbes harboring at least one AMR genetic marker. In some embodiments, the methods comprise detecting AMR genetic markers detected are from the same carrier microbe. In some embodiments, the methods comprise detecting an integrated AMR genetic marker. In some embodiments, the methods comprise detecting an episomal AMR genetic marker.

[0062] In some embodiments, the methods provided herein comprise detecting a carrier microbe harboring a first genetic marker in a sample using a first sequencing assay and then detecting a second genetic marker in the same sample using a second sequencing assay. In some embodiments, the first genetic marker and the second genetic marker are associated with the same phenotype.

[0063] In some embodiments, the methods comprise 1) detecting a carrier microbe harboring an AMR genetic marker in a first sequencing assay, wherein the first sequencing assay is performed on a first aliquot of a sample; and then 2) detecting the AMR genetic marker in asecond sequencing assay, wherein the second sequencing assay is performed on a second aliquot of a sample. In some embodiments, the methods comprise 1) detecting an AMR genetic cassette in the first sequencing assay, wherein the first sequencing assay is performed on a first aliquot of a sample; and then 2) detecting the AMR genetic marker in a second sequencing assay; wherein the second sequencing assay is performed on a second aliquot of a sample. In some embodiments, the methods comprise 1) detecting a carrier microbe and not detecting an AMR genetic marker in the first sequencing assay, wherein the first sequencing assay is performed on a first aliquot of a sample; and then 2) detecting the AMR genetic marker in a second sequencing assay; wherein the second sequencing assay is performed on a second aliquot of a sample. In some embodiments, the methods comprise 1) detecting a carrier microbe and not detecting an AMR gene cassette in the first sequencing assay, wherein the first sequencing assay is performed on a first aliquot of a sample; and then 2) detecting the AMR genetic marker in a second sequencing assay; wherein the second sequencing assay is performed on a second aliquot of a sample. In some embodiments, the methods further comprise providing sufficient amount of the second aliquot for the second sequencing assay. In some embodiments, the second aliquot is the entire remaining sample after the first aliquot has been removed from the sample.

[0064] In some embodiments, the methods comprise detecting an AMR genetic marker harbored by a microbe selected from the group consisting of: Staphylococcus aureus, S. epidermidis, S. lugdunensis, Enterococcus faecalis, E. faecium, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, K pneumoniae, K oxytoca, Proteus mirabilis, P. vulgaris, Salmonella bongori, S. enterica, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter baumannii, and A. calcoaceticus in the second sequencing assay. In some embodiments, the methods comprise performing a second sequencing assay after the first sequencing assay detects a microbe selected from the group consisting of: Staphylococcus aureus, S. epidermidis, S. lugdunensis, Enterococcus faecalis, E. faecium, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, K pneumoniae, K oxytoca, Proteus mirabilis, P. vulgaris, Salmonella bongori, S. enterica, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter baumannii, and A. calcoaceticus.Determinins whether to Perform a Second Sequencing Assay

[0065] In some embodiments, performing a second sequencing assay (e.g., a targeted sequencing assay) is predicated on the first sequencing assay meeting certain criteria or filters. In some embodiments, satisfaction of the criteria can mean that there will be a greater likelihood of detecting an AMR target in the second sequencing assay or of assigning linkage of the AMR marker to the carrier microbe based on the second assay. In some embodiments, the criteria comprise taxa of the potential carrier microbe detected in the first sequencing assay, abundanceof the potential carrier microbe, presence or amount of confounding microbes, presence or absence of an AMR cassette (e.g., SCCmec), indeterminant results, abundance statistically significant above background, microbial sequencing reads spread uniformly across a genome, abundance above a level that could be explained by sequence misattribution from another high abundance microbe, abundance above background expected in healthy samples, or any combination thereof. In some embodiments, criteria that are met before performing a second sequencing assay comprise (1) presence of a microbe known to harbor an AMR marker higher than a threshold value (e.g., a threshold value provided herein) and AMR cassette presence is “indeterminate”; (2) presence of a microbe known to harbor an AMR marker lower than a threshold value (e.g., a threshold value provided herein) and with confounding microbes present below a threshold value (e.g., a threshold value provided herein), and AMR cassette presence indeterminate; or (3) gram negative microbes greater than a threshold value (e.g., a threshold value provided herein).

[0066] In some embodiments, the methods comprise performing a first sequencing assay on a first aliquot of a sample and based on the results of the first sequencing assay, and determining whether to perform the second sequencing assay on a second aliquot of the sample. In some embodiments, the methods comprise 1) detecting nucleic acids from a potential carrier microbe harboring an AMR genetic marker in a first sequencing assay, wherein the first sequencing assay is performed on a first aliquot of a sample; 2) quantifying the nucleic acids from the potential carrier microbe in the first aliquot; 3) determining the likelihood that the potential carrier microbe comprises the AMR genetic marker; and 4) determining to perform an amplification or a second sequencing assay on a second aliquot of the sample to detect the AMR genetic marker. In some embodiments, determining the likelihood in 3) comprises quantifying microbial sequence reads associated with the potential carrier microbe. In some embodiments, determining the likelihood in 3) comprises quantifying sequence reads associated with the AMR genetic marker. In some embodiments, determining the likelihood in 3) comprises comparing the sequence reads associated with the AMR genetic marker to the microbial sequence reads associated with the potential carrier microbe. In some embodiments, determining the likelihood in 3) comprises using a statistical model.

[0067] In some embodiments, detecting an abundance of a microbe in a first sequencing assay that exceeds an abundance threshold value determines whether a second sequencing assay is performed. In some embodiments, the threshold abundance value is at least about 50 MPM, at least about 100 MPM, at least about 150 MPM, at least about 200 MPM, at least about 300 MPM, at least about 400 MPM, at least about 500 MPM, at least about 600 MPM, at least about 700 MPM, at least about 800 MPM, at least about 900 MPM, at least about 1000 MPM, at least about1100 MPM, at least about 1200 MPM, at least about 1300 MPM, at least about 1400 MPM, at least about 1500 MPM, at least about 1600 MPM, at least about 1700 MPM, at least about 1800 MPM, at least about 1900 MPM, at least about 2000 MPM, at least about 2500 MPM, at least about 3000 MPM, at least about 3500 MPM, at least about 4000 MPM, at least about 4500 MPM, at least about 5000 MPM, at least about 5500 MPM, at least about 6000 MPM, at least about 6500 MPM, at least about 7000 MPM, at least about 7500 MPM, at least about 8000 MPM, at least about 8500 MPM, at least about 9000 MPM, at least about 9500 MPM, or at least about 10,000 MPM.

[0068] In some embodiments, the threshold abundance value for gram-positive bacteria is at least about 50 MPM, at least about 100 MPM, at least about 150 MPM, at least about 200 MPM, at least about 300 MPM, at least about 400 MPM, at least about 500 MPM, at least about 600 MPM, at least about 700 MPM, at least about 800 MPM, at least about 900 MPM, at least about 1000 MPM, at least about 1100 MPM, at least about 1200 MPM, at least about 1300 MPM, at least about 1400 MPM, at least about 1500 MPM, at least about 1600 MPM, at least about 1700 MPM, at least about 1800 MPM, at least about 1900 MPM, at least about 2000 MPM, at least about 2500 MPM, at least about 3000 MPM, at least about 3500 MPM, at least about 4000 MPM, at least about 4500 MPM, at least about 5000 MPM, at least about 5500 MPM, at least about 6000 MPM, at least about 6500 MPM, at least about 7000 MPM, at least about 7500 MPM, at least about 8000 MPM, at least about 8500 MPM, at least about 9000 MPM, at least about 9500 MPM, or at least about 10,000 MPM.

[0069] In some embodiments, the abundance threshold value for gram-negative bacteria is at least about 4500 MPM, at least about 5000 MPM, at least about 5500 MPM, at least about 6000 MPM, at least about 6500 MPM, at least about 7000 MPM, at least about 7500 MPM, at least about 8000 MPM, at least about 8500 MPM, at least about 9000 MPM, at least about 9500 MPM, at least about 10,000 MPM, at least about 15,000 MPM, at least about 20,000 MPM, at least about 25,000 MPM, at least about 30,000 MPM, at least about 35,000 MPM, at least about 40,000 MPM, at least about 45,000 MPM, or at least about 50,000 MPM. In some embodiments, the threshold for a carrier microbe harboring / CTX I or / KPC is at least about 5000 MPM, at least about 5500 MPM, at least about 6000 MPM, at least about 6500 MPM, at least about 7000 MPM, at least about 7500 MPM, at least about 8000 MPM, at least about 8500 MPM, at least about 9000 MPM, at least about 9500 MPM, at least about 10,000 MPM, at least about 15,000 MPM, or at least about 20,000 MPM.

[0070] In some embodiments, the abundance threshold value is from 50 MPM to 20,000 MPM, from 50 MPM to 10,000 MPM, from 50 MPM to 9,500 MPM, from 50 MPM to 5,000 MPM, from 100 MPM to 10,000 MPM, from 200 MPM to 8,000 MPM, from 300 MPM to 7,000 MPM,from 400 MPM to 6,000 MPM, from 500 MPM to 5,000 MPM, from 1,000 MPM to 4,000 MPM, from 1,500 MPM to 3,000 MPM, from 2,000 MPM to 2,500 MPM, or from 2,500 MPM to 2,000 MPM.

[0071] In some embodiments, the methods provided herein comprise detecting one or more carrier microbes harboring an AMR genetic marker using a first sequencing assay on a first aliquot of a sample form a subject, and then based on the results from the first sequencing assay, determining to perform an amplification assay or a second sequencing assay on a second aliquot of the same sample. In some embodiments, the methods comprise, after the first sequencing assay on the first aliquot of sample, providing a second aliquot of sample with sufficient amount of cfNA for the amplification assay or the second sequencing assay. In some embodiments, the second aliquot of sample comprises at least 0.05 ng, at least 0.10 ng, at least 0.15 ng, at least 0.20 ng, at least 0.25 ng, at least 0.30 ng, at least 0.35 ng, at least 0.40 ng, at least 0.45 ng, at least 0.50 ng, at least 0.55 ng, at least 0.60 ng, at least 0.65 ng, at least 0.70 ng, at least 0.75 ng, at least0.80 ng, at least 0.85 ng, at least 0.90 ng, at least 0.95 ng, at least 1.00 ng, at least 1.25 ng, at least1.50 ng, at least 1.75 ng, at least 2.00 ng, at least 2.25 ng, at least 2.50 ng, at least 2.75 ng, at least3.00 ng, at least 3.25 ng, at least 3.50 ng, at least 3.75 ng, at least 4.00 ng, at least 4.25 ng, at least4.50 ng, at least 4.75 ng, or at least 5.00 ng of cfNA.

[0072] In some embodiments, the methods comprise, after the first sequencing assay on the first aliquot of sample, providing a second aliquot of sample with sufficient volume for the amplification assay or the second sequencing assay. In some embodiments, the second aliquot of sample comprises at least 50 pl, at least 100 pl, at least 150 pl, at least 200 pl, at least 250 pl, at least 300 pl, at least 350 pl, at least 400 pl, at least 450 pl, at least 500 pl, at least 550 pl, at least 600 pl, at least 650 pl, at least 700 pl, at least 750 pl, at least 800 pl, at least 850 pl, at least 900 pl, at least 950 pl, or at least 1 ml of the sample. In some embodiments, the second aliquot is at least 500 pl of plasma obtained from a human. In some embodiments, the second aliquot is at least 250 pl of plasma obtained from a human. In some embodiments, the second aliquot is at least 100 pl of plasma obtained from a human.

[0073] In some embodiments, the one or more carrier microbes harboring an AMR genetic marker comprise one or more Gram-positive bacteria. In some embodiments, the one or more carrier microbes harboring an AMR genetic marker comprise one or more Gram-negative bacteria. In some embodiments, the one or more Gram-positive bacteria comprise a Staphylococcus spp. In some embodiments, the one or more Gram-positive bacteria comprise one or more Methicillin-resistance carriers. In some embodiments, the one or more Methicillin- resistance carriers comprise In some embodiments, the methicillin-resistance carrier comprises Staphylococcus pseudintermedius, Staphylococcus fleurettii, Staphylococcus epidermidis,Staphylococcus schleiferi, Staphylococcus lugdunensis, Staphylococcus cohnii, Staphylococcus caprae, Staphylococcus warneri, Staphylococcus saprophyticus, Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus capitis, Staphylococcus hominis, Staphylococcus pettenkoferi, or Staphylococcus simulans. For example, the methods provided herein can comprise detecting Staphylococcus aureus (S. aureus) in a high throughput sequencing assay performed on an aliquot of a sample from a patient, wherein the aliquot of the sample comprises mcfNA from the S. aureus.

[0074] n some embodiments, the methods comprise performing an amplification assay or a second sequencing assay on a second aliquot of the same sample based on the results from the first sequencing assay. For example, if the methods detect a quantity of mcfNA from S. aureus below a first threshold (e.g., below 100 MPM, 200 MPM, 300 MPM, 400 MPM, 500 MPM); and 2) detect a quantity of mcfNA from other Staphylococci spp. (a confounding spp.) below a second threshold (e.g., below 1, 5, 10, 15, 20 MPM); the methods can then determine to perform an amplification assay or a second sequencing assay targeting mecA and mecC. In some embodiments, the methods comprise performing the amplification assay or the second sequencing assay when the results from the first sequencing assay is indeterminate. In some embodiments, the methods comprise determining the result from the first sequencing assay is indeterminate and performing a second sequencing assay or an amplification assay if the SCCmec cassette in the sample is likely to be a non-mec SCC, as determined from alignments to alleles known to lack mecA and mecC. In some embodiments, the result from the first sequencing assay is indeterminate if the SCC fragments in the clinical sample have originated from a microbe other than S. aureus, as determined by comparing the abundances of known cross-reacting or interfering species. In some embodiments, the interfering species comprise coagulase-negative staphylococci that harbor sequences highly homologous to SCCmec.

[0075] In some cases, if the methods do not comprise detecting an abundance of sequencing reads from S. aureus over a threshold provided herein, the amplification assay or the second sequencing assay targeting mecA and mecC will be performed. In some cases, if the methods do not comprise detecting a carrier microbe harboring / CTX-M or bla^c above the threshold, the amplification assay or the second sequencing assay targeting / CTX-M or bla^c will be performed. In some cases, if the methods do not comprise detecting a carrier microbe harboring vanA or van B above the threshold, the amplification assay or the second sequencing assay targeting vanA or van B will be performed.

[0076] In some cases, the amplification assay or the second sequencing assay will only be performed if sufficient amount of a second aliquot of the sample is provided. In some cases, the amplification assay or the second sequencing assay will only be performed if detect a lowquantity of mcfNA from a confounding species is detected. In some embodiments, the amplification assay or the second sequencing assay will only be performed if the first sequencing assay detects a quantity of mcfNA from a confounding species below a threshold. In some embodiments, the threshold of mcfNA from a confounding species comprises at most 200 MPM, at most 180 MPM, at most 160 MPM, at most 140 MPM, at most 120 MPM, at most 100 MPM, at most 80 MPM, at most 60 MPM, at most 40 MPM, at most 20 MPM, at most 10 MPM, at most 8 MPM, at most 6 MPM, at most 4 MPM, or at most 2 MPM, or at most 1 MPM.

[0077] In some embodiments, the methods comprise detecting one or more Gram-negative bacteria using the first sequencing assay on the first aliquot of a sample form a subject, wherein the one or more Gram-negative bacteria are potential carrier microbes of an AMR genetic marker, and then based on the results from the first sequencing assay, determining to perform an amplification assay or a second sequencing assay on a second aliquot of the same sample. In some embodiments, the one or more Gram-negative bacteria comprise Enterobacter cloacae complex, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Salmonella enterica, Salmonella bongori, Serratia marcescens, Enterobacter aerogenes, Pseudomonas aeruginosa, Acinetobacter baumannii, Acinetobacter calcoaceticus, or a combination thereof. In some embodiments, the amplification assay or the second sequencing assay is performed only when the methods detect a quantity of mcfNA from the one or more Gram-negative bacteria passes a threshold provided herein.

[0078] In some embodiments, the methods comprise discounting a result from a step of the multi-step process if there are multiple contaminants detected by the step. In some embodiments, the methods comprise discounting a result from a sequencing assay if there are multiple contaminants detected by the sequencing assay. In some embodiments, contaminants comprise contaminating nucleic acids or microbes. In some embodiments, contaminating nucleic acids arise from sample collection, sample site (e.g., skin), or reagents (e.g., buffers, buffer components, water, beads, etc.).Detection ofMRSA based on detection of SCCmec cassette and abundance of S. aureus reads

[0079] In some embodiments, the methods provided herein comprise using abundance of S. aureus combined with detection of a SCCmec cassette in order to identify whether the S. aureus harbors an AMR genetic marker. In some cases, a number of reads mapping to SCCmec is compared to the number of reads mapping to the S. aureus genome and a statistical model is used to determine the likelihood of either the presence or absence of methicillin resistance. In some embodiments, the methods further comprise quantifying sequencing reads from the high throughput sequencing assay, wherein the sequencing reads comprise S. aureus mcfNAsequencing reads. The abundance of the S. aureus mcfNA in the sample can be calculated based on the abundance of the S. aureus mcfNA sequencing reads.

[0080] In some embodiments, the methods comprise detecting SCCmec cassette from the sample and quantifying microbial sequencing reads from the SCCmec cassette. In some embodiments the SCCmec cassette is detected by unbiased HTS; in some embodiments, it is detected by targeted sequencing. The SCCmec cassette is an AMR gene cassette of a known size found in S. aureus. The SCCmec cassette in certain strains of S. aureus can harbor mecA or mecC, which are AMR genes associated with resistance to methicillin. In some embodiments, the SCCmec cassette can be empty and does not comprise the AMR genetic marker of interest. In some embodiments, the methods comprise detecting a non-empty SCCmec cassette.

[0081] The methods provided herein can further comprise determining the likelihood that mecA or mecC is present in the S. aureus in the patient based on the amount of microbial sequencing reads mapped to S. aureus and the amount of microbial sequencing reads from the SCCmec cassette. In some embodiments, the SCCmec cassette in S. aureus is an integrated region of the genome, the sequencing read count from S. aureus and the sequencing read count from the SCCmec cassette can be reliably correlated. For example, when there are more reads (e.g., above the limit of detection) an estimated deduplicated reads (EDR) of >1000, 2000, 3000, 4000 or 5000 can allow the methods to determine whether the SCCmec cassette likely harbors mecA or mecC by analyzing the S. aureus sequencing reads. The methods can compare the SCCmec cassette sequencing reads with the S. aureus genome sequencing reads to determine this likelihood. In some embodiments, if there is a high likelihood that the SCCmec cassette harbors mecA or mecC, the method will determine that the S. aureus is methicillin-resistant S. aureus (MRSA). In some embodiments, if there is a high likelihood that the SCCmec cassette is empty and does not harbor mecA or mecC, the method will determine that the S. aureus is methicillin- susceptible S. aureus (MS SA).

[0082] In some embodiments, a beta-binomial distribution can be used to determine the probability of getting X SCCmec reads given Y S. aureus reads. In some cases, a distribution comprises a distribution of ratio of SCCmec length to S. aureus genome length for MRSA. In some cases, a similar model can be built for MSSA. In some cases, Bayesian interference can be used to calculate probability of MRSA given the observed S. aureus reads and the observed number of SCCmec reads.Detecting AMR Markers using a Targeted Sequencing Assay

[0083] In some embodiments, the methods comprise detecting a nucleic acid (e.g., AMR genetic markers) using a targeted sequencing assay. In some embodiments, the targeted sequencing comprises performing an amplification reaction on a sample using primers specific for targetsequences such as target AMR sequences. In some embodiments, amplicons generated from the amplification reaction can be analyzed in order to determine the abundance of a target sequence (e.g., AMR genetic marker). In some cases, an HTS assay is performed on the amplicons in order to determine the abundance of the target nucleic acid.

[0084] In some embodiments, the methods comprise calculating an estimated deduplicated templates (EDT) from a sequencing assay, e.g., a targeted sequencing assay. As used herein “estimated deduplicated templates” or “EDT” refers to an estimate of the number of original template molecules present prior to amplification. EDT can be calculated by leveraging the primer plus read length or primer plus unique molecular identifier tags to deduplicate PCR amplicons generated from non-amplified nucleic acids.

[0085] In some embodiments, linkage of an AMR marker with a carrier microbe is based on analyzing an EDT value of an AMR marker and the abundance of a carrier microbe detected by HTS. In some embodiments, linkage of an AMR marker with a carrier microbe is based on analyzing an EDT value of an AMR marker and an EDT value of a microbe-specific gene, such as a microbe-specific housekeeping gene.

[0086] An EDT may comprise a calculation of the number of unique template molecules from the amplicon alignments for each targeted marker and from the unique molecular identifiers for the quality control oligonucleotides. An EDT may use a method of deduplicating PCR amplicons. Deduplication of a PCR amplicon may comprise incorporation of unique molecular identifiers. Deduplication of a PCR amplicon may comprise the use of primer+read length or primer+unique molecular identifier tags to deduplicate PCR amplicons. Deduplication of a PCR amplicon may comprise the use of software such as picard or SAMtools to aid in identification of duplicates and removal of duplicates. EDT may be used to calculate the number of original template molecules vs. deduplicating randomly sheared HTS reads.Detecting Microbe-specific Genes using a Targeted Sequencing Assay

[0087] In some embodiments, the methods comprise introducing primers targeting housekeeping genes, amplifying the housekeeping genes to produce amplicons and quantifying the housekeeping genes (e.g., by conducting HTS on the amplicons). In some embodiments, the housekeeping genes are microbe-specific in that identification of the housekeeping gene also enables identification of the corresponding microbe. Housekeeping genes may be chosen from species-specific core protein coding genes and filtered to those where the nucleotide sequence would not cross-react with genomes of other pathogens. In some embodiments, the housekeeping genes target S. aureus, S. epidermidis, and E faecium. The targeted housekeeping genes may be used to determine the presence or absence of the organism they are specific to one or more carriermicrobes. For example, a housekeeping gene targeted to S. aureus may be used in the determination of whether nucleic acids derived from S. aureus are present in a sample.

[0088] In some embodiments, the methods comprise calculating an estimated deduplicated templates (EDT) corresponding to housekeeping genes detected in a sequencing assay, e.g., a targeted sequencing assay. In some embodiments, an EDT value of a housekeeping gene is determined. In some embodiments, linkage of an AMR marker with a carrier microbe is based on analyzing an EDT value of an AMR marker and an EDT value of a microbe-specific gene, such as a microbe-specific housekeeping gene.

[0089] An EDT may comprise a calculation of the number of unique template molecules from the amplicon alignments for each targeted marker and from the unique molecular identifiers for the quality control oligonucleotides. An EDT may use a method of deduplicating PCR amplicons. Deduplication of a PCR amplicon may comprise incorporation of unique molecular identifiers. Deduplication of a PCR amplicon may comprise the use of primer+read length or primer+unique molecular identifier tags to deduplicate PCR amplicons. Deduplication of a PCR amplicon may comprise the use of software such as picard or SAMtools to aid in identification of duplicates and removal of duplicates. EDT may be used to calculate the number of original template molecules vs. deduplicating randomly sheared HTS reads.Linkins an AMR marker with a carrier microbe via unbiased HTS and a targeted sequencing assay

[0090] The methods provided herein can be especially useful for linking an AMR marker associated with a carrier microbe. In some embodiments, targeted sequencing of an AMR marker is based on the sequence of the AMR marker itself (e.g., primers directed to the AMR marker), without giving information about its associated carrier microbe. In some embodiments, linking a carrier microbe with an AMR genetic marker is especially useful for samples in which nucleic acids from multiple carrier microbes are detected, nucleic acids from multiple AMR markers, or a combination thereof. In such cases, it can be challenging to determine which carrier microbe is linked to which AMR genetic marker. In some cases, when multiple species of carrier microbes are detected, the methods provided herein identify which species of the carrier microbe is associated with a particular AMR marker.

[0091] In some embodiments, the methods provided herein comprise linking an AMR genetic marker with its carrier microbe via an unbiased high throughput sequencing (HTS) assay and a targeted sequencing assay. In some embodiments, the methods comprise 1) detecting one or more potential carrier microbes harboring an AMR genetic marker via an unbiased HTS assay; 2) detecting the AMR genetic marker via a targeted sequencing assay; and 3) performing statistical analysis to link the AMR genetic marker with its carrier microbe. In some embodiments, themethods comprise detecting an episomal AMR genetic marker in the targeted sequencing assay. In some embodiments, performing the statistical analysis comprises calculating an abundance of mcfNA from the one or more potential carrier microbes detected in the unbiased HTS assay. In some embodiments, performing the statistical analysis comprises calculating an abundance of mcfNA associated with the AMR genetic marker.

[0092] In some embodiments, the methods comprise linking the AMR genetic marker with its carrier microbe based on the abundance of mcfNA from the one or more potential carrier microbes, the abundance of mcfNA associated with the AMR genetic marker, the copy number of the AMR genetic marker, the abundances of microbial-specific housekeeping genes detected in the targeted sequencing assay, or any combination thereof. In some embodiments, the methods comprise linking the AMR genetic marker with its carrier microbe based on the abundance of mcfNA from the one or more potential carrier microbes and the copy number of the AMR genetic marker. In some embodiments, the methods comprise linking the AMR genetic marker with its carrier microbe based on results from the unbiased HTS assay and results from the targeted sequencing assay.

[0093] In some embodiments, the methods provided herein comprise performing a statistical analysis to link one or more AMR genetic markers to one or more carrier microbes harboring the AMR genetic markers. In some embodiments, the methods comprise linking one AMR genetic marker to one carrier microbe. In some embodiments, the methods comprise linking multiple AMR genetic markers to multiple carrier microbes; for example, the methods can comprise linking one or more carrier microbes to its corresponding AMR marker. In some cases, the statistical analysis comprises a generalized linear model. In some cases, the statistical analysis comprises a maximum-likelihood estimation. In some cases, the statistical analysis comprises a probit model. In some cases, the statistical analysis comprises a maximum-likelihood probit model. Examples of other models that may be used in the statistical analysis include logistic regression, linear probability, linear regression, complimentary log-log, Poisson regression, support vector machine, decision tree, random forest, neural network, gradient boosted model, Bayesian models, hidden Markov models, or any combination thereof.

[0094] In some embodiments, to link the HTS data to the AMR genetic marker detected in the targeted sequencing assay, the methods comprise using a model that uses the observed abundance of the carrier microbes (e.g., MPM), copy number of AMR gene, ratio of AMR gene length to microbe genome length, a measure of PCR reaction efficiency from the abundance of an internal control (PCR WINC), or any combination thereof. With those known variables, in some embodiments, a model is prepared that gives an expected AMR abundance, such as estimateddeduplicated templates or transcripts (EDT)). In some cases, copy number is calculated and used in the statistical analysis. In some cases, copy number is not used in the statistical analysis.

[0095] In some embodiments, the methods comprise using a model (e.g., a model described herein) and / or probabilities for AMR prevalence. In some embodiments, the model or probabilities for AMR prevalence are derived from empirical observations for combinations of observed carrier microbe and AMR gene copy number. In some embodiments, the model or probabilities for AMR prevalence are derived from known observations reported in scientific literature for combinations of observed carrier microbe and AMR gene copy number. In some embodiments, a combination of observed carrier microbe and AMR gene copy number is used to determine the likelihood of this combination given the observed AMR EDT. In some embodiments, thresholds to this final likelihood for microbe-AMR combination are applied in order to determine presence, indeterminate, or absence.

[0096] In some embodiments, the methods comprise performing a statistical analysis to link an AMR genetic marker with a carrier microbe based on detecting or quantifying an AMR marker that is integrated into a chromosome or based on quantifying an AMR marker that is an episomal marker. In some embodiments, such integrated or episomal AMR markers are detected by an unbiased HTS assay or a targeted sequencing assay. In some embodiments, the methods comprise performing a statistical analysis to link an AMR genetic marker with a carrier microbe based on detecting an abundance of an AMR cassette. In some embodiments, the methods comprise detecting an integrated AMR marker (e.g., SCCmec) in a first assay (e.g., unbiased HTS assay) and then detecting a set of integrated or episomal AMR marker in a second assay (e.g., targeted sequencing assay). In some cases, at least 1, 2, 3, 4, 5, 10, 15, or 20 integrated or episomal AMR markers are detected in the targeted sequencing assay. In some embodiments, the methods comprise linking a full set, or a portion of a set, of such AMR markers with carrier microbes detected in a different assay (e.g., unbiased HTS assay).

[0097] In some embodiments, a first assay (e.g., unbiased HTS assay) detects larger genomic targets, such as SCCmec. If the larger genomic targets are known to be integrated in the microbial genome, the methods comprise identifying the linkage to a carrier microbe using a statistical model or without a statistical model. In some embodiments, the methods comprise detecting genes or portions of plasmids by an assay (e.g., unbiased HTS assay) and then using a statistical model to assign linkage without necessarily performing a targeted sequencing assay.

[0098] In some embodiments, the methods comprise detecting an integrated AMR genetic marker that has a copy number that tracks the copy number of a microbial genome; in some cases, the AMR genetic marker is integrated and has one copy per microbial genome. In some cases, multiple AMR genetic markers are integrated into the same microbial genome. In someembodiments, the methods comprise detecting an episomal AMR genetic marker. In some cases, the copy number of the episomal AMR genetic marker dose not present in a one-to-one ratio with the microbial genome (e.g., microbial DNA, RNA), microbial gene, or microbial chromosome. In some embodiments, the methods detect an integrated AMR cassette that may comprise the AMR genetic marker. In some embodiments, the methods detect an episomal AMR cassette. In some embodiments, the methods detect a non-empty AMR cassette. In some embodiments, the methods detect the integrated AMR genetic marker or the integrated AMR cassette in the unbiased HTS assay. In some embodiments, the methods detect the episomal AMR genetic marker or the episomal AMR cassette in the unbiased HTS assay. In some embodiments, the methods detect the episomal AMR genetic marker or the episomal AMR cassette in the targeted HTS assay. In some embodiments, the methods detect the integrated AMR genetic marker or the integrated AMR cassette in the targeted HTS assay. In some embodiments, the methods can detect the AMR genetic marker or the carrier microbes by high throughput sequencing, next generation sequencing (NGS), massively parallel sequencing, or sequencing by synthesis. In some embodiments, the methods detect the AMR genetic marker by an unbiased sequencing assay. In some embodiments, the methods detect the AMR genetic marker by a targeted sequencing assay.

[0099] The general framework of these methods has potential applications for general biomarkers of disease. In some embodiments, a first assay can detect whether disease state is present or absent and a second assay can define the specific disease state (e.g., inflammation) or site of disease (e.g., a site of infection).Linkins an AMR marker with a carrier microbe using a targeted sequencing assay

[0100] In some embodiments, the methods provided herein comprise linking an AMR genetic marker with its carrier microbe via a targeted sequencing assay that detects both the carrier microbe as well as the AMR genetic marker. In some embodiments, the methods comprise 1) detecting the AMR genetic marker via a targeted sequencing assay; 2) detecting one or more microbial-specific genes such as microbe-specific housekeeping genes via the targeted assay; and 3) performing statistical analysis to link the AMR genetic marker with its carrier microbe. In some embodiments, the AMR marker detected by the targeted sequencing assay is an episomal AMR genetic marker. In some embodiments, the AMR marker detected by the targeted sequencing assay is an integrated AMR genetic marker. In some embodiments, performing the statistical analysis comprises calculating a copy number of the AMR genetic marker or calculating abundances of the microbial-specific housekeeping genes detected in the targeted sequencing assay. In some embodiments, a statistical model for linking a carrier microbe with an AMR genetic marker using a targeted assay is similar to a statistical model used for linking acarrier microbe detected by HTS with an AMR marker detected by a targeted assay. Some examples of such models are provided herein. In some embodiments, housekeeping gene abundance (EDT) and AMR-to-housekeeping-gene ratio are used in a statistical model. And in some embodiments, the MPM value in a statistical model described herein can be substituted with the EDT value when using the targeted sequencing assay to detect a carrier microbe and its associated AMR marker.

[0101] In some embodiments, the method comprises detecting microbe-specific housekeeping genes by a targeted sequencing assay. In some cases, the microbe-specific housekeeping genes are detected by HTS or unbiased HTS. In some embodiments, the microbe-specific housekeeping genes are specific for different species of a microbe. In some embodiments, the microbe-specific housekeeping genes are specific for different species of Staphylococcus, e.g., S. aureus and / or S. epidermidis. In some embodiments, the microbe-specific genes comprise A-SA, B-SA, C-SA, D-SA or any combination thereof. In some embodiments, the microbe-specific genes comprise a housekeeping (HK) gene listed in Supplemental Table 6 in the Examples.

[0102] In some embodiments, the methods comprise detecting one or more microbial-specific housekeeping genes in the targeted sequencing assay in order to detect potential carrier microbes. In some embodiments, the methods comprise detecting a carrier microbe based on the detection of the one or more microbial-specific housekeeping genes in the targeted assay. In some embodiments, the methods comprise performing a statistical analysis in order to link a carrier microbe (detected by a housekeeping gene) with an AMR genetic marker.

[0103] In some embodiments, performing the statistical analysis comprises calculating an abundance of the AMR genetic marker, a copy number of the AMR genetic marker, and / or abundances of the microbial-specific housekeeping genes detected in the targeted sequencing assay. In some embodiments, the abundance of the AMR genetic marker or the microbial-specific housekeeping genes comprises a relative abundance, such as an estimated deduplicated template or transcript (EDT). In some embodiments, performing the statistical analysis comprises calculating a ratio of the EDT of AMR genetic marker to the EDT of the microbial-specific housekeeping genes. In some embodiments, performing the statistical analysis comprises detecting a carrier microbe based on the abundances of the one or more microbial-specific housekeeping genes. In some embodiments, the methods comprise linking the AMR genetic marker with its carrier microbe based on the abundance of mcfNA associated with the AMR genetic marker, the copy number of the AMR genetic marker, the abundances of the microbial- specific housekeeping genes detected in the targeted sequencing assay, the ratio of the AMR EDT to housekeeping gene EDT, or any combination thereof. In some embodiments, the methods comprise linking the AMR genetic marker with its carrier microbe based on the copy number ofthe AMR genetic marker and the abundances of the microbial-specific housekeeping genes detected in the targeted assay. In some embodiments, the methods comprise linking the AMR genetic marker with its carrier microbe based only on results from the targeted sequencing assay.

[0104] In some embodiments, a determination of “indeterminate” may be arrived at if the ratio of AMR EDT to housekeeping gene EDT for a carrier microbe departs from the expected ratio. For example, a lack of adequate housekeeping gene EDT would lower confidence in an AMR absence call, making the call indeterminate.Sample Preparation

[0105] In some embodiments, the methods further comprise preparing the sample using any of the sample preparation methods. In some embodiments, the methods further comprise preparing the nucleic acids in the sample for a sequencing assay. In some embodiments, the methods further comprise preparing the nucleic acids in the sample before a sequencing assay. In some embodiments, the methods further comprise preparing the nucleic acids in the sample after a first sequencing assay but before a second sequencing assay. In some embodiments, the methods further comprise performing a sequencing assay without preparing the nucleic acids. In some embodiments, preparing the nucleic acids in the sample comprises extracting, physically enriching, concentrating, amplifying the nucleic acids, or attaching one or more adapter to the nucleic acids. In some embodiments, amplifying the nucleic acids comprises performing a polymerase chain reaction (PCR). In some embodiments, amplifying the nucleic acids comprises attaching one or more adapter to the nucleic acids. In some embodiments, attaching one or more adapter to the nucleic acids comprises performing a ligase reaction or a PCR.

[0106] In some embodiments, the methods provided herein comprise performing a sequencing assay on nucleic acids in a sample that have not been extracted from the sample before the sequencing assay. In some embodiments, the methods provided herein comprise performing a sequencing assay on nucleic acids in a sample that have been extracted from the sample before the sequencing assay. In some embodiments, the methods comprise 1) performing a firsthigh throughput sequencing assay on nucleic acids in a first aliquot of a sample, wherein the nucleic acids have not been extracted from the first aliquot; and 2) performing a second high throughput sequencing assay on nucleic acids in a second aliquot of the sample, wherein the nucleic acids have been extracted from the second aliquot. In some embodiments, the methods comprise 1) performing a first high throughput sequencing assay on nucleic acids in a first aliquot of a sample, wherein the nucleic acids have been extracted from the first aliquot; and 2) performing a second high throughput sequencing assay on nucleic acids in a second aliquot of the sample, wherein the nucleic acids have been extracted from the second aliquot. In some embodiments, the methods comprise 1) performing a first high throughput sequencing assay on nucleic acids in a first aliquotof a sample, wherein the nucleic acids have not been extracted from the first aliquot; and 2) performing a second high throughput sequencing assay on nucleic acids in a second aliquot of the sample, wherein the nucleic acids have not been extracted from the second aliquot. In some embodiments, the methods comprise 1) performing a first high throughput sequencing assay on nucleic acids in a first aliquot of a sample, wherein the nucleic acids have been extracted from the first aliquot; and 2) performing a second high throughput sequencing assay on nucleic acids in a second aliquot of the sample, wherein the nucleic acids have not been extracted from the second aliquot. In some embodiments, the first high throughput sequencing assay is not a targeted sequencing assay. In some embodiments, the second high throughput sequencing assay is a targeted sequencing assay.

[0107] In some embodiments, the methods further comprise extracting nucleic acids from a nucleic acid library before the sequencing assay.

[0108] In some embodiments, the methods further comprise extracting nucleic acids from a sample before the sequencing assay. In some embodiments, the methods provided herein comprise 1) performing a first sequencing assay on nucleic acids in a first aliquot of a sample, wherein the nucleic acids in the first aliquot have not been extracted; 2) extracting nucleic acids from a second aliquot of the sample; and after 2), 3) performing a second sequencing assay on the nucleic acids extracted from the second aliquot. In some embodiments, the methods provided herein comprise 1) performing a first sequencing assay on nucleic acids in a first aliquot of a sample, wherein the nucleic acids in the first aliquot have been extracted; 2) extracting nucleic acids from a second aliquot of the sample; and after 2), 3) performing a second sequencing assay on the nucleic acids extracted from the second aliquot. In some embodiments, the methods provided herein comprise 1) extracting nucleic acids from a first aliquot of a sample; 2) performing a first sequencing assay on the nucleic acids extracted from the first aliquot; and after 2), 3) performing a second sequencing assay on nucleic acids from a second aliquot, wherein the nucleic acids from the second aliquot have not been extracted. In some embodiments, the methods provided herein comprise 1) extracting nucleic acids from a first aliquot of a sample; 2) performing a first sequencing assay on the nucleic acids extracted from the first aliquot; and after 2), 3) performing a second sequencing assay on nucleic acids from a second aliquot, wherein the nucleic acids from the second aliquot have been extracted.

[0109] In some embodiments, the methods provided herein comprise 1) performing a first high throughput sequencing assay on nucleic acids from a first aliquot of a sample in order to detect one or more carrier microbes harboring an AMR genetic marker, wherein the nucleic acids form the first aliquot have not been extracted and the nucleic acids comprise microbial nucleic acids from the one or more carrier microbes; 2) after detecting at least one carrier microbe, extracting-n-nucleic acids from a second aliquot of the sample, thereby producing a concentrated sample comprising the microbial nucleic acids from the carrier microbe; and 3) performing a second high throughput sequencing assay on the nucleic acids from the concentrated sample to detect the AMR marker. In some embodiments, the first high throughput sequencing assay is a non-biased sequencing and not a targeted sequencing assay. In some embodiments, the second high throughput sequencing assay is a targeted sequencing assay.

[0110] In some embodiments, the methods provided herein further comprise attaching one or more adapters to the nucleic acids in an aliquot of the sample before performing a sequencing assay. In some embodiments, the methods comprise attaching the one or more adapters to the nucleic acids that have been extracted from the sample. In some embodiments, the methods comprise attaching the one or more adapters to the nucleic acids that have not been extracted from the sample. In some embodiments, the methods comprise adding the one or more adapters directly to an aliquot of the initial sample. In some embodiments, the one or more adapters are attached to the nucleic acids by a ligase. In some embodiments, the ligase comprises a DNA ligase, a RNA ligase, a DNA / RNA ligase, a splint ligase, a high-fidelity ligase, a thermostable ligase, a truncated ligase, a natural ligase, an engineered ligase, or any combination thereof. In some embodiments, the ligase comprises CircLigase II, CircLigase ssDNA ligase, CircLigase RNA ligase, Thermostable App-DNA / RNA ligase, T4 DNA ligase, T4 RNA ligase 1, T4 RNA Ligase 2, T4 RNA Ligase 2 truncated, Splint ligase, Splint-R ligase, Pfu ligase, any engineered variants thereof, any natural variants thereof, or any combination thereof. In some embodiments, the one or more adapters are attached to the nucleic acids by PCR. In some embodiments, attaching the one or more adapters can comprise using one or more primers comprising an adapter sequence. In some embodiments, the primers comprising an adapter sequence further comprise a sequence targeting a genetic marker provided herein for producing adapted nucleic acids comprising the genetic marker. In some embodiments, the primers comprise an adapter sequence and a sequence targeting an AMR genetic marker for producing adapted nucleic acids comprising the AMR genetic marker.

[0111] In some embodiments, the methods further comprise amplifying nucleic acids and producing amplicons associated with the nucleic acids. In some embodiments, amplifying the nucleic acids comprise amplifying with PCR or a sequencing by synthesis assay. In some embodiments, amplifying nucleic acids comprises generating a nucleic acid library for a sequencing assay provided herein. In some embodiments, the amplified nucleic acids or amplicons are extracted. In some embodiments, amplifying comprises amplifying the nucleic acids comprising the one or more genetic markers provided herein. In some embodiments, amplifying comprises amplifying adapted nucleic acids comprising one or more adapters. Insome embodiments, the methods comprise amplifying nucleic acids comprising the AMR genetic marker to generate amplicons comprising or associated with the AMR genetic marker. In some embodiments, the methods further comprise performing a high throughput sequencing assay on the amplicons comprising the AMR genetic marker to detect the AMR genetic marker. In some embodiments, the methods further comprise extracting the amplicons comprising the AMR genetic marker before the high throughput sequencing assay.

[0112] In some embodiments, the methods comprise performing a first high throughput sequencing assay on nucleic acids that have not been extracted from the sample comprising the nucleic acids. For example, without extracting the nucleic acids from the initial sample, the methods can directly perform a high throughput sequencing assay on the nucleic acids in the initial sample to generate sequence reads, wherein the nucleic acids can comprise microbial nucleic acids from one or more carrier microbe harboring an AMR genetic marker. The first high throughput sequencing assay can detect the microbial nucleic acids from at least one carrier microbe and identify the carrier microbe. After detecting the carrier microbe, the methods further comprise preparing nucleic acid from another portion or aliquot of the same sample. For example, nucleic acids from a second aliquot of the sample can be extracted to provide a concentrated sample comprising the microbial nucleic acids from the carrier microbe. Additionally, the method can further comprise attaching one or more adapters to the nucleic acids in the concentrated sample. The one or more adapters can be attached to the nucleic acids by a ligase, thereby producing adapted nucleic acids. The one or more adapters can also be attached to the nucleic acids by PCR, thereby producing adapted nucleic acids. In some instances, the attaching the one or more adapters can comprise using one or more primers targeting the AMR genetic marker, wherein the primers also contain the adapter sequences, thereby generating adapted nucleic acids comprising the AMR genetic marker. Additionally, the method can further comprise amplifying the adapted nucleic acids by PCR, thereby generating amplicons comprising the AMR genetic marker. In some instances, one or more additional adapters can be attached to the adapted nucleic acids during amplification. The methods can therefore generate adapted amplicons comprising the AMR genetic marker useful for a second sequencing assays to analyze the AMR genetic marker. After this, the methods can further comprise performing a high throughput sequencing assay on the adapted amplicons comprising the AMR genetic marker.Samples

[0113] Disclosed herein in some embodiments are samples derived from subjects. In some embodiments, a sample provided herein can comprise a nucleic acid molecule to be sequencedby a method described herein. As used herein, a “sample” generally refers to any material comprising nucleic acids that has been derived from a subject. A sample can comprise a raw biological sample, such as whole blood. As used herein, the phrase “raw biological sample” refers to an unmanipulated or unprocessed sample obtained from a subject, e.g., a host, containing or presumed to contain target nucleic acids. In some embodiments, a raw biological sample has not been subjected to any extraction methods after being obtained from a subject. In some embodiments, a raw biological sample can be processed or manipulated to produce an initial sample. For example, a raw biological sample can comprise whole blood which is centrifuged to produce an initial sample of plasma for a sequencing assay. As used herein, the term “initial sample” refers to a sample comprising nucleic acids derived from a raw biological sample. In some embodiments, an initial sample can comprise a sample that has been processed or manipulated, such as plasma or serum. In some embodiments, an initial sample can comprise target or desired nucleic acids obtained or extracted from a raw biological sample. In some embodiments, an initial sample can be subjected to a sequencing assay as. In some embodiments, a raw biological sample or an initial sample can be used directly in a sequencing assay without extraction of a nucleic acid. In some embodiments, a nucleic acid as described herein can be extracted from a raw biological sample or an initial sample for use in a sequencing assay. In some embodiments, an extraction method can comprise an alcohol-based extraction, a column purification, a filtration, a size separation, or any combination thereof. As used herein, “removal” or “extraction,” and their cognates, of nucleic acids refers to steps prior to the start of generating or preparing a nucleic acid library that separate nucleic acids from at least one component with which they are normally associated. In some embodiments, removal or extraction of nucleic acids can refer to the process of creating an initial sample from a raw biological sample. For example, without limitation, the fractionation of whole blood into its component parts, such as plasma, can be considered to involve removal or extraction. Similarly, purification or isolation of DNA from a sample (e.g., plasma sample) can be considered extraction. In some embodiments, a nucleic acid extracted from a sample can be subjected to a sequencing assay. In some embodiments, a raw biological sample or an initial sample can comprise a biological sample.

[0114] In some embodiments, a sample can comprise a biological sample obtained or collected from a subject. In some embodiments, a biological sample can comprise cells. In some embodiments, a biological sample can be substantially cell-free. In some embodiments, a biological sample can comprise a biological fluid. In some embodiments, a biological fluid can comprise a bodily fluid of a subject (e.g., blood), a fluid obtained from the subject via a medical procedure (e.g., lavage), or any fluid obtained from processing a biopsy of the subject (e.g., serous fluid). In some embodiments, a biological fluid can comprise a bodily fluid. In someembodiments, a bodily fluid can comprise a whole blood, a plasma, a serum, a lymph, a synovial fluid, a cerebrospinal fluid (CSF), a saliva, a gastric juice, a bile, a pancreatic juice, an intestinal fluid, a respiratory tract mucosal secretion, a semen, a cervical mucus, a vaginal secretion, a urine, a sebum, a breast milk, an amniotic fluid, a pericardial fluid, a pleural fluid, a peritoneal fluid, or any combination thereof. In some embodiments, a biological fluid can be processed from a bodily fluid. For example, blood from a subject can be processed to generate a plasma sample, a serum sample, or a platelet sample. In some embodiments, a biological fluid can comprise a plasma sample. In some embodiments, a biological fluid can comprise a lavage from diagnosing, treating, or cleaning an area of a body of a subject. In some embodiments, a lavage can comprise a bronchoalveolar lavage (BAL), a gastric lavage, a peritoneal lavage, a nasal lavage, a bladder lavage, a rectal lavage, a wound lavage, a joint lavage (arthrocentesis), an eye lavage, a sinus lavage, or any combination thereof. In some embodiments, a biological fluid can comprise an amniotic fluid. In some embodiments, a biological fluid can comprise a BAL. In some embodiments, a biological fluid can comprise a joint lavage. In some embodiments, a biological fluid can comprise a fluid obtained from processing a biopsy of a subject. In some embodiments, a biological fluid can comprise a needle aspiration fluid, a serous fluid, a microdialysis fluid, an exudate fluid, or any combination thereof. As used herein, “plasma” or “blood plasma” refers to the liquid component or fraction of blood. Plasma is generally obtained by spinning a whole blood sample and removing the liquid component.Microbes

[0115] In some embodiments, the methods described herein comprise detecting a microbe or microbial nucleic acids from a microbe. In some embodiments, the microbe comprises a carrier microbe harboring the one or more AMR genetic markers. In some embodiments, the one or more AMR genetic markers are associated with one or more carrier microbes that harbor the one or more AMR genetic markers. In some embodiments, the one or more carrier microbes comprise a virus, a bacterium, a protozoa, a fungus, an archaea, an algae, or any combination thereof. In some embodiments, the one or more carrier microbes comprise a pathogen. In some embodiments, the one or more carrier microbes comprise a commensal microbe.

[0116] In some embodiments, the one or more carrier microbes comprise one or more bacteria. In some embodiments, the one or more bacteria comprise a gram-positive or a gram-negative bacterium. In some embodiments, the one or more bacteria comprise Staphylococcus aureus, S. epidermidis, S. lugdunensis, Enterococcus faecalis, E. faecium, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, K. pneumoniae, K. oxytoca, Proteus mirabilis, P. vulgaris, Salmonella bongori, S. enterica, Serratia marcescens, Pseudomonas aeruginosa,Acinetobacter baumannii, or A. calcoaceticus . In some embodiments, the one or more carrier microbes comprise a methicillin-resistance carrier. In some embodiments, the methicillin- resistance carrier comprises Staphylococcus pseudintermedius, Staphylococcus fleurettii, Staphylococcus epidermidis, Staphylococcus schleiferi, Staphylococcus lugdunensis, Staphylococcus cohnii, Staphylococcus caprae, Staphylococcus warneri, Staphylococcus saprophyticus, Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus capitis, Staphylococcus hominis, Staphylococcus pettenkoferi, or Staphylococcus simulans.

[0117] In some embodiments, the one or more carrier microbes comprise a vancomycin- resistance carrier. In some embodiments, the vancomycin-resi stance carrier comprises Enterococcus faecium, Enterococcus raffinosus, Enterococcus casseliflavus, Enterococcus gallinarum, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, or Streptococcus gallolyticus.

[0118] In some embodiments, the one or more carrier microbes comprise a Gram-negative carbapenem or extended-spectrum beta-lactamase (ESBL) resistance carrier. In some embodiments, the Gram-negative carbapenem or extended-spectrum beta-lactamase (ESBL) resistance carrier comprises Enterobacter cloacae complex, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Salmonella enterica, Salmonella bongori, Serratia marcescens, Enterobacter aerogenes, Pseudomonas aeruginosa, Acinetobacter baumannii, Acinetobacter calcoaceticus, Aeromonas caviae, Aeromonas hydrophila, Citrobacter amalonaticus, Citrobacter freundii, Citrobacter koseri, Pluralibacter gergoviae, Enterobacter hormaechei, Morganella morganii, Pantoea agglomerans, Providencia rettgeri, Providencia stuartii, Shigella flexneri, Shigella sonnei, Serratia liquefaciens, Stenotrophomonas maltophilia, Vibrio cholerae, Kluyvera georgiana, Kluyvera cryocrescens, Kluyvera ascorbata, Klebsiella variicola, Raoultella planticola, Raoultella omithinolytica, Citrobacter braakii, Kluyvera intermedia, Pantoea sp. PSNIH2, Pantoea sp. PSNIH1, or Clavibacter cf. michiganensis LMG 26808.

[0119] In some embodiments, a confounding microbe comprises a carrier microbe. A confounding microbe can refer to any microbe that can harbor nucleic acids that are homologous to the target nucleic acids (e.g., AMR gene or housekeeping gene). In some embodiments, the confounding microbe is a carrier microbe harboring an AMR cassette or an AMR genetic marker.

[0120] As used herein, a “microbe” can refer to a living microorganism or a non-living microscopic entity. In some embodiments, a living microorganism can comprise a bacterium, a protozoa, a fungus, an archaea, an algae, a parasite, or any other living microorganism. In some embodiments, a non-living microscopic entity can comprise a virus, a live virus, a replicatingvirus, or an attenuated virus. In some embodiments, non-host nucleic acids can be derived from a microbe. In some embodiments, target nucleic acids can be derived from a plurality of microbes.

[0121] In some embodiments, a microbe can be pathogenic to a subject (e.g., a pathogen), a commensal microbe of a subject, or a microbe present in a general environment. In some embodiments, a pathogen can comprise any pathogenic or virulent microbe. In some embodiments, a commensal microbe of a subject can comprise a microbe that inhabits any location in or on a subject without causing any symptom of a disease or disorder. In some embodiments, a microbe of a general environment can comprise a microbe at or near a sample collection site or a microbe at or near a location of a subject. In some embodiments, a microbe of a general environment comprises a commensal microbe. In some embodiments, a commensal microbe of a subject may become a pathogen to the subject. In some embodiments, a commensal microbe of a first subject may be a pathogen to a second subject. In some embodiments, a microbe of a general environment of a subject may become a pathogen to a subject. In some embodiments, a microbe of a general environment of a first subject may be a pathogen to a second subject.

[0122] In some embodiments, a pathogen can cause an infection or disease comprising gastrointestinal infections (e.g., Escherichia coli, Salmonella spp., Clostridioides difficile), urinary infections (e.g., Escherichia coli , skin infections (e.g., Staphylococcus aureus, including MRS A), strep throat (or scarlet fever, rheumatic fever) (e.g., Streptococcus pyogenes), tuberculosis (e.g., Mycobacterium tuberculosis), gonorrhea (e.g., Neisseria gonorrhoeae), cholera (e.g., Vibrio cholerae), Lyme disease (e.g., Borrelia burgdorferi), ulcers or stomach cancer (e.g., Helicobacter pylori), syphilis (e.g., Treponema pallidum), anthrax (e.g., Bacillus anthracis), seasonal flu (e.g., Influenza viruses), acquired immunodeficiency syndrome (AIDS) (e.g., Human Immunodeficiency Virus (HIV)), liver infections (e.g., Hepatitis B and Hepatitis C viruses), cervical cancer (e.g., Human Papillomavirus (HPV)), respiratory infections (e.g., SARS- CoV-2), oral or genital herpes, (e.g., Herpes Simplex Virus (HSV-1 and HSV-2)), chickenpox or shingles (e.g., Varicella Zoster Virus), measles (e.g., Measles virus), neurological disorders (e.g., Rabies virus, Trypanosoma brucei), dengue fever (e.g., Dengue virus), Ebola disease (e.g., Ebola virus), candidiasis (e.g., Candida albicans), deep lung infections (e.g., Aspergillus spp., Pneumocystis jirovecii), histoplasmosis (e.g., Histoplasma capsulatum), meningitis (e.g., Cryptococcus neoformans), ringworm (e.g., Trichophyton spp.), malaria (e.g., Plasmodium spp.), intestinal infection (y. .,Ascaris lumbricoides), giardiasis (e.g., Giardia lamblia), amebiasis (e.g., Entamoeba histolytica), toxoplasmosis (e.g., Toxoplasma gondii), leishmaniasis (e.g., Leishmania spp.), schistosomiasis (e.g., Schistosoma spp.), strongyloidiasis (e.g., Strongyloides stercoralis), tapeworm, taeniasis, or cysticercosis (e.g., Taenia solium).

[0123] In some embodiments, commensal microbes can inhabit a gastrointestinal tract, a skin, a respiratory tract, a urogenital tract, or an oral cavity of a subject. In some embodiments, commensal microbes can comprise an endogenous virus (e.g., endogenous retroviruses (ERVs)) of a subject. In some embodiments, a commensal microbe can comprise a Bacteroides fragilis, a Lactobacillus acidophilus, a Bifidobacterium bifidum, an Escherichia coli (non-pathogenic strains), a Staphylococcus epidermidis, a Streptococcus salivarius, a Propionibacterium acnes, a Candida albicans (under normal conditions), a Enterococcus faecalis, a Clostridium difficile (non-toxigenic strains), a Rothia mucilaginosa, a Fusobacterium nucleatum, a Peptostreptococcus anaerobius, a Prevotella melaninogenica, or any combination thereof. In some embodiments, a commensal microbe can comprise Lactobacillus spp., a. Bacleroides spp., & Faecali bacterium prausnitzii, an Escherichia coli, a Clostridium spp., n Enterococcus spp., a Staphylococcus spp., a Candida spp., an Aspergillus spp., a Porcine Endogenous Retroviruses (PERVs), an Eimeria spp., a Bifidobacterium spp., a usobacterium spp., a Simian Immunodeficiency Virus (SIV), a Simian Retrovirus (SRV), a Entamoeba spp, or any combination thereof.

[0124] In some embodiments, a microbe disclosed herein can comprise one or more microbes comprising: Coniosporium, Hantavirus, Talaromyces, Machlomovirus, Betatetravirus, Raoultella, Aeromonas, Ephemerovirus, Empedobacter, Loa, Macluravirus, Stenotrophomonas, Alfamovirus, Rosavirus, Emmonsia, Aggregatibacter, Orthopneumovirus, Weeksella, Nairovirus, Salivirus, Weissella, Mosavirus, Gammapartitivirus, Strongyloides, Passerivirus, Erysipelatoclostridium, Bacillarnavirus, lotatorquevirus, Taenia, Trypanosoma, Olsenella, Cladosporium, Rhizobium, Prevotella, Leclercia, Paracoccus, liarvirus, Lagovirus, Rasamsonia, Plasmodium, Acremonium, Chlamydia, Clonorchis, Vibrio, Bartonella, Nakazawaea, Franconib acter, Anisakis, Norovirus, Nocardia, Solobacterium, Parechovirus, Avenavirus, Orthohepevirus, Aphthovirus, Hepandensovirus, Microbacterium, Lichtheimia, Lomentospora, Achromob acter, Ipomovirus, Tsukamurella, Elizabethkingia, Hepevirus, Seadomavirus, Altemaria, Trueperella, Gammatorquevirus, Bifidobacterium, Chrysosporium, Thogotovirus, Curtovirus, Deltatorquevirus, Balamuthia, Mastrevirus, Bdellomicrovirus, Mupapillomavirus, Pseudozyma, Wickerhamiella, Aquamavirus, Alloscardovia, Thielavia, Idaeovirus, Henipavirus, Coxiella, Haemophilus, Gammacoronavirus, Negevirus Brevibacterium, Peptoniphilus, Alphacarmotetravirus, Nosema, Trichovirus, Arenavirus, Thermomyces, Necator, Waikavirus, Blosnavirus, Jonesia, Tetraparvovirus, Emaravirus, Plectrovirus, Sclerodamavirus, Toxocara, Umbravirus, Burkholderia, Chromobacterium, Paracoccidioides, Brugia, Eragrovirus, Macrococcus, Absidia, Colletotrichum, Inovirus, Phycomyces, Wickerhamomyces, Acidaminococcus, Moraxella, Rothia, Phlebovirus, Slackia, Purpureocillium,Betapapillomavirus, Tupavirus, Cryspovirus, Saksenaea, Erysipelothrix, Kobuvirus, Mimoreovirus, Echinococcus, Mannheimia, Bergeyella, Cyclospora, Xylanimonas, Leptospira, Finegoldia, Curvularia, Cryptosporidium, Babuvirus, Pecluvirus, Lambdatorquevirus, Pythium, Carlavirus, Entomobimavirus, Kocuria, Anaplasma, Ampelovirus, Avihepatovirus, Nepovirus, Rhodococcus, Bordetella, Mischivirus, Scedosporium, Gardnerella, Maculavirus, Trichoderma, Aveparvovirus, Salmonella, Avastrovirus, Copiparvovirus, Trachipleistophora, Clostridioides, Nanovirus, Siccibacter, Leptotrichia, Citrivirus, Odoribacter, Sanguibacter, Novirhabdovirus, Acremonium, Hafnia, Chaetomium, Tenuivirus, Yokenella, Rubulavirus, Varicellovirus, Alphamesonivirus, Sicinivirus, Leuconostoc, Microvirus, Gallantivirus, Morbillivirus, Lolavirus, Pantoea, Hepatovirus, Nupapillomavirus, Metschnikowia, Bamavirus, Kytococcus, Tritimovirus, Tannerella, Respirovirus, Pneumocystis, Dirofilaria, Pediococcus, Lactococcus, Blastomyces, Dianthovirus, Actinobacillus, Teschovirus, Oscivirus, Begomovirus, Potyvirus, Byssochlamys, Alphacoronavirus, Molluscipoxvirus, Lymphocryptovirus, Sapelovirus, Parabacteroides, Pyrenochaeta, Listeria, Senecavirus, Brevidensovirus, Potexvirus, Parvimonas, Flavivirus, Recovirus, Toxoplasma, Yatapoxvirus, Opisthorchis, Trichuris, Cyphellophora, Morganella, Perhabdovirus, Micrococcus, Pequenovirus, Mastadenovirus, Anaeroglobus, Tropheryma, Dolosigranulum, Wolbachia, Lelliottia, Mycoplasma Tobravirus, Shewanella, Paeniclostridium, Erythroparvovirus, Sutterella, Sporopachydermia, Namavirus, Nyavirus, Francisella, Arthroderma, Epsilontorquevirus, Sigmavirus, Amdoparvovirus, Actinomyces, Alphapermutotetravirus, Cardiobacterium, Influenzavirus C, Orthopoxvirus, Poacevirus, Phial ophora, Lactobacillus, Polyomavirus, Debaryomyces, Foveavirus, Bymovirus, Mycoflexivirus, Grimontia, Mucor, Rhytidhysteron, Quadrivirus, Thermoascus, Aureusvirus, Trichosporon, Myceliophthora, Dermacoccus, Dysgonomonas, Pseudoramibacter, Becurtovirus, Gordonia, Sapovirus, Orthobunyavirus, Spiromicrovirus, Pomovirus, Exophiala, Sneathia, Helicobacter, Photorhabdus, Mogibacterium, Betapartitivirus, Avibirnavirus, Ambidensovirus, Oleavirus, Orientia, Deltacoronavirus, Anulavirus, Trichomonasvirus, Budvicia, Geotrichum, Enamovirus, Lachnoclostridium, Schistosoma, Paecilomyces, Panicovirus, Rhizoctonia, Brevibacillus, Beauveria, Pestivirus, Tombusvirus, Cilevirus, Cokeromyces, Peptostreptococcus, Phanerochaete, Proteus, Idnoreovirus, Aspergillus, Pasteurella, Malassezia, Hanseniaspora, Endornavirus, Azospirillum, Velarivirus, Cystovirus, Avisivirus, Bacteroides, Picobirnavirus, Myroides, Circovirus, Arterivirus, Aquaparamyxovirus, Onchocerca, Cosavirus, Kluyveromyces, Fijivirus, Candida, Hepacivirus, Dermabacter, Ourmiavirus, Allexivirus, Enterobacter, Acidovorax, Bracorhabdovirus, Carmovirus, Pluralibacter, Coltivirus, Fonsecaea, Streptobacillus, Corynebacterium, Macrophomina, Marburgvirus, Comovirus, Fabavirus, Alphanodavirus, Cellulomonas, Enterobius, Catabacter, Moellerella, Nakaseomyces,Cucumovirus, Valsa, Deltapartitivirus, Plesiomonas, Pseudomonas, Torovirus, Cuevavirus, Hypovirus, Trichomonas, Influenzavirus D, Giardiavirus, Crinivirus, Tepovirus, Sakobuvirus, Cyberlindnera, Paenalcaligenes, Bafinivirus, Rymovirus, Pegivirus, Yarrowia, Treponema, Borreliella, Rubivirus, Aureobasidium, Angiostrongylus, Filobasidium, Photobacterium, Rhizopus, Orthoreovirus, Ustilago, Simplexvirus, Aquareovirus, Protoparvovirus, Propionibacterium, Sprivivirus, Hunnivirus, Apophysomyces, Meyerozyma, Alphapapillomavirus, Candida, Brucella, Gallivirus, Dinovernavirus, Anaerobiospirillum, Eubacterium, Tatlockia, Terri sporobacter, Quaranjavirus, Sobemovirus, Dicipivirus, Arcanobacterium, Macanavirus, Atopobium, Vesi virus, Lodderomyces, Dinomavirus, Betatorquevirus, Kerstersia, Aparavirus, Neisseria, Agrobacterium, Edwardsiella, Labyrnavirus, Totivirus, Actinomadura, Tobamovirus, Influenzavirus B, Mandarivirus, Anaerococcus, Kunsagivirus, Naegleria, Campylobacter, Veillonella, Yamadazyma, Filobasidiella, Oerskovia, Penicillium, Anncaliia, Leptosphaeria, Pneumovirus, Psychrobacter, Isavirus, Granulicatella, Torradovirus, Cladophialophora, Influenzavirus A, Ophiostoma, Aerococcus, Ureaplasma, Etatorquevirus, Bocaparvovirus, Megasphaera, Reptarenavirus, Comamonas, Capnocytophaga, Alphatorquevirus, Syncephalastrum, Wallemia, Betacoronavirus, Hyphopichia, Nocardiopsis, Legionella, Trichinella, Paraburkholderia, Mammarenavirus, Echinostoma, Sphingobacterium, Enterovirus, Methanobrevibacter, Ochroconis, Cheravirus, Pasivirus, Enterococcus, Mycoreovirus, Tospovirus, Betanodavirus, Phytoreovirus, Enterocytozoon, Ferlavirus, StemphyliumFilifactor, Leishmaniavirus, Gemella, Bromovirus, Alloiococcus, Cunninghamella, Cronobacter, Oribacterium, Orbivirus, Chrysovirus, Cripavirus, Tatum ella, Pandoraea, Ogataea, Dracunculus, Volvariella, flavirus, Benyvirus, Rhadinovirus, Histoplasma, Rahnella, Morococcus, Verticillium, Janibacter, Gyrovirus, Alphapartitivirus, Mycobacterium, Roseomonas, Varicosavirus, Chryseobacterium, Parapoxvirus, Rhizomucor, Aureimonas, Levivirus, Leishmania, Luteovirus, Cypovirus, Ochrobactrum, Microsporum, Piscihepevirus, Ceratocystis, Sporothrix, Vesiculovirus, Cupriavidus, Cryptococcus, Metapneumovirus, Alphanecrovirus, Eikenella, Brevundimonas, Escherichia, Leifsonia, Schizophyllum, Granulibacter, Gordonibacter, Lachancea, Madurella, Ophiovirus, Phellinus, Nebovirus, Acanthamoeba, Fusobacterium, Pichia, Verruconis, Ehrlichia, Tibrovirus, Higrevirus, Wohlfahrtiimonas, Rhinocladiella, Neorickettsia, Sadwavirus, Roseobacter, Sequivirus,Pannonibacter, Rotavirus, Turicella, Cardiovirus, Propionimicrobium, Furovirus, Naumovozyma, Closterovirus, Fluoribacter, Zeavirus, Clavispora, Megrivirus,Gammapapillomavirus, Rickettsia, Polemovirus, Corynespora, Encephalitozoon, Shimwellia, Fusarium, Yersinia, Capronia, Delftia, Victorivirus, Marafivirus, Kluyvera, Iteradensovirus, Isoptericola, Vitivirus, Roseolovirus, Conidiobolus, Abiotrophia, Babesia, Phoma,Sanguibacteroides, Staphylococcus, Rhodotorula, Zetatorquevirus, Hymenolepis, Fasciola, Cytorhabdovirus, Cardoreovirus, Memnoniella, Trichophyton, Mitovirus, Phaeoacremonium, Providencia, Lysinibacillus, Giardia, Oligella, Streptomyces, Paraclostridium, Ralstonia, Coccidioides, Brambyvirus, Biatriospora, Allolevivirus, Acinetobacter, Starmerella, Omegatetravirus, Porphyromonas, Avulavirus, Streptococcus, Arcobacter, Topocuvirus, Mamastrovirus, Ancylostoma, Bomavirus, Capillovirus, Alphavirus, Tymovirus, Nucleorhabdovirus, Diaporthe, Chlamydiamicrovirus, Tumcurtovirus, Saccharomyces, Riemerella, Betanecrovirus, Clostridium, Mobiluncus, Cercospora, Mamavirus, Mortierella, Aquabimavirus, Xanthomonas, Dependoparvovirus, Ebolavirus, Neofusicoccum, Borrelia, Leminorella, Klebsiella, Blastocystis, Alcaligenes, Citrobacter, Eggerthella, Cedecea, Serratia, Penstyldensovirus, Bacillus, Laribacter, Wuchereria, Hordeivirus, Cytomegalovirus, Actinomucor, Ascaris, Shigella, Vittaforma, Torulaspora, Kingella, Oryzavirus, Polerovirus, Tremovirus, Erbovirus, Entamoeba, Lyssavirus, Paenibacillus, Facklamia, Kappatorquevirus, Metarhizium, Stachybotrys, Okavirus, Botrexvirus, Thetatorquevirus, or Basidiobolus. In some embodiments, a microbe disclosed herein can comprise one or more microbes disclosed in the Drawings or the Examples.Cell-Free Nucleic acids

[0125] In some embodiments, the nucleic acids described herein comprise cell-free nucleic acids (cfNAs). Cell-free nucleic acids are generally fragments of nucleic acids that float freely outside of cells in any body fluid of a subject. In some embodiments, cfNA can comprise plasma cfNA, serum cfNA, whole-blood cfNA, cerebrospinal fluid (CSF) cfNA, saliva cfNA, bronchoalveolar lavage (BAL) cfNA, urine cfNA, amniotic cfNA, fetal cfNA, synovial fluid cfNA, lymphatic cfNA, or any combination thereof. In some cases, cfNA comprise circulating cfNA in a subject’s bloodstream. In some embodiments, the nucleic acids comprise circulating cfDNA, circulating cfRNA, cfDNA, cfRNA, circulating DNA, circulating RNA, or any combination thereof. In some embodiments, a sample of a body fluid can comprise a cfNA.

[0126] The cfNA described herein are, in some embodiments, nucleic acids that, when floating within a body fluid of a subject, are not encapsulated by a cell. In some embodiments, a cfNA comprises nucleic acids that are not encapsulated by a human cell, not encapsulated by a microbial cell, or not encompassed by either a human cell or a microbial cell. The cfNAs can be free-floating, such as cfDNA fragments in plasma. In some embodiments, cfNA includes vesicle- associated cfNA, such as cfNA associated with exosomes, extracellular vesicles, microvesicles, apoptotic bodies, or any combination thereof. In some embodiments, cfNA do not comprise vesicle-associated cfNA, such as cfNA associated with exosomes, extracellular vesicles,microvesicles, apoptotic bodies, or any combination thereof. cfNAs can also be associated with proteins or other cellular constituents, outside of an intact cell. For example, cfNA can comprise free-floating nucleosome-associated cfNA. cfNAs can arise from various biological processes, including cell death (apoptosis, necrosis) or active secretion.

[0127] In some embodiments, cfNA can comprise viral nucleic acids that are not encapsulated by a capsid, that are fragmented, or a combination thereof. Of note, the methods provided herein can, in some embodiments, be practiced using viral nucleic acids derived from whole viruses floating in a bodily fluid.

[0128] In some embodiments, cfNA can be alternatively referred to as free-circulating nucleic acids. In some embodiments, a cfNA can originate from cell death and other processes that release fragments of nucleic acids into a bloodstream or other biological fluid. In some embodiments, a cfNA can be derived from any source of nucleic acids provided herein. In some embodiments, cfNA present in a raw biological sample can be isolated from genomic nucleic acid in the raw biological sample by processing the raw biological sample into an initial sample by removing intact cells. In some embodiments, removing intact cells can comprise centrifuging or filtering a raw biological sample to produce a cell-free fraction of a biological fluid comprising cfNA. In some cases, the removal of intact cells may use a technique targeting a specific cell type. For example, centrifugation at a relative low RPM can remove human or mammalian cells. In some cases, centrifugation at a higher RPM can be used to remove microbial cells such as bacterial cells. In some cases, centrifugation can be performed at an even higher speed (e.g, via ultracentrifugation) in order to remove viral particles. In some cases, afaster spin can be performed after an initial removal (e.g., by centrifugation) of mammalian cells. In some embodiments, a sample can comprise non-host nucleic acids. In some embodiments, non-host nucleic acids can comprise microbial nucleic acids. In some embodiments, microbial nucleic acids can comprise microbial cell-free nucleic acid (mcfNA). In some embodiments, the phrase “target nucleic acids” as used herein can refer to target cfNA. In some embodiments, the phrase “target nucleic acids” as used herein can refer to target mcfNA. In some embodiments, an mcfNA can be derived from one or more kingdoms, divisions, classes, orders, families, genuses, species and / or strain of microbe. In some embodiments, a mcfNA can be derived from a prokaryotic or a eukaryotic microbe. In some embodiments, an mcfNA can comprise a bacterial cfNA, a fungal cfNA, a viral cfNA, a protozoan cfNA, an archaeal cfNA, an algal cfNA, or any combination thereof. In some embodiments, a sample can comprise a non-microbial nucleic acid (e.g., a non- microbial cell-free nucleic acid). In some embodiments, a sample can comprise mcfNAs from one or more species of microbes. In some embodiments, a sample can comprise mcfNAs from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 species of microbes.

[0129] In some embodiments, a sample can comprise a mixture of nucleic acids. In some embodiments, a sample can comprise target nucleic acids (e.g., target cfNAs) and / or non-target nucleic acids. In some cases, the cfNAs comprise cfNA derived from a subject or microbial genome. In some cases, the cfNAs are derived from a housekeeping gene, e.g., a human or mammalian housekeeping gene, a microbial housekeeping gene, a microbe-specific housekeeping gene, and / or a microbe non-specific housekeeping gene.

[0130] In some embodiments, a sample can further comprise contaminant nucleic acids. In some embodiments, contaminant nucleic acids can comprise nucleic acids from a general environment (e.g., a sample collection site). In some embodiments, contaminant nucleic acids are introduced during any step of sample processing. In some embodiments, the contaminant nucleic acids comprise contaminating microbial nucleic acids, contaminating nucleic acids from a different sample, contaminating host nucleic acids, or any combination thereof.

[0131] In some embodiments, cell-free nucleic acids (cfNAs) can comprise a mixture of cfNAs. In some embodiments, a mixture of cfNAs can comprise cfNAs originated from one or more organisms. In some embodiments, a mixture of cfNAs can comprise microbial nucleic acids (e.g., mcfNAs) originated from one or more species of microbes. In some embodiments, an mcfNA can comprise a bacterial -derived cfNA, a fungal-derived cfNA, a viral-derived cfNA, a protozoan-derived cfNA, an archaeal-derived cfNA, an algal-derived cfNA, or any combination thereof.

[0132] In some embodiments, a cfNA can comprise a double-stranded nucleic acid (dsNA), a single-stranded nucleic acid (ssNA), nicked double-stranded, or a combination thereof. In some embodiments, a cfNA can comprise a cell-free DNA (cfDNA), a cell-free RNA (cfRNA), a cell- free DNA-RNA hybrid (cfDNA-RNA), or a combination thereof. In some embodiments, hcfNA can comprise host cell-free DNA (hcfDNA), host cell-free RNA (hcfRNA), host cell-free DNA- RNA hybrid (hcfDNA-RNA), or a combination thereof. In some embodiments, mcfNA can comprise microbial cell-free DNA (mcfDNA), microbial cell-free RNA (mcfRNA), microbial cell-free DNA-RNA hybrid (mcfDNA-RNA), or any combination thereof.

[0133] As used herein, a cell-free samples is generally a sample devoid, or almost devoid, of cells. In some instances, the cell-free sample is devoid of human cells (e.g., intact human cells). In some instances, the cell-free sample is devoid of microbial cells (e.g., intact microbial cells). In some instances, the cell-free sample is devoid of microbial and human cells. In some instances, the cell-free sample is devoid of a particular type of microbial cell or virus, while comprising adifferent type of microbial cell or virus. For example, the cell-free sample can, in some embodiments, be devoid of intact bacterial cells while containing intact viruses. In some instances, the cell-free sample is devoid of all types of microbes, including eukaryotic or prokaryotic cells. The cell-free sample can be obtained from a biological sample provided herein. In some instances, the cell-free sample is a plasma, which has been processed in order to remove blood cells, subject cells, and / or intact microbes, or fragments thereof. In some instances, the cell-free sample can be obtained by a sample preparation process, such as centrifuging, ultracentrifuging, or filtering a biological sample.

[0134] In some embodiments, a cfNA can comprise a host nucleic acid, a non-host nucleic acid, a target nucleic acid, or a combination thereof. In some embodiments, a cfNA can be derived from a host (host cell free nucleic acids or “hcfNA”) or a non-host. In some embodiments, a hcfNA can be derived from nuclear nucleic acids, mitochondria nucleic acids, exosomal nucleic acids, fetal nucleic acids, or any combination thereof. In some embodiments, a sample can comprise a host nucleic acid (e.g., a host cell-free nucleic acid). In some embodiments, a host is any subject provided herein.

[0135] In some embodiments, the nucleic acids from a sample can be extracted and / or enriched to generate enriched nucleic acids. In some embodiments, the enriched nucleic acids comprise degraded nucleic acids, ultra-short nucleic acids, single stranded nucleic acids, double stranded nucleic acids, nicked nucleic acids, microbial cell-free nucleic acids (mcfNA), subject’s cell-free nucleic acids, circulating tumor nucleic acids (ctNA), mitochondrial nucleic acids (mtNA), or any combination thereof. In some embodiments, the methods comprise enriching for degraded nucleic acids, ultra-short nucleic acids, single stranded nucleic acids, or nicked double stranded nucleic acids. As used herein, “degraded nucleic acid” refers to fragments of DNA and RNA that are released into circulation due to cell death, turnover, or pathological processes. Degraded nucleic acids can originate from various sources, including natural sources or artificial sources. In some embodiments, the degraded nucleic acids originate from normal cellular apoptosis, necrosis, or disease-related processes such as cancer or infections. In some embodiments, the degraded nucleic acids originate from factors during laboratory handling, including, but not limited to, sample degradation due to prolonged storage, or temperature fluctuations. In some embodiments, ultrashort nucleic acids comprise nucleic acids less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 60 bp, less than 50 bp, less than 40 bp, or less than 30 bp in length. In some embodiments, at least 70%, 75%, 80%, 85%, 90%, or 95% of the mcfNA are degraded nucleic acids.Sizes of cfNAs

[0136] In some embodiments, a cfNA as disclosed herein or fragments thereof can be approximately less than about 10 bp, less than about 15 bp, less than about 20 bp, less than about 25 bp, less than about 30 bp, less than about 35 bp, less than about 40 bp, less than about 45 bp, less than about 50 bp, less than about 55 bp, less than about 60 bp, less than about 65 bp, less than about 70 bp, less than about 75 bp, less than about 80 bp, less than about 85 bp, less than about 90 bp, less than about 95 bp, less than about 100 bp, less than about 105 bp, less than about 110 bp, less than about 115 bp, less than about 120 bp, less than about 125 bp, less than about 130 bp, less than about 135 bp, less than about 140 bp, less than about 145 bp, less than about 150 bp, less than about 155 bp, less than about 160 bp, less than about 165 bp, less than about 170 bp, less than about 175 bp, less than about 180 bp, less than about 185 bp, less than about 190 bp, less than about 195 bp, or less than about 200 bp long.

[0137] In some embodiments, cfNAs provided herein or fragments thereof can be approximately about 10 bp, about 15 bp, about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 105 bp, about 110 bp, about 115 bp, about 120 bp, about 125 bp, about 130 bp, about 135 bp, about 140 bp, about 145 bp, about 150 bp, about 155 bp, about 160 bp, about 165 bp, about 170 bp, about 175 bp, about 180 bp, bout 185 bp, about 190 bp, about 195 bp, or about 200 bp long. In some embodiments, the cfNAs provided herein comprise ultra short cfNAs. As used herein, “ultra short nucleic acids” refer to subnucleosomal nucleic acids or fragments shorter than subnucleosomal nucleic acids. In some embodiments, ultra short cfNAs can be from about 10 bp to about 100 bp long. In some embodiments, the ultra short cfNAs can be from about 30 bp to about 80 bp long. In some embodiments, the ultra short cfNAs can be from about 40 bp to about 60 bp long. In some embodiments, the ultra short cfNAs are about 50 bases long.

[0138] In some embodiments, mcfNA can be present at higher concentrations relative to hcfNA at lengths that fall outside a nucleosomal interval. In some embodiments, mcfNA can be enriched relative to hcfNA by enriching for cfNA of less than 180bp, less than 170bp, less than 160bp, less than 150bp, less than 140bp, less than 130bp, less than 120bp, less than HObp, less than lOObp, less than 90bp, less than 80bp, less than 70bp, less than 60bp, less than 50bp, less than 40bp, less than 30bp, or less than 20bp. In some embodiments, enriching for mcfNA can comprise enriching for cfNA between 10-180 bp.

[0139] In some embodiments, a cfNA can comprise any nucleic acid that is not encapsulated by a cell (e.g., a eukaryotic or microbial cell). In some embodiments, a cfNA can originate from anynucleic acids. In some embodiments, a cfNA can comprise a plurality of chemical forms of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or DNA / RNA hybrid. In some embodiments, nucleic acids can comprise a plurality of structural forms of DNA, RNA, or DNA / RNA hybrid. In some embodiments, a cfNA can comprise linear nucleic acids or circular nucleic acids. In some embodiments, a cfNA can comprise single stranded nucleic acids (ssNA), double strand nucleic acids (dsNA) or hybrid nucleic acids. In some embodiments, nucleic acids can be from a genome of an organism or an organelle of a cell (e.g., an exosome or a mitochondria). In some embodiments, a cfNA can comprise a mitochondrial DNA, an intercellular signal nucleic acid, an exogenous nucleic acid, a DNA enzyme, a RNA enzyme, a food-derived nucleic acid, any metabolic form of nucleic acid-based therapeutic, or any combination thereof. In some embodiments, a cfNA can be derived from a member selected from the group consisting of genomic DNA, cDNA, mRNA, cRNA, tRNA, ribosomal RNA, miRNA, siRNA, nuclear DNA, nuclear RNA, mitochondrial DNA, mitochondrial RNA, exosomal DNA, exosomal RNA, fetal DNA, fetal RNA, plasmids, vectors, and any combination thereof.

[0140] In some embodiments, nucleic acids can comprise a mixture of nucleic acids from various sources. In some embodiments, nucleic acids can be derived from a plurality of biological fluids. In some embodiments, nucleic acids can be from a plurality of organisms. In some embodiments, nucleic acids can be from a subject. In some embodiments, nucleic acids can be from one or more species of microbes. In some embodiments, nucleic acids can comprise environmental nucleic acids. In some embodiments, environmental nucleic acids can comprise any nucleic acid at or near a sample collection site, or any nucleic acid introduced by personnel, equipment or a reagent used in collecting and / or processing a sample from a subject.Process control molecules

[0141] As used herein, the phrase “process control molecules” refers to molecules that are added to a sample before or during nucleic acid library generation to aid in the identification or quantification of nucleic acids in a sample. In some embodiments, process control molecules can comprise nucleic acids. In some embodiments, process control molecules can comprise synthetic nucleic acids. In some embodiments, process control molecules are separate from and not integrated in the target molecules. In some embodiments, process control molecules can have special features such as specific sequences, lengths, GC content, degrees of degeneracy, degrees of sequence diversity, different secondary, tertiary, or quaternary structures, and / or known starting concentrations. In some embodiments, process control molecules can be used for normalizing the signal in a sample to account for variations in sample processing or to control process performance. In some embodiments, process control molecules can include whole assayintemal control (WINC) molecules. In some embodiments, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 unique WINC molecules are spike in the sample. In some embodiments, process control molecules can include sample identifiers. In some embodiments, process control molecules can comprise dephosphorylation control molecules, denaturation control molecules, and / or ligation control molecules. In some embodiments, multiple different types or sets of control molecules can be added to a sample.

[0142] As used herein, the phrase “adapter attachment control molecule” refers to a control molecule that allows monitoring of the efficiency of an adapter attachment reaction. An adapter attachment reaction can be ligation-based, TdT-based, template-switching-based, primer- extension-based, amplification-based, or a combination thereof.

[0143] As used herein, the phrase “degradation assessment molecules” refers to a control molecule used to evaluate sample and spiked sample integrity during processing.

[0144] As used herein, the phrase “spiked initial sample” refers to an initial sample to which process control molecules (or synthetic spike-ins) have been added prior to the start of generating a sequencing library.

[0145] As used herein, “sequence diversity controls” refers to degenerate pools, or pools of nucleic acids with diverse sequences, which degenerate pools can often be used for diversity assessment, abundance calculation, and / or determination of information transfer efficiency /

[0146] As used herein, “size controls,” “length controls,” “GC Spike-in Panel” or “GC size / length controls” refers to nucleic acids that are size or length or GC-content markers, which can be used for abundance normalization, development, and / or analysis purposes and other purposes.

[0147] As used herein, “ID Spike(s)” refers to identification spikes that can be used, for example without limitation, for sample identification tracking, cross-contamination detection, reagent tracking, and / or reagent lot tracking.Sample Preparation and Processing

[0148] In some embodiments, a sample comprising nucleic acids can be prepared prior to a sequencing assay. In some embodiments, a raw biological sample comprising whole blood can be processed by centrifugation to generate an initial sample of plasma.

[0149] In some embodiments, whole blood can be collected in a K2-EDTA tube. In some embodiments, whole blood draws are not pooled. In some embodiments, a tube can be gently inverted multiple times after draw. In some embodiments, a tube can be centrifuged for about 1200 RCF (g), about 1400 RCF (g), about 1600 RCF (g), or more after draw in order to separateplasma from the blood. The centrifugation can occur at ambient temperature. In some cases, the centrifugation occurs for greater than 5 minutes, 7 minutes, 10 minutes, 15 minutes or 20 minutes. In some embodiments, for tubes containing less than 4 mL a tube manufacturer’s instruction and centrifugation speed and time can be used. In some embodiments, the plasma fraction can be transferred into a new tube. In some embodiments, the plasma is subjected to centrifugation a second time to remove residual cells (e.g., mammalian cells and microbial cells). The additional centrifugation can be conducted at, e.g., about 1400 RCF (g), about 1600 RCF (g), about 1800 RCF (g), about 2000 RCF (g), or more.

[0150] In some embodiments, at least 0.4 ml, at least 0.5 ml, at least 0.7 ml, or at least 1.0 ml of plasma can be transferred into a sterile polypropylene tube, with care taken to not disturb a buffy coat when transferring. In some embodiments, a tube can be labelled with a patient’s first and last name, a unique identifier (DOB or MRN), and / or a date and time of specimen collection. In some embodiments, if a specimen is unlikely to reach a testing facility within 96 hours of collection it can be frozen directly in K2-EDTA after centrifugation. In some embodiments, if a gel plug does not rise to separate cells from plasma, then a tube can be re-centrifuged at a higher speed. In some embodiments, a specimen tube can be shipped to a testing facility.

[0151] In some embodiments, the methods comprise analyzing a cell-free sample. In some embodiments, the methods comprise performing a sequencing assay on the cell-free sample. In some embodiments, the methods comprise preparing a cell-free sample from a biological sample for the sequencing assay. In some instances, the methods comprise centrifuging a biological sample to generate a cell-free sample. In some embodiments, the cell free sample is plasma. In some instances, the methods comprise centrifuging a biological sample to generate a cell-free sample devoid of or almost devoid of human cells. In some instances, the methods comprise centrifuging a biological sample to generate a cell-free sample devoid of or almost devoid of non-human cells, such as a microbe. In some embodiments, the methods described herein comprise processing the sample comprising nucleic acids to maximize collection of target nucleic acids. In some embodiments, the methods can enrich for or maximize the collection of degraded nucleic acids, ultra-short nucleic acids, single stranded nucleic acids, double stranded nucleic acids, nicked nucleic acids, or rare nucleic acids.

[0152] In some embodiments, a nucleic acid can be extracted from a sample. In some embodiments, an extraction can comprise separating nucleic acids from other cellular components and contaminants that can be present in a sample. In some embodiments, a nucleic acid can be extracted from a sample using a liquid extraction (e.g., a Trizol, a DNAzol) technique. In some embodiments, an extraction can be performed by phenol chloroform extraction or precipitation by organic solvents (e.g., ethanol, or isopropanol). In some embodiments, anextraction can be performed using a nucleic acid-binding column, a nucleic acid-binding spin column, or a combination thereof. In some cases, an extraction of a cell-free nucleic acid can involve filtration or ultra-filtration. In some embodiments, a nucleic acid can be extracted or purified by use of magnetic beads that bind nucleic acids. In some embodiments, compositions of a binding buffer can be adjusted to control a strength of bonds between functional groups and a nucleic acid, allowing for controlled and reversible binding. In some embodiments, a nucleic acid can be released from a magnetic particle with an elution buffer.

[0153] In some embodiments, the methods described herein comprise enriching a population of cfNA. In some embodiments, enriching a population of cfNA comprises bioinformatically enriching or physically enriching. In some embodiments, enriching a population of cfNA comprises isolating, extracting, or selectively amplifying a desired population of cfNA (e.g., target cfNA) from the initial sample. In some embodiments, enriching a population of cfNA does not comprise isolating or extracting the desired population of cfNA from the initial sample. In some embodiments, enriching a population of cfNA does not comprise amplifying the desired population of cfNA. In some embodiments, enriching a population of cfNA comprises removing the undesired cfNA population (e.g., non-target cfNA or contaminants) from the initial sample. In some embodiments, enriching cfNA can comprise differentiating and / or selecting the cfNA by one or more characteristics comprising size, sequence, GC content, secondary structure, biological source, or protein-binding.

[0154] In some embodiments, the methods comprise enriching microbial cfNA (mcfNA) in the biological sample. In some embodiments, the methods provided herein comprise enriching for at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the mcfNA in the biological sample. In some embodiments, enriching mcfNA comprises enriching cfNA that are less than about 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, 200 bp, 210 bp, 220 bp, 230 bp, 240 bp, or 250 bp in length. In some embodiments, enriching mcfNA comprises amplifying the nucleic acids in the initial sample with primers containing non-human nucleic acid sequences. In some embodiments, enriching mcfNA comprises removing non-microbial cfNA. In some embodiments, enriching mcfNA comprises removing nucleosome-bound cfNA. In some embodiments, the methods comprise enriching non-microbial cfNA (e.g., host cfNA) in the biological sample. In some embodiments, the methods comprise enriching mammalian cfNA in the biological sample.

[0155] In some embodiments, the methods comprise enriching cfNA by size selection. In some embodiments, size selection can comprise removing nucleic acids not in the desired size range. In some embodiments, the desired size range comprises an artificial or engineered threshold. Insome embodiments, size selection comprises separating nucleic acids by size via chromatography (e.g., size-exclusion chromatography), electrophoresis (e.g., gel or capillary electrophoresis), centrifugation (e.g., density -gradient centrifugation), filtration (e.g., membrane ultrafiltration), magnetic bead-based methods (e.g., SPRI beads), affinity-based methods (e.g., streptavidin beads), or any combination thereof. In some embodiments, the methods described herein can comprise discriminating or differentiating cfNA by size and / or selectively isolating or extracting the cfNA of the desired size range. In some embodiments, the methods comprise differentiating microbial cfNA from the non-microbial cfNA (e.g., host cfNA) in the sample and selectively removing the non-microbial cfNA in the sample by size. In some embodiments, the non- microbial cfNA comprises mammalian cfNA (e.g., human or animal cfNA).

[0156] In some embodiments, the methods can comprise selectively removing nucleic acid fragments greater than about 500 bp, about 450 bp, about 400 bp, about 350 bp, about 300 bp, about 250 bp, about 200 bp, about 150 bp, about 140 bp, about 130 bp, about 120 bp, about 110 bp, about 100 bp, about 90 bp, about 80 bp, about 70 bp, or about 60 bp in length. In some embodiments, the methods can comprise selectively enriching nucleic acid fragments at most about 20 bp, about 30 bp, about 40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 160 bp, about 170 bp, about 180 bp, about 190 bp, about 200 bp, about 210 bp, about 220 bp, about 230 bp, about 240 bp, or about 250 bp in length.

[0157] In some embodiments, the methods can comprise selectively enriching nucleic acid fragments of about 10 bp to about 20 bp, about 10 bp to about 30 bp, about 10 bp to about 40 bp, about 10 bp to about 50 bp, about 10 bp to about 60 bp, about 10 bp to about 70 bp, about 10 bp to about 80 bp, about 10 bp to about 90 bp, about 10 bp to about 100 bp, about 10 bp to about 110 bp, about 10 bp to about 120 bp, about 10 bp to about 130 bp, about 10 bp to about 140 bp, about 10 bp to about 150 bp, about 10 bp to about 160 bp, about 10 bp to about 170 bp, about 10 bp to about 180 bp, about 10 bp to about 190 bp, about 10 bp to about 200 bp, about 10 bp to about 210 bp, about 10 bp to about 220 bp, about 10 bp to about 230 bp, about 10 bp to about 240 bp, or about 10 bp to about 250 bp in length. In some embodiments, the methods can comprise selectively enriching nucleic acid fragments of about 20 bp to about 250 bp, about 20 bp to about 200 bp, about 20 bp to about 150 bp, about 20 bp to about 100 bp, about 20 bp to about 90 bp, about 20 bp to about 80 bp, about 20 bp to about 70 bp, about 20 bp to about 60 bp, about 20 bp to about 50 bp, about 30 bp to about 250 bp, about 30 bp to about 200 bp, about 30 bp to about 150 bp, about 30 bp to about 100 bp, about 30 bp to about 90 bp, about 30 bp to about 80 bp, about 30 bp to about 70 bp, about 30 bp to about 60 bp, about 30 bp to about 50 bp, about 40 bp to about 250 bp, about 40 bp to about 200 bp, about 40 bp to about 150 bp, about 40 bp toabout 100 bp, about 40 bp to about 90 bp, about 40 bp to about 80 bp, about 40 bp to about 70 bp, about 40 bp to about 60 bp, or about 40 bp to about 50 bp in length.Exemplary Process 1 - double-stranded cfDNA

[0158] In some embodiments, the methods provided herein comprise performing process 1 to prepare a sample for high throughput sequencing assay. Process 1 provides an example of preparing a sequencing library from the double-stranded cfDNA in the original sample. In some embodiments, a control molecule can be added to an initial sample of plasma to generate a spiked plasma sample. In some embodiments, nucleic acid extraction can be performed on a spiked plasma sample to generate purified and concentrated cfDNA.

[0159] In some embodiments, a library preparation process can be performed on a purified and concentrated cfDNA sample. In some embodiments, the library preparation can comprise attaching (e.g., by ligation) double-stranded adapters to double-stranded cfDNA. In some embodiments, library preparation can comprise performing unbiased amplification on the sample.

[0160] In some embodiments, a sample preparation method does not comprise extracting nucleic acids from a raw or initial sample. For example, in some cases, nucleic acids can be extracted during or following library preparation, if at all.

[0161] In some embodiments, an adapter pair can be attached to cfDNA fragments in a sample such as by ligation or PCR amplification. In some embodiments, a pair of adapters can comprise a p5 adapter that is attached to a 5’ end of a molecule and a p7 adapter that is attached to a 3’ end of a molecule. In some embodiments, a p5 and p7 sequence can allow a nucleic acid library to bind and generate clusters on a flow cell.

[0162] In some cases, the cfDNA can be attached to adapters comprising identifier sequences that can differentiate between multiple samples. In some embodiments, the multiple samples comprise a plurality of patient samples and / or control samples (e.g., positive control, negative control). In some embodiments, samples can be pooled after barcoding, then sequenced, then demultiplexed to assign each cluster to its sample.Exemplary Process 2- double-stranded cfDNA and single-stranded cfDNA

[0163] In some embodiments, the methods provided ...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for performing a multi-step assay for detecting nucleic acids in a sample from a subject, the method comprising:(a) providing a first aliquot of the sample from the subject, wherein the sample comprises nucleic acids comprising a first target nucleic acid and a second target nucleic acid that is different from the first target nucleic acid;(b) performing a sequencing assay on the nucleic acids to produce sequence reads comprising sequence reads associated with the first target nucleic acid;(c) analyzing the sequence reads, thereby obtaining an identification of the first target nucleic acid;(d) after the identification of the first target nucleic acid is obtained, providing a second aliquot of the sample comprising nucleic acids comprising the first target nucleic acid and the second target nucleic acid;(e) introducing primers into the second aliquot or to nucleic acids derived from the second aliquot wherein the primers specifically target the second target nucleic acid and do not target the first target nucleic acid; and(f) conducting an amplification reaction on the second target nucleic acid with the primers, thereby amplifying the second target nucleic acid and producing amplicons associated with the second target nucleic acid.

2. The method of claim 1, further comprising performing a high-throughput sequencing assay on the amplicons associated with the second target nucleic acid.

3. The method of any one of the preceding claims, wherein the second target nucleic acid is not detected by the sequencing assay in (b).

4. The method of any one of the preceding claims, wherein the first nucleic acid is associated with a genome of an organism but not a phenotype of interest of the organism and the second nucleic acid is associated with the phenotype of interest of the organism.

5. The method of any one of the preceding claims wherein the first target nucleic acid comprises at least two, at least three, at least four, at least five, at least ten, at least 15, at least 20, or at least 25 first target nucleic acids; or the second target nucleic acid comprises at least two, at least three, at least four, at least five, at least ten, at least 15, at least 20, or at least 25 second target nucleic acids.

6. The method of any one of the preceding claims wherein the first target nucleic acid or the second target nucleic acid are not negative or positive controls for any step of the multi-step assay.

7. The method of any one of the preceding claims, wherein the primers comprise multiple primers targeting multiple target nucleic acids.

8. The method of any one of the preceding claims, wherein the first target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae.

9. The method of any one of the preceding claims, wherein the second target nucleic acid comprises nucleic acids from an animal, a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae.

10. The method of any one of the preceding claims, wherein the second target nucleic acid comprises a cancer marker.

11. The method of any one of the preceding claims, wherein the first target nucleic acid is associated with a carrier microbe harboring a target genetic marker and the second target nucleic acid comprises a sequence associated with the target genetic marker.

12. The method of claim 11, where the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae.

13. The method of any one of the preceding claims, wherein the target genetic marker comprises an antimicrobial resistance (AMR) genetic marker.

14. A method for performing a multi-step sample processing procedure for detecting nucleic acids in a sample from a subject:(a) providing a first aliquot of the sample from the subject, wherein the sample comprises one or more first nucleic acids, wherein the one or more first nucleic acids comprise one or more microbial nucleic acids derived from one or more carrier microbes harboring an antimicrobial resistance (AMR) genetic marker;(b) performing a sequencing assay on the one or more first nucleic acids to produce sequence reads comprising microbial nucleic acid sequence reads from the one or more carrier microbes;(c) detecting the one or more carrier microbes by analyzing the microbial nucleic acid sequence reads;(d) after at least one carrier microbe is detected, providing a second aliquot of the sample from the subject, wherein the second aliquot comprises second nucleic acids from at least one AMR genetic marker;(e) annealing primers targeting at least one AMR genetic marker to the second nucleic acids; and(f) amplifying the AMR genetic marker in an amplification reaction to produce amplicons associated with the at least one AMR genetic marker.

15. A method for assaying nucleic acids in a sample from a subject comprising:(a) providing a first aliquot of the sample from the subject, wherein the sample comprises one or more first nucleic acids, wherein the one or more first nucleic acids comprise one or more microbial nucleic acids derived from one or more carrier microbes harboring an antimicrobial resistance (AMR) genetic marker;(b) performing a sequencing assay on the one or more first nucleic acids to produce sequence reads comprising microbial nucleic acid sequence reads from the one or more carrier microbes;(c) detecting the one or more carrier microbes by analyzing the microbial sequence reads;(d) providing a second aliquot of the sample from the subject, wherein the second aliquot comprises second nucleic acids, wherein the second aliquot comprises second nucleic acids from at least one AMR genetic marker;(e) annealing primers to the second nucleic acids that target the at least one AMR genetic marker, wherein the primers that target the at least one AMR genetic marker are annealed prior to amplification of nucleic acids in the sample; and(f) conducting an amplification reaction on the second nucleic acids using the primers targeting the at least one AMR genetic marker, thereby amplifying at least one AMR genetic marker and producing amplicons associated with the at least one AMR genetic marker.

16. A method for performing a multi-step sample processing procedure for detecting nucleic acids in a sample from a subject:(a) providing a first aliquot of the sample from the subject, wherein the sample comprises one or more first nucleic acids, wherein the one or more first nucleic acids comprise one or more microbial nucleic acids derived from one or more carrier microbes harboring an antimicrobial resistance (AMR) genetic marker;(b) performing a sequencing assay on the one or more first nucleic acids to produce sequence reads comprising microbial nucleic acid sequence reads from the one or more carrier microbes;(c) detecting the one or more carrier microbes by analyzing the microbial nucleic acid sequence reads;(d) after at least one carrier microbe is detected, providing a second aliquot of the sample from the subject, wherein the second aliquot comprises second nucleic acids from at least one AMR genetic marker;(e) annealing primers to the second nucleic acids that target at least one AMR genetic marker; and(f) amplifying the AMR genetic marker and producing amplicons associated with the at least one AMR genetic marker.

17. A method for assaying nucleic acids in a sample from a subject comprising:(a) providing a first aliquot of the sample from the subject, wherein the first aliquot comprises first nucleic acids, and the first nucleic acids comprise an AMR genetic marker and a gene cassette comprising the AMR genetic marker derived from one or more carrier microbes harboring the AMR genetic marker;(b) performing a sequencing assay on the first nucleic acids to produce sequence reads comprising microbial nucleic acid sequence reads from the one or more carrier microbes;(c) detecting the AMR genetic marker or the gene cassette comprising the AMR genetic marker in the sample and quantifying microbial sequence reads from the AMR genetic marker and / or microbial sequence reads from the gene cassette;(d) after and based on the quantification, providing a second aliquot of the sample from the subject, wherein the second aliquot comprises second nucleic acids, wherein the second nucleic acids comprise the AMR genetic marker;(e) introducing primers targeting the AMR genetic marker to the second nucleic acids, wherein the primers targeting the AMR genetic marker are introduced prior to amplification of nucleic acids in the second aliquot; and(f) conducting an amplification reaction on the second nucleic acids using the primers targeting the AMR genetic marker, thereby amplifying the AMR genetic marker and producing amplicons associated with the AMR genetic marker.

18. The method of any one of the preceding claims, further comprising in (d) comparing an abundance of the microbial sequence reads from the AMR genetic marker to a threshold value.

19. The method of any one of the preceding claims, further comprising in (d) comparing an abundance of the microbial sequence reads from the gene cassette to a threshold value.

20. The method of any one of the preceding claims, further comprising providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the AMR genetic marker is below the threshold.

21. The method of any one of the preceding claims, further comprising providing a second aliquot for the amplification reaction when the abundance of the microbial sequence reads from the gene cassette is below the threshold.

22. The method of any one of the preceding claims, further comprising in (d) quantifying mcfNA sequencing reads from the one or more microbes.

23. The method of any one of the preceding claims, further comprising comparing the abundance of the mcfNA sequencing reads from the one or more microbes to a threshold.

24. The method of any one of the preceding claims, further comprising providing a second aliquot for the amplification reaction when the abundance of the mcfNA sequencing reads from the one or more microbes is below the threshold.

25. The method of any one of the preceding claims, further comprising performing high- throughput sequencing on the amplicons associated with the AMR genetic marker.

26. The method of any one of the preceding claims, wherein the sequencing assay on the first nucleic acids comprises a high-throughput sequencing assay.

27. The method of any one of the preceding claims, further comprising conducting a polymerase chain reaction (PCR) to amplify the AMR genetic marker, thereby producing amplicons associated with the AMR genetic marker.

28. The method of any one of the preceding claims, wherein the PCR comprises multiplex PCR, random PCR (rPCR), non-biased PCR, Nested PCR, Hot Start PCR, or Assembly PCR.

29. The method of any one of the preceding claims, further comprising attaching an adapter sequence to the second nucleic acids.

30. The method of any one of the preceding claims, wherein the primers comprise an adapter sequence.

31. The method of any one of the preceding claims, further comprising physically manipulating the sample to produce a fraction of cfNA enriched for degraded cfNA, wherein the fraction of cfNA comprises the AMR genetic marker.

32. The method of any one of the preceding claims, wherein the degraded cfNA comprises ultra short cfNA, single stranded cfNA, or nicked double stranded cfNA.

33. The method of any one of the preceding claims, wherein the ultra short cfNA comprises cfNA fragments that are less than 100 nucleotides in length.

34. The method of any one of the preceding claims, wherein the ultra short cfNA comprises cfNA fragments that are from 30 nucleotides to 70 nucleotides in length.

35. The method of any one of the preceding claims, wherein physically manipulating the sample comprises performing size selection of the nucleic acids in the sample.

36. The method of any one of the preceding claims, wherein performing size selection using a method selected from the group consisting of: chromatography, size-exclusion chromatography, electrophoresis, gel electrophoresis, automated electrophoresis,capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation, filtration, membrane ultrafiltration, magnetic beads, and affinity-based beads.

37. The method of any one of the preceding claims, wherein the second aliquot comprises at least 500 pl of plasma.

38. The method of any one of the preceding claims, wherein detecting the carrier microbe comprises determining an abundance of the carrier microbe.

39. The method of any one of the preceding claims, wherein the abundance is expressed as molecules of mcfNA per microliter of sample (MPM).

40. The method of any one of the preceding claims, further comprising calculating an AMR gene copy number from the amplification or sequencing of the AMR genetic marker.

41. The method of any one of the preceding claims, further comprising linking the AMR genetic marker to the carrier microbe using the abundance of the mcfNA from the carrier microbe and the AMR gene copy number.

42. The method of any one of the preceding claims, where the AMR gene copy number is an episomal gene copy number.

43. The method of any one of the preceding claims, comprising introducing at least 200 primers targeting the plurality of AMR genetic markers into the second aliquot of the sample.

44. The method of any one of the preceding claims, further comprising introducing primers targeting housekeeping genes into the second aliquot of the sample.

45. The method of any one of the preceding claims, further comprising determining antimicrobial resistance of the microbe infecting the subject.

46. The method of any one of the preceding claims, wherein the sample comprises plasma.

47. The method of any one of the preceding claims, wherein the first nucleic acids are DNA.

48. The method of any one of the preceding claims, wherein the second nucleic acids are DNA.

49. The method of any one of the preceding claims, wherein the sample is not subjected to a process that primarily causes cell lysis.

50. The method of any one of the preceding claims, further comprising preparing a library from the amplicons associated with the AMR genetic marker .

51. The method of any one of the preceding claims, wherein the primers are added directly to the second aliquot.

52. The method of any one of the preceding claims, wherein the sample comprises cell-free nucleic acids.

53. The method of any one of the preceding claims, wherein the sample comprises cell-free DNA.

54. The method of any one of the preceding claims, wherein the first nucleic acids comprise microbial cell-free nucleic acids.

55. The method of any one of the preceding claims, wherein the second nucleic acids comprise microbial cell-free nucleic acids.

56. The method of any one of the preceding claims, wherein intact microbes are not actively lysed prior to performing the sequencing assay.

57. A method for preparing a cell-free DNA fraction from a subject infected with a microbe useful for analyzing an antimicrobial resistance (AMR) genetic marker, comprising:(a) providing a sample from the subject, wherein:(i) the sample comprises cell-free nucleic acids (cfNA) from the subject and microbial nucleic acids from the microbe infecting the subject;(ii) the cfNA comprises degraded cfNA, single-stranded cfNA and intact cfNA; and (iiii) the microbe harbors an AMR genetic marker;(b) physically manipulating the sample in order to produce a fraction of cfNA enriched for degraded cfNA and single-stranded cfNA, wherein the fraction of cfNA comprises microbial nucleic acids comprising a genetic locus associated with the AMR genetic marker;(c) performing a sequencing assay on the fraction of cfNA enriched for the degraded cfNA and single-stranded cfNA; and(d) analyzing the genetic locus associated with the AMR genetic marker.

58. The method of any one of the preceding claims, wherein the microbial nucleic acids comprise microbial cell-free nucleic acids (mcfNA) from the microbe.

59. The method of any one of the preceding claims, wherein the microbial nucleic acids comprise nucleic acids associated with a microbial cell.

60. The method of any one of the preceding claims, wherein (c) comprises enriching for at least at least 75%, at least 80%, at least 85%, or at least 90% of the mcfNA in the sample.

61. The method of any one of the preceding claims, wherein the degraded cfNA comprise ultra short cfNA, single stranded cfNA, nicked double stranded cfNA, or any combination thereof.

62. The method of any one of the preceding claims, wherein the degraded cfNA comprise cfNA fragments that are less than 100 nucleotides in length.

63. The method of any one of the preceding claims, wherein the degraded cfNA comprise cfNA fragments that are from 30 nucleotides to 70 nucleotides in length.

64. The method of any one of the preceding claims, wherein physically manipulating the sample comprises performing size selection of the nucleic acids in the sample.

65. The method of claim 64, wherein performing size selection using a method selected from the group consisting of: chromatography, size-exclusion chromatography, electrophoresis, gel electrophoresis, automated electrophoresis, capillary electrophoresis, high-throughput size selection, centrifugation, density-gradient centrifugation, filtration, membrane ultrafiltration, magnetic beads, and affinity -based beads.

66. The method of any one of the preceding claims, further comprising generating sequence reads from the cfNA from the subject, wherein the sequence reads comprise microbial sequencing reads derived from the microbe infecting the subject.

67. The method of any one of the preceding claims, wherein the sequence reads further comprise microbial sequencing reads from one or more carrier microbe.

68. The method of any one of the preceding claims, further comprising calculating an abundance of the mcfNA from the microbe in the sample and an abundance of the mcfNA from the one or more carrier microbe.

69. The method of any one of the preceding claims, wherein the abundance is expressed as molecules of mcfNA per microliter of sample (MPM).

70. The method of any one of the preceding claims, wherein the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample.

71. The method of any one of the preceding claims, further comprising calculating an AMR gene copy number for each of the one or more carrier microbe.

72. The method of any one of the preceding claims, where the AMR gene copy number is an episomal gene copy number.

73. The method of any one of the preceding claims, further comprising detecting one or more carrier microbe from the sequencing assay.

74. The method of any one of the preceding claims, further comprising identifying the one or more carrier microbe as the microbe infecting the subject.

75. The method of any one of the preceding claims, further comprising linking the AMR genetic marker to one of the carrier microbes or the microbe infecting the subject.

76. The method of any one of the preceding claims, further comprising determining antimicrobial resistance of the microbe infecting the subject.

77. A method for determining antimicrobial resistance of a microbe infecting a subject, comprising:(a) providing a sample comprising nucleic acids from the subject, wherein(b) the nucleic acids comprise microbial nucleic acids from one or more potential carrier microbes harboring an antimicrobial resistance (AMR) genetic marker;(c) performing a sequencing assay on the nucleic acids from the subject to generate sequence reads and detecting the one or more potential carrier microbe based on the sequence reads;(d) if a carrier microbe is detected in (c), performing an amplification reaction on an aliquot of the sample, wherein the amplification reaction targets one or more AMR genetic markers in order to provide a copy number for the one or more AMR genetic markers; and(e) conducting a statistical analysis to link one or more of the AMR genetic markers to one of the one or more potential carrier microbe in the subject, thereby determining that a potential carrier microbe comprises an AMR genetic marker.

78. The method of any one of the preceding claims, further comprising obtaining an abundance of the one or more carrier microbes based on an abundance of carrier microbe sequences in the sample.

79. The method of any one of the preceding claims, wherein conducting a statistical analysis comprises using the abundance of the microbial nucleic acids of the one or more potential carrier microbes.

80. The method of any one of the preceding claims, wherein conducting a statistical analysis comprises using the AMR gene copy number.

81. The method of any one of the preceding claims, where the AMR gene copy number is an episomal gene copy number.

82. The method of any one of the preceding claims, wherein the abundance is expressed as molecules of mcfNA per microliter of sample (MPM).

83. The method of any one of the preceding claims, wherein the abundance is expressed as molecules of mcfNA per 100 nanoliters of sample.

84. The method of any one of the preceding claims, further comprising calculating a probability of each of the one or more potential carrier microbes being the microbe harboring the AMR genetic marker, thereby identifying the potential carrier microbe as the microbe infecting the subject.

85. The method of any one of the preceding claims, further comprising determining the antimicrobial resistance of the microbe infecting the subject based on the calculated probability.

86. A method for determining antimicrobial resistance of a microbe infecting a subject, comprising:(a) providing a first aliquot of the sample comprising first nucleic acids, wherein the first nucleic acids comprise microbial nucleic acids derived from a potential carrier microbe harboring an antimicrobial resistance (AMR) genetic marker;(b) detecting the potential carrier microbe by analyzing the first nucleic acids;(c) after the potential carrier microbe is detected, providing a second aliquot of the sample comprising second nucleic acids, wherein the second nucleic acids comprise microbial nucleic acids from the potential carrier microbe harboring an AMR genetic marker;(d) conducting an amplification reaction on the second nucleic acids to detect the AMR genetic marker; and(e) determining the antimicrobial resistance of the microbe infecting the subject based on the AMR genetic marker detection.

87. The method of any one of the preceding claims, wherein the detecting comprises performing a sequencing assay.

88. The method of any one of the preceding claims, wherein the sequencing assay comprises a high-throughput sequencing assay.

89. The method of any one of the preceding claims, wherein the amplification reaction comprises introducing primers targeting a plurality of AMR genetic markers.

90. The method of any one of the preceding claims, wherein the amplification reaction produces amplicons associated with the AMR genetic marker.

91. The method of any one of the preceding claims, further comprising performing high- throughput sequencing on the amplicons associated with the AMR genetic marker.

92. The method of any one of the preceding claims, wherein the sample comprises plasma.

93. The method of any one of the preceding claims, wherein the first nucleic acids are DNA.

94. The method of any one of the preceding claims, wherein the second nucleic acids are DNA.

95. The method of any one of the preceding claims, wherein the sample does not undergo cell lysis.

96. The method of any one of the preceding claims, wherein intact microbes are not actively lysed.

97. The method of any one of the preceding claims, wherein the amplicons associated with the AMR genetic marker undergoes direct library preparation.

98. The method of any one of the preceding claims, wherein the sample comprises cell-free nucleic acids.

99. The method of any one of the preceding claims, wherein the sample comprises cell-free DNA.

100. The method of any one of the preceding claims, wherein the first nucleic acids comprise cell-free nucleic acids.

101. The method of any one of the preceding claims, wherein the second nucleic acids comprise cell-free nucleic acids.

102. The method of any one of the preceding claims, wherein (d) comprises adding primers targeting a plurality of AMR genetic markers into the second aliquot of the sample.

103. The method of any one of the preceding claims, comprising adding at least 200 primers targeting a plurality of AMR genetic markers into the second aliquot of the sample.

104. The method of any one of the preceding claims, further comprising introducing primers targeting housekeeping genes into the second aliquot of the sample.

105. The method of any one of the preceding claims, wherein the second aliquot of the sample comprises at least 500 pl of plasma.

106. The method of any one of the preceding claims, wherein detecting the pathogen comprises detecting the abundance of mcfNA from the pathogen in the sample over a threshold.

107. The method of any one of the preceding claims, further comprising calculating a positive percent agreement (PPA), negative percent agreement (NPA), diagnostic yield (DY), or any combination thereof.

108. The method of any one of the preceding claims, further comprising detecting cfDNA from one or more housekeeping genes.

109. The method of any one of the preceding claims, further comprising spiking one or more control molecules into the sample at a known concentration.

110. The method of any one of the preceding claims, wherein the one or more control molecules are synthetic oligonucleotides.

111. The method of any one of the preceding claims, wherein the control molecules comprise whole assay internal control (WINC) molecules.

112. The method of any one of the preceding claims, further comprising spiking at least 25,000 unique WINC molecules at known concentrations.

113. The method of any one of the preceding claims, further comprising generating a report listing the carrier microbes or pathogen detected in the subject.

114. The method of any one of the preceding claims, wherein the report further comprises the abundance of microbial cell-free DNA (mcfDNA) from microbes detected in the sample or the antimicrobial resistance of the microbes infecting the subject.

115. The method of any one of the preceding claims, wherein the sample comprises a biological sample obtained from the subject.

116. The method of any one of the preceding claims, wherein the biological sample is a whole blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a synovial fluid sample, a urine sample, a stool sample, a bronchoalveolar lavage sample, or any combination thereof.

117. The method of any one of the preceding claims, wherein the sample is a plasma sample.

118. The method of any one of the preceding claims, where the AMR genetic marker comprises a gene, a genetic element, a genetic cassette, an allele, a mutation, or any combination thereof.

119. The method of any one of the preceding claims, where the AMR genetic marker is associated with one or more genes selected from the group consisting of: SCCmec, mecA, mecC, vanA, vanB, blacrx-M, blctKPC, OXA-48-like, OXA-23, NDM, VIM, IMP, or mcr-1.

120. The method of any one of the preceding claims, where the AMR genetic marker provides resistance to an anti-microbial agent selected from the group consisting of: methicillin, vancomycin, cephalosporin, carbapenem, and oxyimino-cephalosporin / aztreonam resistance.

121. The method of any one of the preceding claims, where the microbe infecting the subject or the carrier microbe comprises a virus, a bacterium, a protozoa, a fungus, an archaea, or an algae.

122. The method of any one of the preceding claims, where the microbe infecting the subject or the carrier microbe comprises a gram-positive bacterium or a fungus.

123. The method of any one of the preceding claims, where the microbe infecting the subject or the carrier microbe is a microbe listed in Table 1.

124. The method of any one of the preceding claims, where the microbe infecting the subject or the carrier microbe harbors at last two AMR genetic markers.

125. The method of any one of the preceding claims, wherein the antimicrobial resistance is phenotypic antimicrobial resistance.

126. The method of any one of the preceding claims, wherein the sequencing comprises nextgeneration sequencing or a sequencing method beyond next generation sequencing.

127. The method of any one of the preceding claims, wherein the sequencing comprises sequencing by synthesis.

128. The method of any one of the preceding claims, wherein the subject is an animal.

129. The method of any one of the preceding claims, wherein the subject is a human.

130. The method of any one of the preceding claims, wherein the subject has an infection by the microbe infecting the subject harboring the AMR genetic marker.

131. The method of any one of the preceding claims, further comprising administering an anti- infective agent to the subject.

132. The method of any one of the preceding claims, wherein the subj ect has been treated with an anti-infective agent for an infection.

133. The method of any one of the preceding claims, further comprising adjusting the antimicrobial agent received by the subject at least in part based on the antimicrobial resistance of the microbe infecting the subject.

134. The method of any one of the preceding claims, further comprising determining antimicrobial resistance of a microbe.

135. The method of claim 134, wherein the microbe comprises a microbe infecting the subject.

136. The method of claim 135, wherein the microbe comprises a carrier microbe.

137. The method of any one of the preceding claims wherein the method comprises determining whether an AMR gene is carried by a microbe infecting the subject or by a carrier microbe.

138. The method of any one of claims 134-137, wherein the determining comprises linking an AMR genetic marker to one of the carrier microbes or the microbe infecting the subject.

139. The method of any one of claims 134-138, wherein the determining comprises determining a copy number of the AMR genetic marker in the microbe.

140. The method of claim 138 or 139, wherein the linking comprises performing a statistical analysis on the sequence reads.

141. The method of claim 140, wherein the statistical analysis comprises a generalized linear model, a maximum-likelihood estimation, a probit model, a logistic regression, a linear probability, a linear regression, a complimentary log-log, a Poisson regression, a support vector machine, a decision tree, a random forest, a neural network, a gradient boosted model, a Bayesian model, a hidden Markov model, or any combination thereof.

142. The method of any one of claims 134-141, wherein the method further comprises comparing the sequence read data to the amplification data.

143. The method of any one of claims 134-142, wherein the method further comprises calculating estimated deduplicated templates (EDT) for an AMR gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library.

144. The method of any one of claims 134-143, wherein the method further comprises calculating an EDT for a housekeeping gene, wherein calculating EDT comprises calculating a number of unique DNA sequencing reads from a microorganism present in a sequenced library.

145. The method of claim 143 or 144, further comprising determining a presence of an organism in a sample by calculating a ratio of an AMR estimated deduplicated templates (EDT) to a housekeeping gene EDT.

146. The method of any one of the preceding claims, wherein the method further comprises administering an alternative therapy to the subject if an AMR marker is detected.

147. The method of any one of the preceding claims, wherein the method further comprises detecting methicillin-resistant Staph aureus by detecting SCCmec, mecA, or mecC.

148. The method of any one of the preceding claims, wherein the method further comprises detecting methicillin-resistant Staph aureus and administering ceftaroline, ceftobiprole, vancomycin, linezolid, daptomycin, ceftobipole or combination thereof to the subject in order to treat the methicillin-resistant staph aureus.

149. The method of any one of the preceding claims, wherein the method further comprises detecting vancomycin resistant enterococcus by detecting VanA or VanB.

150. The method of any one of the preceding claims, wherein the method further comprises detecting vancomycin resistant enterococcus and administering linezolid or daptomycin to the subject in order to treat the vancomycin resistant enterococcus.

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