WO2020041449A1 - Methods and compositions for tracking sample quality - Google Patents

Abstract

Reagents for storage, lysis or purification of a biological sample comprising a spike-in standard are provided. Method for using such standards to assess and monitor samples are also provided.

Description

DESCRIPTION

METHODS AND COMPOSITIONS FOR TRACKING SAMPLE QUALITY

[0001] This application claims the benefit of United States Provisional Patent Application No. 62/720,250, filed August 21, 2018, the entirety of which is incorporated herein by reference.

BACKGROUND

1. Field

[0002] The present invention generally relates to biochemistry and molecular biology. More specifically, the invention relates to methods and compositions for assessing or tracking the quality of measurements upon a biological sample as well as changes in the condition of the sample itself.

2. Description of Related Art

[0003] A variety of protocols have been developed for purification of biological molecules, such as proteins and nucleic acids. However, the processes of isolation, storage and purification of such molecules can all potentially lead to degradation or loss of specific types of molecules. To date there have not been adequate methodologies to assess the loss or degradation of such molecules, which has hampered the ability to collect accurate information about the same. Thus, there is a need in the art for methods assessing loss or degradation of biomolecules during isolation, storage and purification. SUMMARY

[0004] In a first embodiment the invention provides a reagent for storage, lysis or purification of a biological sample comprising a spike-in standard. In some aspects, the biological sample comprises bacteria, archaea, fungi, eurkaryote, viruses, DNA, RNA, proteins, metabolites, lipids, and/or carbohydrates. In several aspects, the spike-in standard comprises varying sizes of DNA, RNA (including miRNA), modified DNA (e.g., including non -natural or chemically modified nucleotides) and modified RNA (e.g., including non - natural or chemically modified nucleotides). In further aspects, the spike-in standard comprises a cell (or a plurality of cells), such as a microbes (e.g., Eubacteria, Archaebacterial, yeast or fungi) or viruses. In other aspects, the biological sample is a fecal sample, sputum sample, saliva sample, or blood sample. In specific aspects, the biological sample is a human biological sample. In certain aspects, the spike-in standard is a cellular spike-in standard. In alternative aspects, the spike-standard comprises 2 or 3 microbes. In a further aspect, the microbes may be Treupera radiovictrix, Imtechella halotolerans , and/ or Allobacillus halotolerans .

[0005] In additional aspects, wherein the spike-in standard is further defined as a nucleic acid spike-in standard. In certain aspects, the nucleic acid spike-in standard comprises artificial nucleic acids any known organism. In certain aspects, the nucleic acid spike-in standard comprises artificial nucleic acids exhibiting minimal similarity to any known organisms or only having significant similarity to nucleic acids of organisms not relevant to the sample of interest. In some specific aspects, the nucleic acid spike-in standard comprises one or more nucleic acid molecules comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOS: 1-30. In several aspects, the nucleic acid spike-in standard enables collection, preservation, and/or transportation of the biological sample without nucleic acid degradation, microbial growth gene induction, and/or epigenetic change. In some aspects, the nucleic acid spike-in standard comprises at least 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 2000 bp RNA. In some aspects, the nucleic acid spike-in standard comprises between about 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 2000 bp RNA. In a particular aspect, the RNA is miRNA. In certain specific aspects, the spike-in standard may comprise a 17, 18, 19, 20, 21, 22, 23, 24 or 25 base pair miRNA. In other aspects, the nucleic acid spike-in standard comprises a double-stranded DNA (dsDNA). For example, the dsDNA can comprise at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 or 3000 bp dsDNA. In further aspects, the dsDNA comprises between about 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 2000 bp of dsDNA.

[0006] In further aspects, the nucleic acid spike-standard comprises log dilutions. In some aspects, the log dilutions comprise 100 ng, 10 ng, 1 ng, 10 ² ng, l0 ³ ng, 10 ⁴ ng, and/or 10 ⁵ ng, 10 ⁶ ng, l0 ⁷ ng, l0 ⁸ ng, l0 ⁹ ng of DNA or RNA, especially l0 ³ ng, 10 ⁴ ng, and/or 10 ⁵ ng. In alternative aspects, the nucleic acid spike-in standard may be GC-rich or AT-rich wherein the DNA comprises a range of GC content, especially including about 35, about 50, or about 65% GC-content. In other aspects, the DNA or RNA is epigenetically modified DNA or RNA. In particular aspects, the epigenetically modified DNA or RNA comprises at least 10% 5-methylcytosine methylation, but can range from 0%-l0%, 10%— 20%, 20%-30%, 30%- 40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, and 90%-l00% methylated DNA. In another aspect, the biological sample has undergone storage, transport, purification, library preparation, and/or processing.

[0007] In a further embodiment there is provided a container comprising the reagent of the embodiment and aspects described above. In certain aspects, the container is further defined as a swab and collection tube, blood vacutainer, fecal scoop tube, or saliva collection device.

[0008] In still a further embodiment, the invention provides a kit comprising the reagent of the embodiment and aspects described above and a primer or probe that can be used to quantify the amount or integrity of the nucleic acid molecule in a sample. In several aspects, the reagent is provided in a swab and collection tube, blood vacutainer, fecal scoop tube, or saliva collection device. In one aspect there is provided a kit comprising a sealed tube comprising a stabilization reagent and at least a first spike-in standard. In some aspects, the sealed container comprises a stabilization reagent with an effective amount of a guanidinium salt to inhibit nuclease activity and at least a first spike-in standard, that is a RNA or DNA and is present in a known quantity and has substantially no sequence similarity any mammalian or microbial nucleic acid sequence. In some aspects, the tube comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different spike-in standards, that are RNA or DNA and are each present in a known quantity and have substantially no sequence similarity any mammalian or microbial nucleic acid sequence. In a further aspects, the sealed container comprises a stabilization reagent with an effective amount of a guanidinium salt to inhibit nuclease activity and at least a first spike- in standard, that is a microbe cell or cell lysate and that is present in a known quantity. In some aspects, the tube comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different spike-in standards, that are different a microbe cells or cell lysates and are each present in a known quantity.

[0009] In yet still another embodiment there is provided a method for assessing the quality of a biological sample comprising: (a) adding a spike-in control of known quantity to a biological sample; (b) sequencing the biological sample comprising the spike-in control; (c) performing bioinformatics analysis to obtain relative abundance of molecule(s) of interest; and (d) converting the relative abundance of the molecule(s) of interest into absolute quantification of the molecule(s) of interest. In additional aspects, the method further comprises extracting DNA from the biological sample comprising the spike-in control prior to step (b). In some aspects, the method further comprises preparing a library from the extracted DNA. In other aspects, the sequencing is DNA sequencing. In certain aspects, converting comprises generating a standard curve of the abundance of the molecule of interest versus abundance of the spike-in standard. In another aspect, converting comprises subtracting abundance of the spike-in standard from abundance of the molecule(s) of interest. In further aspects, the biological sample has undergone storage, transport, purification, library preparation, and/or processing.

[0010] In an additional aspect, the method comprises assessing quality changes in the biological sample at collection, purification, and analysis stages. In several aspects, analysis may comprise PCR and/or sequencing. In other aspects, assessing quality may comprise measuring degradation of the molecule(s) of interest. In a particular aspect, the molecule(s) of interest are derived from microbes, cells, or any biological material containing DNA or RNA. In certain specific aspects, assessing measurement quality comprises determining a measurement integrity quotient (MIQ) score. In still further aspects, the method comprises using a nucleic acid spike-in standard to assess bias in sequencing. In another particular aspect, the molecule(s) of interest is a virus. In some aspects, the virus may be human immunodeficiency virus (HIV) or herpes simplex virus (HSV).

[0011] In still further another embodiment, there is provided a nucleic acid molecule comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1-30. In specific aspects, the nucleic acid molecule may be a DNA or a RNA molecule. In several aspects, the nucleic acid molecule comprises a label.

[0012] Yet still a further embodiment provides a reagent for storage, lysis or purification of biological molecules comprising a nucleic acid molecule of the embodiments and aspects described above. In an additional aspect, the reagent comprises a known concentration of the nucleic acid molecule ( e.g the spike-in standard). In further aspects, the reagent inactivates organisms preventing growth (e.g., to allow for safe transport). Additionally, in some aspects, the reagent protect nucleic acids from degradation over an extended period of time. For example, in some aspects, less than 10%, 5% or 1% degradation is exhibited over a period of 3 month, 6month, 1 year, 3 years, 5 years or 10 years. In certain aspects, the reagent can be defined as providing less than 5% degradation of spike-in standard over a period of 1 year. [0013] In a further embodiment, the invention provides a kit comprising the reagent of the embodiments and aspects described above, and a primer or probe that can be used to quantify the amount or integrity of the nucleic acid molecule in a sample.

[0014] In certain embodiments, the present disclosure provides methods for purifying unbiased RNA from blood sample comprising obtaining a biological sample, dissolved in a lysis reagent; lysing the sample dissolved in the lysis reagent; and purifying RNA from the mixture, wherein the purifying does not involve a RNA precipitation step. In some aspects, the method allows for purification of nucleic acid with a G/C bias. Thus, in some aspects, the lysis and purification involves the additional of a spike-in standard with a known G/C content or a plurality of standards with a range of known G/C contents. In still further aspects, the method allows for sequencing of quantitation of nucleic acids from microbes without a bias for one microbe other another. Thus, in some aspects, the lysis and purification involves the additional of a spike-in standard of a known microbe or a plurality of known microbes.

[0015] In additional aspects, the method further comprises selectively analyzing micro RNAs from the purified RNA, wherein the purified RNAs provide a representative population of the RNA content of the original sample.

[0016] In some aspects, the biological sample is a liquid sample, a tissue sample, or a blood sample. In particular aspects, the blood sample is whole blood, plasma, serum, or huffy coat. In some aspects, obtaining the blood sample comprises collecting blood in a tube comprising the lysis reagent.

[0017] In certain aspects, the lysis reagent inactivates one or more microbes and nucleases in the blood sample. In some aspects, the one or more microbes comprise a virus, bacteria, and/or yeast. In certain aspects, the virus is influenza, ebola, HIV, influenza or HSV. In some aspects, the bacteria is E. coli, B. subtilis, L. fermentum, E. faecalis, L. monocytogenes, P. aeruginosa, S. enterica, or S. aureus. In certain aspects, the yeast is C. neoformans and/or S. cerevisiae.

[0018] In some aspects, lysing, storage, and purifying are performed at 20-30°C, such as between about 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C and 30°C. In certain aspects, the lysing or storage involves an incubation of period of at least 1 minute, such as for about 10 minutes to 2 hour, particularly about 5 minutes to 1 hour (e.g., 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, or 50 minutes). In some aspects, the incubation step comprises storing the sample at less than 10 degrees C ( e.g ., 9°C, 8°C, 7°C, 6°C, 5°C, or 4°C), for at least one day, such as for 24-72 hours, such as 2 days, 3 days, 4 days, or 5 days. In still further aspects, the incubation step can involve storage of the sample at less than 10 degrees for at least a one week, two weeks, a month two month, six months or a year. In aspects the storage may be at ambient temperature for up to one week, two weeks, a months, two months, three months, six months, 1 year or 2 years.

[0019] In certain aspects, the lysis agent and the sample are mixed at 1: 1 vokvol ratio. In some aspects, the lysis agent and sample are mixed at a vol of 0.7-1.5 of lysis agent to vol of 0.7-1.5 of sample, such as 0.7: 1, 0.8:1, 0.9: 1, 1 :0.7, 1:0.8, 1 :0.9, 1 :1.1, 1 : 1.2, 1: 1.3, 1: 1.4, 1 : 1.5, 1.1 : 1, 1.2: 1, 1.3: 1, 1.4: 1, or 1.5: 1 vokvol of lysis agent to sample.

[0020] In specific aspects, the lysis agent comprises a chaotropic salt. In one specific aspect, the chaotropic salt is a guanidinium salt. In additional aspects, the lysing step further comprises proteinase K digestion.

[0021] In some aspects, the lysing step further comprises agitation of the sample with one or more bead. In particular aspects, the one or more bead is a plurality of beads. In certain aspects, the plurality of beads are comprised of beads of different materials, sizes, or different shapes or the combination thereof. In some aspects, the beads are substantially spherical and comprise an average diameter of between 0.01 and 1.0 mm, such as 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, or 0.09 mm. In specific aspects, the beads of different sizes comprise beads that are between 0.25 and 0.75 mm (e.g., 0.3, 0.4, 0.5, 0.6 or 0.7 mm) and beads that are between 0.05 and 0.25 mm (e.g., 0.06, 0.07, 0.08, 0.09, 0.1, or 0.2 mm) in diameter. In particular aspects, the bead is substantially spherical. In some aspects, the bead is composed of a substantially non reactive material. In one specific aspect, the bead is composed of a ceramic. In some embodiments beads of two different diameters are used in the lysis, In a preferred embodiment 0.1 mm and 0.5 mm ceramic beads are used in a 1:2, 1 :3, or 1: 1 ratio.

[0022] In certain aspects, the purifying step comprises applying the mixture to a silica spin column to bind the RNA to said column. In some aspects, the mixture is diluted in an equal volume of isopropanol prior to applying said sample to the column.

[0023] In additional aspects, purifying further comprises performing DNase I digestion. In some aspects, purifying further comprises removal of the chaotropic salt. In certain aspects, purifying further comprises washing the column with a buffer comprising ethanol or isopropanol. In particular aspects, purifying does not comprise alcohol precipitation of the RNA or phase separation. In some aspects, purifying comprises eluting the RNA into RNase-free water. In certain aspects, the purified RNA is essentially free of DNA. In some aspects, the purified RNA comprises micro RNA, small interfering RNA, and/or piwi RNA. In certain aspects, the purified RNA comprises RNA molecules less than 200 nucleotides in length.

[0024] In some aspects, analyzing micro RNAs comprises performing microarray analysis, single cell assays, northern blotting, or qRT-PCR. In particular aspects, analyzing micro RNAs comprises constructing a library for miRNA sequencing and performing next generation miRNA sequencing on said library. In some aspects, constructing a library comprises ligating adaptors to each end of the micro RNAs. In specific aspects, the adaptors comprise barcodes. In certain aspects, the method further comprises performing Nanostring nCounter analysis on the sequencing results.

[0025] In certain aspects, the method further comprises performing unbiased miRNA functional enrichment analysis. In some aspects, the analysis comprises using a target prediction program, gene annotation data, and applying statistical analysis.

[0026] As used herein,“essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0027] As used herein in the specification and claims,“a” or“an” may mean one or more. As used herein in the specification and claims, when used in conjunction with the word “comprising”, the words“a” or“an” may mean one or more than one. As used herein, in the specification and claim,“another” or“a further” may mean at least a second or more.

[0028] As used herein in the specification and claims, the term“about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. [0029] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating certain embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be beher understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0031] FIG. 1: Flow-chart outlining the steps for the generation of spike-in sequences with minimal collision risk against known, sequenced genomes (SEQ ID NO: 31-32 shown). [0032] FIG. 2: The microbial composition profiles of a 10 mg fecal sample before and after subtracting the taxa of the spike-in microbes.

[0033] FIG. 3: Microbial composition profile of a sputum sample with ZymoBIOMICS™ Spike-in Control II.

[0034] FIG. 4: The standard curve that plots the defined DNA abundance (ng) against the percentage abundance (%) for the three spike-in microbes.

[0035] FIG. 5 : HIV RNA spike-in control with saliva shield mixture.

[0036] FIG. 6: HSV DNA spike-in tracking stability in DNA/RNA shield with stool at

10% m /v.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. The Present Embodiments

[0037] DNA and RNA sequencing is an important tool for microbial identification and profiling, including microbiome analysis and pathogen identification. Although this technique is quantitative, microbial abundance is commonly reported as percentage abundance because the common workflows require PCR-based amplifications and library normalizations, which can introduce biases.

[0038] Often PCR controls may be included in a specific assay, but this would not account for problems with a failed purification of DNA that resulted in no or low DNA. It would also not account for poor purity from a failed purification. A PCR control would also not account for sample degradation during collection, transportation and storage. Meaning a failure in any one of these steps could lead to a false negative and risk the safety of a patient. Furthermore, even if a purification spike-in control is implemented the sample collection still remains an unknown. This present method provides method which enable complete confidence in sample handling from collection to analyses to maximize confidence in a test and the safety of a patient.

[0039] The instant application provides for the first time a method assessing or tracking the quality of biological sample to access if there may be bias both as to quantity or diversity (e.g., a purification or processing protocol from microorganisms or liquid or solid tissues or cells from essentially any organism). Provided herein are internal standards (or controls) that can be used to provide a read-out degradation of loss of biomolecules especially DNA, RNA or modified DNA and RNA over time, cellular lysis inefficiencies, losses of nucleic acids during purification, bias associated library preparation or sequencing etc.

[0040] Thus, in certain embodiments, the present disclosure provides methods to enable absolute microbial cell quantification by DNA/RNA sequencing. The methods comprise adding whole microbial cell spike-in controls of known quantities into the sample of interest, sequencing the sample, and then converting the relative abundance quantification into absolute quantification.

[0041] The methods provided herein can be used to determine the quality of a biological sample, such as after transportation or storage. The sample may be assessed after purification or processing to identify any degradation in quality, such as due to lysis inefficiency or failed purification. In addition, the present methods may be used to track sample quality in library preparation, such as bias during preparation. In certain aspects microbe(s) to be included a Spike-In Standard include but are not limited to: (1) a microbe well characterized genome; (2) a plurality of different microbes with different lysis properties, genome GC- content, genome complexity and different growth conditions. Different types of Spike-In Standards for varying applications, for example where the Spike-In goal is absolute auantification a known genome; and known quantity of microbe may be used.

[0042] In some aspects, fidelity to a known biological input sample (standard) may be assessed by determining a Measurement Integrity Quotient (MIQ) score that grades the measurement relative to the known quantities or ratios of different components of the standard sample while taking into account manufacturing tolerances within the standard itself.

MIQ Score

[0043] The Measurement Integrity Quotient (MIQ) Score, is a metric designed to simplify the detection of bias or lack thereof in sample preparation or analysis down to a single number. The MIQ score is composed of a mathematical formula with a software package to implement it in an analytic pipeline. The statistical basis for the MIQ score is the root mean square of errors calculation, a statistical method for assessing errors in measurement or analysis. The MIQ score calculation takes this formula and adds a modification to account for manufacturing tolerances in the known standard as well as some transformations to allow for a possible high score of 100, which would indicate that the ratio of components detected from a sample of known composition and manufacturing tolerance is the same as the expected ratios, with any deviation from the expected being fully explainable within the manufacturing tolerances of the known composition sample.

[0044] The MIQ score calculation starts by determining percent error while accounting for manufacturing tolerance. This is achieved by dividing the observed quantity or relative abundance of the known sample component by the expected quantity or relative abundance and multiplying the quotient by 100. This percent of expected is then converted to a percent deviation by subtracting 100. This value is then compared to the manufacturing tolerance as a percentage, with any deviation that is less than the manufacturing tolerance being set to zero any deviation outside the range of manufacturing tolerance being adjusted to its distance from the range of manufacturing tolerance.

[0045] This percent of error is calculated for every component of the known standard as described above. These values are then squared and the square root of the mean of the errors squared is calculated, yielding a root mean square of errors that has an adjustment for manufacturing tolerances built-in. This value is then subtracted from 100 so that a maximum score of 100 indicates no errors that could not be accounted for by manufacturing tolerances while any lower score indicates some potential errors that could not be accounted for in this manner. The final score may be a negative value, which can either be set to zero for ease of understanding or left as the raw negative value.

[0046] The computational component of the MIQ Scoring system implements the above calculation by taking in one set of data containing key: value pairs of component: expected value and another key: value pair set containing component: observed value. This software package may also take in multiple sets of expected key: value pairs for different analysis or handling conditions if different expected values are predicted, in which case the conditions must also be supplied. The output from this software will always include a single floating-point decimal number that is the MIQ score itself.

[0047] Software for MIQ scoring may also be designed to include the production of plots such as bar plots or radar plots with the components grouped or organized in such a manner as to facilitate the user being able to quickly identify known or expected potential patterns of bias in their observed sample composition versus the expected composition.

Biological Samples

[0048] As used herein a“biological sample” refers to any sample that comprises a organic molecules produced by biological systems. For example, in some aspects, the biological sample comprises DNA and/or RNA.

[0049] The biological sample may comprise, but is not limited to, bacteria, archaea, fungi, eurkaryote, viruses, DNA, RNA {including, miRNA), modified nucleic acids both naturally occurring (e.g., epigenetic modification including DNA Methylation wherein each fragment of DNA contains a different amount of 5-methylcytosine methylation ranging from 1-100% such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%) or artificial such as Locked Nucleic Acids or log distributed DNA or RNA, proteins, metabolites, lipids, and/or carbohydrates.

[0050] The absolute level of at least one molecule of interest can be measured in a biological sample. A biological sample may comprise a cell, milk, blood, serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebral spinal fluid, tears, urine, feces, saliva, sputum, virus, tissue, plants, or combinations thereof.

[0051] For example, a tissue sample can be removed from a subject by conventional biopsy techniques. In another example, a blood sample can be removed from a subject, and white blood cells can be isolated for RNA extraction by standard techniques. The blood or tissue sample is preferably obtained from the subject prior to initiation of radiotherapy, chemotherapy or other therapeutic treatment. A corresponding control tissue or blood sample can be obtained from unaffected tissues of the subject, from a normal human individual or population of normal individuals, or from cultured cells corresponding to the majority of cells in the subject's sample.

Spike-in standards of the embodiments

[0052] As used herein the term“quality” relative to a biological sample refers to the level of degradation of components in the sample relative to when the components were comprised in a biological system, such as a cell. For example, assessing the quality of RNA and/or DNA can comprise assessing the level of partial degradation of RNA and/or DNA polymers. In some aspects, the assessing quality comprises assessing partial degradation of RNA and/or DNA from a spike-in standard.

[0053] As used herein the term“quantity” relative to a biological sample refers to the level of a component present in the sample relative to when the components were comprised in biological system, such as a cell. For example, assessing the quantity of RNA and/or DNA can comprise assessing the level of degradation or loss of RNA and/or DNA polymers in a sample.

[0054] As used herein the terms“spike-in control” and“spike-in standard” are used interchangeably.

[0055] As used herein a spike-in standard that is a nucleic acid and has“substantially no sequence similarity” to a reference nucleic sequence ( e.g a mammalian or microbial nucleic acid sequence), refers to a spike-in standard with a sequence that does not hybridize to a refence sequence in conditions used for hybridization in polymerase chain reaction. For example, a sequence that does not hybridize to a reference sequence under standard salt conditions at a temperature above 40 °C, 45 °C or 50 °C.

[0056] A standard sample for benchmarking sample processing and analysis can be any sample or set of samples designed to be detected by the final assay and present in known absolute quantities or at least known ratios with some known amount of manufacturing tolerance. Knowing these values, one can analyze the standard sample after putting it through the pipeline as its own sample and determine if the standard components being detected by the assay match the expected composition within the known manufacturing tolerance. Any deviation from the expected quantities or ratios between components that cannot be explained within manufacturing tolerances is presumed to be due to bias or other defects in sample handling or analysis. In some aspects, a spike-in standard of the embodiments is present in the same reaction containing a biological sample and is assayed along with the sample. In further aspects, the spike-in standard is in a reaction different from the biological sample and is assessed in that reaction.

[0057] A spike-in sample is similar in concept to standard sample with a few key differences: The standard sample can be composed of any component or set of components that are detectable by the final assay and are useful for detecting errors in sample processing or analysis (e.g. lysis, purification, library preparation, bioinformatics). A spike-in sample is designed to be added to a sample of known source and unknown composition and is thus limited to components that are detectable by the final assay, but have essentially no chance of being mistaken for any potential component of the unknown sample itself. This allows for later removal of any signal or data created by the spike-in sample, leaving behind only signal or data from the unknown sample. A spike-in sample can be designed with multiple components present in known absolute quantities or known ratios in order to detect potential bias or processing flaws with individual sample-by-sample resolution, rather than for an entire cohort, which is the limit of a standard sample. Additionally, if a spike-in sample is present in known absolute quantities, it provides a potential means to absolutely quantify components of the unknown sample.

[0058] In one embodiment, the present disclosure provides a DNA/RNA shield comprising a spike-in nucleic acid standard. The DNA/RNA shield can be used as a nucleic acid preservation reagent that enables samples to be collected, preserved, and transported at ambient temperature without nucleic acid degradation, microbial growth gene induction, or epigenetic change. As such, the DNA/RNA shield offers the unique opportunity to stabilize nucleic acid standards from the point of collection to serve as a control for an entire assay from sample collection, nucleic acid purification, through analyses by methods including but not limited to PCR or sequencing. The internal control can enable understanding of many parameters that affect downstream analyses especially for diagnostics including the sample integrity post transportation/storage, sample purity and efficacy of the purification, or as a positive control for the analytical method used such as PCR and address detection limits. The internal controls can also be created to be GC rich or AT rich to assess biases associated with various techniques such as next generation sequencing.

[0059] The DNA/RNA shield™ in all its embodiments can contain artificial nucleic acids with no known matching sequences based upon the best available databases. For instance, 3-5 species of DNA could be aliquoted at varying concentrations with a preferred embodiment being a log series dilution (e.g. lxl0 ³ ng/ml, lxl0 ⁴ ng/ml, lxl0 ⁵ ng/ml, lxlO ⁵ ng/ml, lxlO ⁶ ng/ml). The log dilution would allow for a detection limit of an assay to be understood taking into account all processes beginning from sample collection. For instance, if the target analyte DNA-X at a concentration of lxlO ⁶ ng/ml was used as the detection limit for a PCR assay and the assay failed to detect the target it would mean that the assay was likely compromised. Since internal control accounts for all steps from collection through analyses it can be concluded reliably that the patient’s sample is compromised, and a new collection should be made.

[0060] In some aspects, the present disclosure further provides a DNA/RNA shield collection device, such as a swab and collection tube filled with 1 ml of DNA/RNA shield reagent. Other collection devices may include a blood vacutainer, fecal scoop tubes, and saliva collection devices etc. All of these devices can contain spike-in standards.

[0061] The DNA and RNA spike-in standard may comprise a range of sizes with preferred embodiments includingl,000, 2,000, and 3,000 bp double-stranded DNA. The RNA spike-in standard may comprise a range of sizes with preferred embodiments including 500, 1000, and 2000 bp RNA. For miRNA, the miRNA standard can comprise a range of sizes with preferred embodiments including 17, 20, and 25 bp miRNA. The templates may be purely artificial sequences not matching any known organisms or alternatively may directly correspond to known organism depending on the need of a particular assay. However, the preferred embodiment is sequences which are unnatural - having been determined by BLAST search or similar method to possess the least similarity to known naturally-occurring sequences. Each DNA fragment can contain a different amount of GC content (e.g., 35%, 50%, 65%). The standards can each be a log dilution of the next (e.g., l0 ³ ng, 10 ⁴ ng, and 10 ⁵ ng of DNA). [0062] The sequences and primers can be designed for a qPCR ready target of 150 bp. Primers can be designed and validated in advance for 56, 58, 60, 62, 64, and 66 degrees Celsius wherein each amplifies the 150 bp sequence within each DNA spike to serve as a prebuilt multiplexable PCR, qPCR, to be paired with existing tests, including but not limited to diagnostics.

[0063] In some aspects, the spike-in standard is a celluar spike-in control. The celluar spike-in control can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more microbes. The microbes may be artificially modified organisms to create uniqueness from the background or alternatively be organisms that are foreign to the sample types of interest. For example, for human samples of low biomass, the microbes may be Treupera radiovictrix, Imtechella halotolerans, and Allobacillus halotolerans . For human samples of higher biomass, the microbes may be Imtechella halotolerans and Allobacillus halotolerans, such as in Table 1.

[0064] In some aspects the spike-in standard may include may include microbes from the Table 2 below.

[0065] Additional bacteria suitable for use in a Spike-In standard include but are not limited to: Acetobacter aurantius, Acinetobacter species: Acinetobacter baumannii, Acinetobacter calcoaceticus , Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter Iwoffli, Acinetobacter radioresistens, Acinetobacter septicus, Acinetobacter schindleri, Acinetobacter ursingii; Actinomyces species: Actinomyces bovis, Actinomyces bowdenii, Actinomyces canis, Actinomyces cardiffensis , Actinomyces catuli, Actinomyces coleocanis, Actinomyces dentalis, Actinomyces denticolens, Actinomyces europaeus, Actinomyces funkei, Actinomyces georgiae, Actinomyces gerencseriae, Actinomyces graevenitzii, Actinomyces hongkongensis, Actinomyces hordeovulneris, Actinomyces howellii, Actinomyces humiferus, Actinomyces hyovaginalis, Actinomyces israelii, Actinomyces marimammalium, Actinomyces meyeri, Actinomyces naeslundii, Actinomyces nasicola, Actinomyces neuii, Actinomyces odontolyticus, Actinomyces oricola, Actinomyces radicidentis, Actinomyces radingae, Actinomyces slackii, Actinomyces streptomycini, Actinomyces suimastitidis, Actinomyces suis, Actinomyces turicensis, Actinomyces urogenitalis, Actinomyces vaccimaxillae, Actinomyces viscosus; Actinobacillus species: Actinobacillus actinomycetemcomitans, Actinobacillus arthritidis, Actinobacillus capsulatus, Actinobacillus delphinicola, Actinobacillus equuli, Actinobacillus hominis, Actinobacillus indolicus, Actinobacillus lignieresii, Actinobacillus minor, Actinobacillus muris, Actinobacillus pleuropneumoniae, Actinobacillus porcinus, Actinobacillus rossii, Actinobacillus scotiae, Actinobacillus seminis, Actinobacillus succinogenes, Actinobacillus suis, Actinobacillus ureae; Aeromonas species: Aeromonas allosaccharophila, Aeromonas bestiarum, Aeromonas bivalvium, Aeromonas encheleia, Aeromonas enteropelogenes , Aeromonas euchrenophila, Aeromonas hydrophila, Aeromonas ichthiosmia, Aeromonas jandaei, Aeromonas media, Aeromonas molluscorum, Aeromonas popoffii, Aeromonas punctata, Aeromonas salmonicida, Aeromonas schubertii, Aeromonas sharmana, Aeromonas simiae, Aeromonas sobria, Aeromonas veronii; Afipia fells, Agrobacterium species: Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium rubi, Agrobacterium tumefaciens ; Agromonas species, Alcaligenes species: Alcaligenes aquatilis, Alcaligenes eutrophus, Alcaligenes faecalis, Alcaligenes latus, Alcaligenes xylosoxidans; Alishewanella species, Alterococcus species, Anaplasma phagocytophilum, Anaplasma marginale, Aquamonas species, Arcanobacterium haemolyticum, Aranicola species, Arsenophonus species, Azotivirga species, Azotobacter vinelandii, Azotobacter chroococcum, Bacillary dysentery (Shigellosis), Bacillus species: Bacillus abortus (Brucella melitensis biovar abortus), Bacillus anthracis (Anthrax), Bacillus brevis, Bacillus cereus, Bacillus coagulans, Bacillus fusiformis, Bacillus globigii, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus natto, Bacillus stearothermophilus , Bacillus subtilis, Bacillus sphaericus, Bacillus thuringiensis; Bacteroides species: Bacteroides forsythus (Tannerella forsythensis), Bacteroides acidifaciens , Bacteroides distasonis (reclassified as Parabacteroides distasonis), Bacteroides gingivalis, Bacteroides gracilis, Bacteroides fragilis, Bacteroides oris, Bacteroides ovatus, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides stercoris, Bacteroides suis, Bacteroides tectus, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bartonella species: Bartonella alsatica, Bartonella bacilliformis , Bartonella birtlesii, Bartonella bovis, Bartonella capreoli, Bartonella clarridgeiae, Bartonella doshiae, Bartonella elizabethae, Bartonella grahamii, Bartonella henselae (cat scratch fever), Bartonella koehlerae, Bartonella muris, Bartonella peromysci, Bartonella quintana, Bartonella rochalimae, Bartonella schoenbuchii, Bartonella talpae, Bartonella taylorii, Bartonella tribocorum, Bartonella vinsonii spp. Arupensis, Bartonella vinsonii spp. Berkhoffli, Bartonella vinsonii spp. Vinsonii, Bartonella washoensis; BCG (Bacille Calmette-Guerin) , Bergeyella zoohelcum (Weeksella zoohelcum), Bifidobacterium bifldum, Blastobacter species, Blochmannia species, Bordetella species: Bordetella ansorpii, Bordetella avium, Bordetella bronchiseptica, Bordetella hinzii, Bordetella holmesii, Bordetella parapertussis, Bordetella pertussis (Whooping cough), Bordetella petrii, Bordetella trematum; Borrelia species: Borrelia burgdorferi, Borrelia afzelii, Borrelia anserina, Borrelia garinii, Borrelia valaisiana, Borrelia hermsii, Borrelia Parkeri, Borrelia recurrentis ; Bosea species, Bradyrhizobium species, Brenneria species, Brucella species: Brucella abortus, Brucella canis, Brucella melitensis, Brucella neotomae, Brucella ovis, Brucella suis, Brucella pinnipediae; Buchnera species, Budvicia species, Burkholderia species: Burkholderia cepacia (Pseudomonas cepacia), Burkholderia mallei (Pseudomonas mallei/Actinobacillus mallei), Burkholderia pseudomallei (Pseudomonas pseudomallei); Buttiauxella species, Calymmatobacterium granulomatis, Campylobacter species: Campylobacter coli, Campylobacter concisus, Campylobacter curvus, Campylobacter fetus, Campylobacter gracilis, Campylobacter helveticus, Campylobacter hominis, Campylobacter hyointestinalis , Campylobacter insulaenigrae, Campylobacter jejuni, Campylobacter lanienae, Campylobacter lari, Campylobacter mucosalis, Campylobacter rectus, Campylobacter showae, Campylobacter sputorum, Campylobacter upsaliensis; Capnocytophaga canimorsus (Dysgonic fermenter type 2), Corynebacterium species, Cardiobacterium hominis, Cedecea species, Chlamydia species: Chlamydia trachomatis (Lymphogranuloma venereum), Chlamydia muridarum, Chlamydia suis; Chlamydophila species: Chlamydophila pneumoniae, Chlamydophila psittaci (Psittacosis), Chlamydophila pecorum, Chlamydophila abortus, Chlamydophila felis, Chlamydophila caviae; Citrobacter species: Citrobacter amalonaticus, Citrobacter braakii, Citrobacter farmeri, Citrobacter freundii, Citrobacter gillenii, Citrobacter intermedius, Citrobacter koseri aka Citrobacter diversus, Citrobacter murliniae, Citrobacter rodentium, Citrobacter sedlakii, Citrobacter werkmanii, Citrobacter youngae; Clostridium species: Clostridium botulinum, Clostridium difficile, Clostridium novyi, Clostridium septicum, Clostridium tetani (Tetanus), Clostridium welchii (Clostridium perfringens); Corynebacterium species: Corynebacterium diphtheriae (Diphtheria), Corynebacterium amycolatum, Corynebacterium aquaticum, Corynebacterium bovis,

Corynebacterium equi, Corynebacterium flavescens, Corynebacterium glutamicum, Corynebacterium haemolyticum, Corynebacterium jeikeiun (corynebacteria of group JK), Corynebacterium minutissimum (Erythrasma) , Corynebacterium parvum (also called

Propionibacterium acnes), Corynebacterium pseudodiptheriticum (also called

Corynebacterium hofmannii), Corynebacterium pseudotuberculosis (also called

Corynebacterium ovis), Corynebacterium pyogenes, Corynebacterium urealyticum (corynebacteria of group D2), Corynebacterium renale, Corynebacterium striatum, Corynebacterium tenuis (Trichomycosis palmellina, Trichomycosis axillaris), Corynebacterium ulcer ans, Corynebacterium xerosis; Coxiella burnetii (Q fever), Cronobacter species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis; Delftia acidovorans (Comamonas acidovorans), Dickeya species, Edwardsiella species, Eikenella corr odens, Enterobacter species: Enterobacter aerogenes, Enterobacter cloacae, Enterobacter sakazakii; Enterococcus species: Enterococcus avium, Enterococcus durans, Enterococcus faecalis (Streptococcus faecalis/Streptococcus Group D), Enterococcus faecium, Enterococcus solitarius, Enterococcus galllinarum, Enterococcus maloratus; Ehrlichia chaffeensis, Erysipelothrix rhusiopathiae, Erwinia species, Escherichia species: Escherichia adecarboxylata, Escherichia albertii, Escherichia blattae, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris; Ewingella species, Flavobacterium species: Flavobacterium aquatile, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium flevense, Flavobacterium gondwanense, Flavobacterium hydatis, Flavobacterium johnsoniae, Flavobacterium pectinovorum, Flavobacterium psychrophilum, Flavobacterium saccharophilum, Flavobacterium salegens, Flavobacterium scophthalmum, Flavobacterium succinans; Francisella tularensis (Tularaemia), Francisella novicida, Francisella philomiragia, Fusobacterium species: Fusobacterium necrophorum (Lemierre syndrome/Sphaerophorus necrophorus), Fusobacterium nucleatum, Fusobacterium polymorphum, Fusobacterium novum, Fusobacterium mortiferum, Fusobacterium varium; Gardnerella vaginalis, Gemella haemolysans, Gemella morbillorum (Streptococcus morbillorum), Grimontella species, Haemophilus species: Haemophilus aegyptius (Koch- Weeks bacillus), Haemophilus aphrophilus, Haemophilus avium, Haemophilus ducreyi (Chancroid), Haemophilus felis, Haemophilus haemolyticus, Haemophilus influenzae (Pfeiffer bacillus), Haemophilus paracuniculus, Haemophilus parahaemolyticus, Haemophilus parainfluenzae, Haemophilus paraphrophilus (Aggregatibacter aphrophilus), Haemophilus pertussis, Haemophilus pittmaniae, Haemophilus somnus, Haemophilus vaginalis; Hafnia species, Hafnia alvei, Helicobacter species: Helicobacter acinonychis, Helicobacter anseris, Helicobacter aurati, Helicobacter bilis, Helicobacter bizzozeronii, Helicobacter brantae, Helicobacter Canadensis, Helicobacter canis, Helicobacter cholecystus, Helicobacter cinaedi, Helicobacter cynogastricus , Helicobacter felis, Helicobacter fennelliae, Helicobacter ganmani, Helicobacter heilmannii (Gastrospirillum hominis), Helicobacter hepaticus, Helicobacter mesocricetorum, Helicobacter marmotae, Helicobacter muridarum, Helicobacter mustelae, Helicobacter pametensis, Helicobacter pullorum, Helicobacter pylori (stomach ulcer), Helicobacter rappini, Helicobacter rodentium, Helicobacter salomonis, Helicobacter trogontum, Helicobacter typhlonius, Helicobacter winghamensis; Human granulocytic ehrlichiosis (Anaplasma phagocytophilum/Ehrlichia phagocytophila) , Human monocytotropic ehrlichiosis (Monocytic ehrlichiosis/Ehrlichia chaffeensis), Klebsiella species: Klebsiella granulomatis (Calymmatobacterium granulomatis) , Klebsiella mobilis, Klebsiella ornithinolytica, Klebsiella oxytoca, Klebsiella ozaenae, Klebsiella planticola, Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Klebsiella singaporensis, Klebsiella terrigena, Klebsiella trevisanii, Klebsiella variicola; Kingella kingae, Kluyvera species, Lactobacillus species: Lactobacillus acetotolerans, Lactobacillus acidifarinae, Lactobacillus acidipiscis, Lactobacillus acidophilus (Doderlein bacillus), Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylophilus, Lactobacillus amylotrophicus, Lactobacillus amylovorus, Lactobacillus animalis, Lactobacillus antri, Lactobacillus apodemi, Lactobacillus aviarius, Lactobacillus bifermentans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus camelliae, Lactobacillus casei, Lactobacillus catenaformis, Lactobacillus ceti, Lactobacillus coleohominis, Lactobacillus collinoides, Lactobacillus composti, Lactobacillus concavus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus diolivorans, Lactobacillus equi, Lactobacillus equigenerosi, Lactobacillus farraginis, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus formicalis, Lactobacillus fructivorans, Lactobacillus frumenti, Lactobacillus fuchuensis, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus ghanensis, Lactobacillus graminis, Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacillus harbinensis, Lactobacillus hayakitensis, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus homohiochii, Lactobacillus iners, Lactobacillus ingluviei, Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis, Lactobacillus kefuranofaciens, Lactobacillus keflri, Lactobacillus kimchii, Lactobacillus kitasatonis, Lactobacillus kunkeei, Lactobacillus leichmannii, Lactobacillus lindneri, Lactobacillus malefermentans, Lactobacillus mali, Lactobacillus manihotivorans, Lactobacillus mindensis, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus namurensis, Lactobacillus nantensis, Lactobacillus oligofermentans, Lactobacillus oris, Lactobacillus panis, Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracollinoides, Lactobacillus parafarraginis , Lactobacillus parakeflri, Lactobacillus paralimentarius, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus psittaci, Lactobacillus rennini, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rimae, Lactobacillus rogosae, Lactobacillus rossiae, Lactobacillus ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus satsumensis, Lactobacillus secaliphilus, Lactobacillus sharpeae, Lactobacillus siliginis, Lactobacillus spicheri, Lactobacillus suebicus, Lactobacillus thailandensis, Lactobacillus ultunensis, Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus versmoldensis, Lactobacillus vini, Lactobacillus vitulinus, Lactobacillus zeae, Lactobacillus zymae; Leclercia species, Legionella species: Legionella adelaidensis, Legionella anisa, Legionella beliardensis , Legionella birminghamensis, Legionella bozemanii, Legionella brunensis, Legionella busanensis, Legionella cherrii, Legionella cincinnatiensis, Legionella donaldsonii, Legionella drancourtii, Legionella drozanskii, Legionella erythra, Legionella fairfleldensis , Legionella fallonii, Legionella feeleii, Legionella geestiana, Legionella genomospecies, Legionella gratiana, Legionella gresilensis, Legionella hackeliae, Legionella impletisoli, Legionella israelensis, Legionella jamestowniensis,‘Candidatus Legionella jeonii’, Legionella jordanis, Legionella lansingensis , Legionella londiniensis, Legionella longbeachae, Legionella lytica, Legionella maceachernii, Legionella micdadei, Legionella moravica, Legionella nautarum, Legionella oakridgensis, Legionella parisiensis, Legionella pneumophila, Legionella quateirensis , Legionella quinlivanii, Legionella rowbothamii, Legionella rubrilucens, Legionella sainthelensi, Legionella santicrucis, Legionella shakespearei, Legionella spiritensis, Legionella steigerwaltii, Legionella taurinensis, Legionella tucsonensis, Legionella wadsworthii, Legionella waiter sii, Legionella worsleiensis, Legionella yabuuchiae; Leminorella species, Leptospira species: Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira alexanderi, Leptospira weilii, Leptospira genomospecies 1, Leptospira borgpetersenii, Leptospira santarosai, Leptospira inadai, Leptospira fainei, Leptospira broomii, Leptospira licerasiae, Leptospira biflexa, Leptospira meyeri, Leptospira wolbachii, Leptospira genomospecies 3, Leptospira genomospecies 4, Leptospira genomospecies 5; Lepromatous leprosy (Danielssen-Boeck disease), Leptospira canicola, Leptospira hebdomadis, Leptospirosis (Weil disease/Leptospira icterohaemorrhagiae/Leptospira interrogans serovar icterohaemorrhagiae), Leptotrichia, Leuconostoc species: Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc durionis, Leuconostoc fallax, Leuconostoc flculneum, Leuconostoc fructosum, Leuconostoc garlicum, Leuconostoc gasicomitatum, Leuconostoc gelidum, Leuconostoc inhae, Leuconostoc kimchii, Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc pseudoflculneum, Leuconostoc pseudomesenteroides; Listeria species: Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes (Listeriosis), Listeria seeligeri, Listeria welshimeri ; Methanobacterium extroquens, Microbacterium multiforme, Micrococcus species: Micrococcus antarcticus, Micrococcus flavus, Micrococcus luteus, Micrococcus lylae, Micrococcus mucilaginosis, Micrococcus roseus, Micrococcus sedentarius; Mobiluncus, Moellerella species, Morganella species, Moraxella species: Moraxella atlantae, Moraxella boevrei, Moraxella bovis, Moraxella canis, Moraxella caprae, Moraxella catarrhalis (Branhamella catarrhalis), Moraxella caviae, Moraxella cuniculi, Moraxella equi, Moraxella lacunata, Moraxella lincolnii, Moraxella nonliquefaciens , Moraxella oblonga, Moraxella osloensis, Moraxella saccharolytica; Morganella morganii, Mycobacterium species: Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium agri, Mycobacterium aichiense, Mycobacterium alvei, Mycobacterium arupense, Mycobacterium asiaticum, Mycobacterium aubagnense, Mycobacterium aurum, Mycobacterium austroafricanum, Mycobacterium avium (Battey disease/Lady Windermere syndrome), Mycobacterium avium paratuberculosis (implicated in Crohn's disease in humans and Johne's disease in sheep), Mycobacterium avium silvaticum, Mycobacterium avium “hominissuis”, Mycobacterium colombiense, Mycobacterium boenickei, Mycobacterium bohemicum, Mycobacterium bolletii, Mycobacterium botniense, Mycobacterium bovis (Bovine tuberculosis), Mycobacterium branderi, Mycobacterium brisbanense, Mycobacterium brumae, Mycobacterium canariasense, Mycobacterium caprae, Mycobacterium celatum, Mycobacterium chelonae, Mycobacterium chimaera, Mycobacterium chitae, Mycobacterium chlorophenolicum, Mycobacterium chubuense, Mycobacterium conceptionense, Mycobacterium confluentis, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium cosmeticum, Mycobacterium diernhoferi, Mycobacterium doricum, Mycobacterium duvalii, Mycobacterium elephantis, Mycobacterium fallax, Mycobacterium farcinogenes, Mycobacterium flavescens, Mycobacterium florentinum, Mycobacterium fluoroanthenivorans, Mycobacterium fortuitum, Mycobacterium fortuitum subsp. Acetamidolyticum, Mycobacterium frederiksbergense, Mycobacterium gadium, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gilvum, Mycobacterium goodii, Mycobacterium gordonae (Mycobacterium aquae), Mycobacterium haemophilum, Mycobacterium hassiacum, Mycobacterium heckeshornense, Mycobacterium heidelbergense, Mycobacterium hiberniae, Mycobacterium hodleri, Mycobacterium holsaticum, Mycobacterium houstonense, Mycobacterium immunogenum, Mycobacterium interjectum, Mycobacterium intermedium, Mycobacterium intr acellular e, Mycobacterium kansasii, Mycobacterium komossense, Mycobacterium kubicae, Mycobacterium kumamotonense, Mycobacterium lacus, Mycobacterium lentiflavum, Mycobacterium leprae (causes leprosy or Hansen disease/Hanseniasis), Mycobacterium lepraemurium, Mycobacterium madagascariense, Mycobacterium mageritense, Mycobacterium malmoense, Mycobacterium marinum (Fish tank granuloma), Mycobacterium massiliense, Mycobacterium microti, Mycobacterium monacense, Mycobacterium monteflorense, Mycobacterium moriokaense, Mycobacterium mucogenicum, Mycobacterium murale, Mycobacterium nebraskense, Mycobacterium neoaurum, Mycobacterium neworleansense, Mycobacterium nonchromogenicum, Mycobacterium novocastrense, Mycobacterium obuense, Mycobacterium palustre, Mycobacterium parafortuitum, Mycobacterium parascrofulaceum, Mycobacterium parmense, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium phocaicum, Mycobacterium pinnipedii, Mycobacterium porcinum, Mycobacterium poriferae, Mycobacterium pseudoshottsii, Mycobacterium pulveris, Mycobacterium psychrotolerans, Mycobacterium pyrenivorans, Mycobacterium rhodesiae, Mycobacterium saskatchewanense, Mycobacterium scrofulaceum, Mycobacterium senegalense, Mycobacterium seoulense, Mycobacterium septicum, Mycobacterium shimoidei, Mycobacterium shottsii, Mycobacterium simiae, Mycobacterium smegmatis, Mycobacterium sphagni, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium the rmoresistibile, Mycobacterium tokaiense, Mycobacterium triplex, Mycobacterium triviale, Mycobacterium tuberculosis (major cause of human tuberculosis), Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii’, Mycobacterium tusciae, Mycobacterium ulcerans (causes Bairnsdale ulcer/Buruli ulcer), Mycobacterium vaccae, Mycobacterium vanbaalenii, Mycobacterium wolinskyi, Mycobacterium xenopi; Mycoplasma species: Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma phocacerebrale, Mycoplasma pneumoniae, Nanukayami (Seven-day fever/Gikiyami), Neisseria species: Neisseria gonorrhoea (Gonococcus /Gonorrhea), Neisseria meningiditis (Meningococcus), Neisseria sicca, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria polysaccharea, Neisseria subflava; Nitrobacter species, Nocardia species: Nocardia asteroides, Nocardia brasiliensis , Nocardia caviae; Noma (cancrum oris/gangrenous stomatitis), Obesumbacterium, Oligotropha species, Orientia tsutsugamushi (Scrub typhus), Oxalobacter formigenes, Pantoea species: Pantoea agglomerans, Pantoea ananatis, Pantoea citrea, Pantoea dispersa, Pantoea punctata, Pantoea stewartii, Pantoea terrea; Pasteurella species: Pasteurella aerogenes, Pasteurella anatis, Pasteurella avium, Pasteurella bettyae, Pasteurella caballi, Pasteurella canis, Pasteurella dagmatis, Pasteurella gallicida, Pasteurella gallinarum, Pasteurella granulomatis , Pasteurella langaaensis, Pasteurella lymphangitidis , Pasteurella mairii, Pasteurella multocida, Pasteurella pneumotropica, Pasteurella skyensis, Pasteurella stomatis, Pasteurella testudinis, Pasteurella trehalosi, Pasteurella tularensis, Pasteurella ureae, Pasteurella volantium ; Pediococcus species: Pediococcus acidilactici, Pediococcus cellicola, Pediococcus claussenii, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus ethanolidurans, Pediococcus inopinatus, Pediococcus parvulus, Pediococcus pentosaceus, Pediococcus stilesii; Peptostreptococcus species: Peptostreptococcus anaerobius, Peptostreptococcus asaccharolyticus , Peptostreptococcus harei, Peptostreptococcus hydrogenalis, Peptostreptococcus indoliticus, Peptostreptococcus ivorii, Peptostreptococcus lacrimalis, Peptostreptococcus lactolyticus , Peptostreptococcus magnus, Peptostreptococcus micros, Peptostreptococcus octavius, Peptostreptococcus prevotii, Peptostreptococcus tetradius, Peptostreptococcus vaginalis; Photorhabdus species, Photorhizobium species, Plesiomonas shigelloides, Porphyromonas gingivalis, Pragia species, Prevotella, Propionibacterium species: Propionibacterium acnes, Propionibacterium propionicus; Proteus species: Proteus mirabilis, Proteus morganii, Proteus penneri, Proteus rettgeri, Proteus vulgaris; Providencia species: Providencia friedericiana, Providencia stuartii; Pseudomonas species: Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas anguilliseptica, Pseudomonas argentinensis, Pseudomonas borbori, Pseudomonas citronellolis, Pseudomonas flavescens, Pseudomonas mendocina, Pseudomonas nitroreducens, Pseudomonas oleovorans, Pseudomonas pseudoalcaligenes, Pseudomonas resinovorans, Pseudomonas straminea, Pseudomonas aurantiaca, Pseudomonas aureofaciens, Pseudomonas chlororaphis, Pseudomonas fragi, Pseudomonas lundensis, Pseudomonas taetrolens, Pseudomonas Antarctica, Pseudomonas azotoformans, Pseudomonas brassicacearum, Pseudomonas brenneri, Pseudomonas cedrina, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas gessardii, Pseudomonas libanensis, Pseudomonas mandelii, Pseudomonas marginalis, Pseudomonas mediterranea, Pseudomonas meridiana, Pseudomonas migulae, Pseudomonas mucidolens, Pseudomonas orientalis, Pseudomonas panacis, Pseudomonas proteolytica, Pseudomonas rhodesiae, Pseudomonas synxantha, Pseudomonas thivervalensis, Pseudomonas tolaasii, Pseudomonas veronii, Pseudomonas denitriflcans , Pseudomonas pertucinogena, Pseudomonas cremoricolorata, Pseudomonas fulva, Pseudomonas monteilii, Pseudomonas mosselii, Pseudomonas oryzihabitans , Pseudomonas parafulva, Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas balearica, Pseudomonas luteola, Pseudomonas stutzeri, Pseudomonas amygdale, Pseudomonas avellanae, Pseudomonas caricapapayae, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas flcuserectae, Pseudomonas meliae, Pseudomonas savastanoi, Pseudomonas syringae, Pseudomonas viridiflava, Pseudomonas abietaniphila, Pseudomonas acidophila, Pseudomonas agarici, Pseudomonas alcaliphila, Pseudomonas alkanolytica, Pseudomonas amyloderamosa, Pseudomonas asplenii, Pseudomonas azotiflgens, Pseudomonas cannabina, Pseudomonas coenobios, Pseudomonas congelans, Pseudomonas costantinii, Pseudomonas cruciviae, Pseudomonas delhiensis, Pseudomonas excibis, Pseudomonas extremorientalis, Pseudomonas frederiksbergensis, Pseudomonas fuscovaginae, Pseudomonas gelidicola, Pseudomonas grimontii, Pseudomonas indica, Pseudomonas jessenii, Pseudomonas jinjuensis, Pseudomonas kilonensis, Pseudomonas knackmussii, Pseudomonas koreensis, Pseudomonas lini, Pseudomonas lutea, Pseudomonas moraviensis, Pseudomonas otitidis, Pseudomonas pachastrellae, Pseudomonas palleroniana, Pseudomonas papaveris, Pseudomonas peli, Pseudomonas perolens, Pseudomonas poae, Pseudomonas pohangensis, Pseudomonas psychrophila, Pseudomonas psychrotolerans, Pseudomonas rathonis, Pseudomonas reptilivora, Pseudomonas resiniphila, Pseudomonas rhizosphaerae, Pseudomonas rubescens, Pseudomonas salomonii, Pseudomonas segitis, Pseudomonas septica, Pseudomonas simiae, Pseudomonas suis, Pseudomonas thermotolerans, Pseudomonas tremae, Pseudomonas trivialis, Pseudomonas turbinellae, Pseudomonas tuticorinensis, Pseudomonas umsongensis, Pseudomonas Vancouver ensis, Pseudomonas vranovensis, Pseudomonas xanthomarina; Rahnella species, Ralstonia species: Ralstonia basilensis, Ralstonia campinensis, Ralstonia eutropha, Ralstonia gilardii, Ralstonia insidiosa, Ralstonia mannitolilytica, Ralstonia metallidurans, Ralstonia paucula, Ralstonia pickettii, Ralstonia respiraculi, Ralstonia solanacearum, Ralstonia syzygii, Ralstonia taiwanensis; Raoultella species, Rhodoblastus species, Rhodopseudomonas species, Rhinos cleroma, Rhizobium radiobacter, Rhodococcus equi, Rickettsia species: Rickettsia africae, Rickettsia akari, Rickettsia australis, Rickettsia conorii, Rickettsia felis, Rickettsia japonica, Rickettsia mooseri, Rickettsia prowazekii (Typhus fever), Rickettsia rickettsii, Rickettsia siberica, Rickettsia typhi, Rickettsia conorii, Rickettsia africae, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae ; Rothia dentocariosa, Salmonella species: Salmonella arizonae, Salmonella Bongori, Salmonella enterica, Salmonella enteriditis, Salmonella paratyphi, Salmonella typhi (Typhoid fever), Salmonella typhimurium, Salmonella salamae, Salmonella arizonae, Salmonella diarizonae, Salmonella houtenae, Salmonella indica; Samsonia species, Serratia species: Serratia entomophila, Serratia flcaria, Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia marcescens, Serratia odoriferae, Serratia plymuthica, Serratia proteamaculans, Serratia quinivorans, Serratia rubidaea, Serratia ureilytica ; Shewanella putrefaciens, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Sodalis species, Spirillum species: Spirillum minus rat bite fever, Staphylococcus species: Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus felis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus pettenkoferi, Staphylococcus saprophyticus , Staphylococcus schleiferi, Staphylococcus simulans, Staphylococcus vitulus, Staphylococcus warneri, Staphylococcus xylosus; Stenotrophomonas species: Stenotrophomonas acidaminiphila, Stenotrophomonas dokdonensis, Stenotrophomonas koreensis, Stenotrophomonas maltophilia, Stenotrophomonas nitritireducens , Stenotrophomonas rhizophila; Streptobacillus species: Streptobacillus moniliformis (Streptobacillary rat bite fever); Streptococcus species: Streptococcus Group A, Streptococcus Group B, Streptococcus agalactiae, Streptococcus aginosus, Streptococcus avium, Streptococcus bovis, Streptococcus canis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus milleri, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus parasanguinis, Streptococcus suis, Streptococcus thermophilus, Streptococcus vestibularis, Streptococcus viridans, Streptococcus uberis, Streptococcus zooepidemicus; Tatumella species, Trabulsiella species, Treponema species: Treponema carateum (Pinta), Treponema denticola, Treponema endemicum (Bejel), Treponema pallidum (Syphilis), Treponema pertenue (Yaws); Tropheryma whipplei (Whipple disease), Tuberculoid leprosy, Ureaplasma urealyticum, Veillonella, Vibrio species: Vibrio aerogenes, Vibrio aestuarianus, Vibrio agarivorans, Vibrio albensis, Vibrio alginolyticus , Vibrio brasiliensis, Vibrio calviensis, Vibrio campbellii, Vibrio chagasii, Vibrio cholerae (Cholera), Vibrio cincinnatiensis , Vibrio Comma, Vibrio coralliilyticus , Vibrio crassostreae, Vibrio cyclitrophicus, Vibrio diabolicus, Vibrio diazotrophicus, Vibrio ezurae, Vibrio flscheri, Vibrio fluvialis, Vibrio fortis, Vibrio furnissii, Vibrio gallicus, Vibrio gazogenes, Vibrio gigantis, Vibrio halioticoli, Vibrio harveyi, Vibrio hepatarius, Vibrio hispanicus, Vibrio ichthyoenteri, Vibrio kanaloae, Vibrio lentus, Vibrio litoralis, Vibrio logei, Vibrio mediterranei, Vibrio metschnikovii, Vibrio mimicus, Vibrio mytili, Vibrio natriegens, Vibrio navarrensis, Vibrio neonatus, Vibrio neptunius, Vibrio nereis, Vibrio nigripulchritudo, Vibrio ordalii, Vibrio orientalis, Vibrio pacinii, Vibrio parahaemolyticus , Vibrio pectenicida, Vibrio penaeicida, Vibrio pomeroyi, Vibrio ponticus, Vibrio proteolyticus, Vibrio rotiferianus, Vibrio ruber, Vibrio rumoiensis, Vibrio salmonicida, Vibrio scophthalmi, Vibrio splendidus, Vibrio superstes, Vibrio tapetis, Vibrio tasmaniensis , Vibrio tubiashii, Vibrio vulnificus, Vibrio wodanis, Vibrio xuii; Vogesella indigofera, Wigglesworthia species, Wolbachia species, Xenorhabdus species, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, and Yokenella species.

[0066] Additional food-bourne bacteria suitable for use in a Spike-In standard include, but are not limited to Aeromonas hydrophilia, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, enteropathogenic Escherichinia coli such as 0157.H7 (E Coli), Listeria monocytogenes, Salmonella, Shigella, Staphylococcus aureus, Vibrio (e.g., parahaemolyticus) and Yersinia enterocolitica. [0067] In brief, the present example the methods can comprise the following steps for absolute quantification of a mixed microbial community using next generation sequencing: (1) add a cellular spike-in control, (2) DNA extraction, (3) library prep, (4) DNA sequencing, and (5) bioinformatics analysis. In the first step of an exemplary method, a cellular spike-in control is added into the sample of interest. The sample of interest is prepared in a defined amount (e.g., 10 mg of feces, 100 mΐ of saliva or 200 mΐ of blood). For a specific sample type, the defined amount can be customized and optimized. 50 mΐ of the ZymoBIOMICS™ Spike-in Control I (High Microbial Load) or ZymoBIOMICS™ Spike-in Control II (Low Microbial Load) is added to the sample. The sample is then mixed, such as by inversion or pipetting. In the second step, total DNA is extracted from the sample using a DNA extraction protocol that ensure complete unbiased lysis of microbes (e.g., ZymoBIOMICS DNA Miniprep kit, ZymoBIOMICS-96 MagBead kit). If the DNA extraction process cannot lyse all microbes equally or preferentially lyses certain microbes (e.g., Gram-negative bacteria), the bias can be carried to the quantification of the relative abundance and eventually the absolute abundance. This is particularly important for large bias as the quantification becomes irrational on the whole and especially for each unique taxon. In the third step, the library is prepared/ Depending on sequencing platforms (e.g., Illumina, Ion Torrent, PacBio and Nanopore) and the type of library (amplicon sequencing or shotgun sequencing), the library preparation process varies. Two library preparation processes that are exemplary for Illumina platforms comprise 16S rRNA gene targeted sequencing where the library preparation is performed using the Quick- 16S NGS library prep kit and shotgun metagenomics sequencing where the library preparation is performed with Kapa HyperPlus kit or Illumina Nextera with customized adapters that are compatible with Illumina sequencing. In the present example, the 16S library is then sequenced on Illumina MiSeq using the 600-cycle kit and the shotgun library is then sequenced using Illumina HiSeq. For the bioinformatics analysis, data from 16S and shotgun sequencing are analyzed by any relevant bioinformatics tools to convert information into composition profiles (i.e., microbes identified and their relative abundance). The 16S sequencing data may be analyzed using an internal bioinformatics pipeline that confers species-level resolution. Centrifuge, a bioninformatics program or suite of programs, may be used to analyze shotgun sequencing data with the NCBI RefSeq genome database as reference.

[0068] In one embodiment, the present methods may be used to determine the confidence of negative conclusions. For instance, sample integrity could be determined to be maintained if the yields were high from a sample by A260, and the spike-in control nucleic acids and the positive control amplified via qPCR, but the target of interest (DNA) being amplified within the sample and the negative control did not amplify.

[0069] The methods may be used for built-in assessment of PCR inhibitors or impurities. For example, the presence of a PCR inhibitor would be determined if the spike in control nucleic acid, negative control, and the target of interest did not amplify, the A260 indicated there was substantial DNA present, and the positive control amplified.

[0070] In another embodiment, the methods may be used for assessment of sample transportation integrity. If the yields were very low from A260, then the spike-in control nucleic acid, negative control, and the target of interest would not amplify while the positive control would amplify. This would indicate that the sample was compromised during shipment, such as high temperature shipping conditions.

[0071] The present methods also allow for built-in assessment of detection limits individualized for each sample. By using qPCR of targets within a sample, at least that level of detection can be determined with certainty based on both shipping conditions and purification inefficiencies etc.

[0072] Using next-generation sequencing, it can be determined if a specific detection limit is achieved by absolute quantification systems built-in.

[0073] Thus, the methods provided herein can be used to control the total process from collection of the biological sample to the conclusions. The methods have an absolute quantification system built-in to the process which allows for assessment of detection limits individualized for each sample and assessment of PCR inhibitor or impurities.

[0074] So specific spike-in standards contemplated herein include, but are not limited to:

• A spike-in standard comprised in a sealed container comprising Bacillus subtilis (G+) Listeria monocytogenes (G+) Staphylococcus aureus (G+) Enterococcus faecalis (G+) Lactobacillus fermentum (G+) Salmonella enterica ( G- ) Escherichia coli ( G- ) Pseudomonas aeruginosa ( G- ) Saccharomyces cerevisiae and Cryptococcus neoformans microbial cells or cell lysate in a preservation reagent ( e.g comprising a chaotropic salt). In some aspects, each of the microbes are present in a known amount in the sealed container. For example, in some aspects, each microbe is present in a different amount (such as each differing from one another 10 fold in a log distribution). In some aspects, the seal container further comprises a biological sample, optionally wherein the biological sample is comprises on a collection matrix (e.g., a swab).

• A spike-in standard comprised in a sealed container comprising Imtechella halotolerans and Allobacillus halotolerans microbial cells or cell lysate in a preservation reagent (e.g., comprising a chaotropic salt). In some aspects, each of the microbes are present in a known amount in the sealed container. In some aspects, the seal container further comprises a biological sample, optionally wherein the biological sample is comprises on a collection matrix (e.g., a swab).

• A spike-in standard comprised in a sealed container comprising Truepera radiovictrix, Imtechella halotolerans and Allobacillus halotolerans microbial cells or cell lysate in a preservation reagent (e.g., comprising a chaotropic salt). In some aspects, each of the microbes are present in a known amount in the sealed container (e.g., 10³-10⁵ log distribution). In some aspects, the seal container further comprises a biological sample, optionally wherein the biological sample is comprises on a collection matrix (e.g., a swab).

• A spike-in standard comprised in a sealed container comprising Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Enterococcus faecalis, Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis, and Saccharomyces cerevisia microbial cells or cell lysate in a preservation reagent (e.g., comprising a chaotropic salt). In some aspects, each of the microbes are present in a known amount in the sealed container. For example, in some aspects, each microbe is present in a different amount (such as each differing from one another 10 fold in a log distribution). In some aspects, the seal container further comprises a biological sample, optionally wherein the biological sample is comprises on a collection matrix (e.g., a swab).

II. Reagents and Kits

[0075] Kits may comprise suitably aliquoted reagents of the present disclosure, such as a spike-in standard. Additional components that may be included in a kit according to the present disclosure include, but are not limited to, one or more wash buffer, an elution buffer, a proteinase composition, DNase and/or RNase inhibitors, DNase or RNase enzymes, oligonucleotide primers, reference samples (e.g., samples comprising known amounts of DNA or RNA) as positive or negative controls, distilled water, DEPC-treated water, probes, sample vials, polymerase, and instructions for nucleic acid purification. In certain further aspects, additional reagents for DNA and/or RNA clean-up may be included.

[0076] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing reagent containers in close confinement for commercial sale. Such containers may include cardboard containers or injection or blow-molded plastic containers into which the desired vials are retained.

[0077] When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being preferred. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

[0078] In some aspects, a kit composed of collection devices containing a DNA/RNA preservation reagent ( e.g . DNA/RNA Shield) and a previously described spike-in control, such as cells, microbes, viruses, DNA and RNA. In some aspects, the spike-in standard is provided in a tube comprising a preservation reagent and configured for the collection of swab samples, fecal samples, blood sample, saliva samples, urine samples or tissue samples. In a specific example, there is provided a sealed container comprising: a preservation reagent (e.g., comprising a chaotropic salt), one or more spike-in standards of the embodiments and a biological sample. In a further example, there is provided a sealed container comprising: a preservation reagent (e.g., comprising a chaotropic salt), one or more spike-in standards of the embodiments and a biological sample comprises on a collection matrix (e.g., a collection swab). In a specific example, there is provided a sealed container comprising: a preservation reagent (e.g., comprising a chaotropic salt), one or more spike-in standards of the embodiments wherein the sealed container (e.g., sealed with a cap that can be punctured by a needle) is comprised in a pressure lower than atmospheric pressure. Optionally, this example can further comprise a blood sample and an anti-coagulant. In a further specific example, there is provided a sealed container comprising: a preservation reagent (e.g., comprising a chaotropic salt), one or more spike-in standards of the embodiments and a fecal sample comprised on a sample collection matrix (e.g., a swab). In a specific example, there is provided a sealed container comprising: a preservation reagent (e.g., comprising a chaotropic salt), one or more spike-in standards of the embodiments and a saliva. In a specific example, there is provided a sealed container comprising: a preservation reagent ( e.g a urine collection buffer comprising a chaotropic salt), one or more spike-in standards of the embodiments and a urine sample. In a specific example, there is provided a sealed container comprising: a preservation reagent (e.g., comprising a chaotropic salt), one or more spike-in standards of the embodiments and a solid tissue sample.

[0079] In a further aspects, a method of the embodiments, comprises measuring a standard, before purification to assess the quality of the sample prior to processing and analyses. This includes methods such as using a fluorophore or otherwise observable compound attached to a spike-in nucleic acids that would give a read on the sample prior to any other action by a simple method absorbance or fluorescence reader.

III. Examples

[0080] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Development and Characterization of Spike-in Standards for Microbiome Samples

[0081] A preparation of ZymoBIOMICS™ Spike-in Standard I (High Microbial Load) was loaded into 100 pL of 10% (w/v) stool suspended in DNA/RNA Shield, which is equivalent to a 10 mg stool sample. One prep of ZymoBIOMICS™ Spike-in Standard I has 5x10⁷ cells for each of the two microbes, Allobacillus halotolerans and Imtechella halotolerans , which are alien to the human microbiome.

[0082] The result of the 16S sequencing data of this sample was shown in the first Taxa bar plot in FIG. 2. The abundance of the two spike-in microbes in the overall sample as determined by 16S copy number was shown as 5.63% A. halotolerans and 2.67% /. halotolerans . The original composition profile of the sample was obtained by subtracting the two spike-in taxa, as shown in the 2^nd Taxa bar plot in FIG. 2.

[0083] To calculate the total cell number in this fecal sample (10 mg) from the abundance of/ halotolerans :

[0084] Assuming there were three 16S copies per bacterial genome and one genome per cell:

Total 16S copies

Total cells = = 1.8 x 10⁹

[0085] Similarly, the total cell number calculated from the abundance of A. halotolerans was 1.95 x 10⁹.

[0086] In another study, a preparation of ZymoBIOMICS Spike-in Control II was spiked into a sputum sample of 200 pL. 1 prep of ZymoBIOMICS Spike-in Control II contains 10⁵ and 10⁴ and 10³ cells of Treupera radiovictrix, Imtechella halotolerans, and Allobacillus halotolerans, respectively (Table 2). The microbial composition of this sample was determined by shotgun metagenomics sequencing. The abundance was determined as percentage abundance of total DNA (Figure 2) because Centrifuge reports abundance based on total reads assigned to each microbe. A standard curve (Figure 3) was generated by plotting defined DNA abundance (ng) of the three microbes against their percentage DNA abundance generated by Centrifuge. The standard curve equation is y = 0.0318x, which means 1% of DNA measured by Centrifuge equals to 0.0318 ng of DNA in this case.

[0087] This standard equation was then used to calculate the abundance of other containing microbes. Take S. aureus for example.

Its DNA abundance = 65.3% x 0.0318 ng/% = 2.08 ng 2.0871$ x 9.26 x 10 ^bp/ng

Cell number = = 6.9 x 10⁵cells

2.08 x 10⁶bp/cell [0088] Table 2: Microbial Composition of ZymoBIOMICS™ Spike-in Control II.

¹ 16S copies = cells ^c 16S copy number per cell/genome.

² Total genomic DNA (pg) = cells x genome size (bp/genome) x DNA unit conversion constant (ng/bp)/l000. DNA unit conversion constant (ng/bp) = 1.079 x 10 ¹².

[0089] Table 3. The abundance table of some selected microbes found in the sputum sample.

Example 2 - Methods for the Generation of Spike-in Sequences

[0090] The following describes a method for the generation of spike-in sequences with minimal collision risk against known, sequenced genomes (see FIG. 1). First, a large number of random DNA sequences are generated with a given GC content. The sequences are generated with the appropriate number of GCs and ATs. These sequences are created by randomly adding a G or a C (in the case of GC) or an A or a T (in the case of AT) the appropriate number of times, which is determined by multiplying the desired final sequence count by a factor of 10 raised to the power of the number of selection steps. The two sequences are then concatenated and shuffled. The resulting sequences should have the appropriate GC content, with a normal distribution of G vs. C and A vs. T randomly ordered.

[0091] Next, the sequences are measured for their longest homopolymer run. The sequences with their longest homopolymer run lengths in the in the bottom 10% are kept, while the remaining 90% are discarded. In order to remove sequences comprising long runs of G/C or A/T, both A and T are assigned to be W, and both G and C are assigned to be S. The sequences are analyzed once again and those with the their longest homopolymer run lengths in the bottom 10% are again kept, while the remaining 90% of sequences are discarded. Sequences are then compressed using the bzip (Burrows-Wheeler based) algorithm and the ratios of compressed to uncompressed are measured. As more complex sequences are desired and as compressibility should be inverse to complexity, sequences in the bottom 10% of compressibility are kept and the remaining 90% of sequences are discarded. The remaining sequences are searched within the BLAST database with no species specified in order to compare the sequence to all genomes with sequence data deposited on the BLAST server (e.g., NCBI). Sequences in the bottom 10% for matching any known genome are kept, while the remaining 90% of sequences are discarded. The final sequences are optimal for spike-in creation with as they have high complexity, low homopolymer runs, and show minimal matching to any known genomes. Selected final sequences are shown below, each sequence comprising 500 bases (70% GC content, SEQ ID NOS: 1-10; 50% GC content, SEQ ID NOS: 11-20; 30% GC content, SEQ ID NOS: 21-30).

SEQ ID NO : 1 :

GCCGCGCCTACCCCTGT CACCCGGGCCCAGT CCGCCTCAGCCCCCTGTAGGGCGCTGCCGCC

AGT CGGCAGGGGCAATCCCCGGGGTGGGCCAGGCCTCGGGGCGGAAAACTACGT GGTATACG

GCGCGGGGCTACTCCGCCGACCCTTCGCGGCGGTTAGGAGGGTAGCACGGGGGCTACTGAAC

CGGCGCCGGAAGCACTTAACGGCCCAGAAGGGCTCTTCCCGCACACGCGTTCTCTGCACGAG

TTGGGCCCTCCCAGCCCCCTAGCCAGACCCGCGCTTGTTGGCTACCGCGCCACAGGCGGTGC

GCCAGCTCTATGGAGTCGCCGCGTCGGCCGGCACCTGAAAGCCGGACACGTCAGGACTTTTG

AGT GGAGCGAGGTCCCCTGGCCCATGGAGGGGTTGGATGGGGAGCCCCACGCAGTACAACGC

ATTTCCGCGGGGGACCTACGCCGGCTGGCCCGCTGTCGACTGCTAGGGGT CATACGGGGCCT

GGGC

SEQ ID NO : 2 :

GCCGGACCGGGCCGACGCGCCCAGCCGTTGTAAAT GTTAAGGCTACCTTCACGCCTGGGTTC

GAGGCTCCGGCCGCATAACTAGGTCCT GCCCGAGGTAGGGTAGAACGGCGGTGGATAGCCCC

AGGGGCGT CCCCCTCGGTGTCGCAGGAGGGCCGTCCCGGGTGCTT GAGTGGTGAAGGCCGCC

CCCTCCTAGGGCTGAGGGCGCGGCACCCCGCGGGCAGAAGATGCGTCCAGTCGTTGCTACCT

TGGGGCCGGCTTATGTCGGACCGGGAGCCGAGCGTTCCAAGGGCGTCGCCATCTAGGTGTGA

ACCGACCT CCCGCCCCGTATCCTCCAGCCTCAGATTCAATACGGAGGCTTTGCCCTGGCGCG

GAGCCGTGCCCATGCCACACGGTCTCCCAACATGCGCAGGCCCGAGGGGAGGCCCCGGGATT

GCGGACCCGCGACCTCGGGGCACGGGTACTCCGCCCGCCACGGCAGGGGGACCCCGAGCGCG

CCCC

SEQ ID NO : 3 :

CCCCAGCGGCGCAGCGGTCACGAGGGT GAGGTTGAAGCAGATCAACCGTGATTCGGGGGCCC

TCGCGCTAGCGTTGGGGCGTTAACGCCACCGCGGGACCTCGCCCGAAACGCGTGCGAGCCGG

CGAAGACT CTAGGCCGCCCACCGGTCCTGGGTCGT GGCGT CCGAGTCTCCCCGCAACGCCAC GCT CCCCGCATAGACCCCGCT GGTCTGCCCGCGGGTGCCACGCCCATGGAAGACTGCGCGGG

GACCGCGT GGGGGAGCGCGCCTAGTACACGGTGCCCGGGCGGATGTGCGGCGAGATCCCGCC

GACATGCCAGCGTCTTGCGCCAGATGCGGCGTCGGTCGACTTCGTTGAGT CCCCGGGCT CGC

CCCTAGAGGGGGTCCGGGGCTACGACGGTAT CCCGCCCAAGGCGGGTGACCAGCCCAGGATC

AGGCCCGACGTGGCTTCCGGTTCCGAGCCTTTTGCCCAAT GCTTATCCACCAACACGGCGTA

GGGT

SEQ ID NO: 4:

GCCATAGGGACCGTGGGGACAGCAACGATCAAGGCCCTCCACGCGTTTTGCCTGCTGCAGCG

GGCCACAACGCAATACGCCCCGCTCAGCCGACTGACGCGTACGGCGCCTACCCCAGCCCTAC

CGGCTCGCATTGAATACGCCTTGTGTGGCGCGGCT GGAGTTCCGAGTCTGCTACAGGGGCGG

CCCCTGGAGCGGACCCGCGCCGACCGCGAGCGGAGTGGGAACCCGCGGGACGGGCAGGCACT

CGGTGCTGCGCGTATAAAGCGCGGTCAAGGT CCCCGGTCT CGGGT GGGCAGCAT CGGAGATC

CCCTGGTGCGCCGCTGGCCCCGAGTGT GGTCGGCAGGGTATTCACGCTGGTGTGCCGCGCAC

GCCCAGGGCCCGCGATTTTGCCCCGGCGTCGCCAGGCGGGCCCGT GTACCGAGCTGCCGGGC

ACGTCACGCCTCTGACAGCCT GCAGAAAACCCGGGGTCGTATGGCGGGGCCCTGCCGTCATC

ACT C

SEQ ID NO: 5:

GCGATCGGGTGCGTGGGCCGT CGCAGCCTGAGCTCCGATACAACCCAGCGGGGCTTAAT CGC GCGGCGCGTTCCGGAGCGCCCGCAACTTTTCAGCCAGCAT GGCGATTCCCGAGCGGGGACCT TGCCATCCACACTTTCT GTCGCTGACCACGGGCGAACGGGCCCCCGTGCGAGACCCCAGACC GCGCTTAGTCGGGGCTCCGCAGGGGAACCCCGGGCCGCAGCCGTACGCCGAGGATGCGCGGG TCGCGCCGATGGGATGGTCGCT GAT GGGGCTT GGGT CTTGGGATCGCGCCAGGCTGTGCTGA CCCGCGCT CTGGGACGCGCGCCAGACCCCCT CGGCCCGGCAGCCCCGTTCGACGGGACGGGC TGGCCCGGGGGCTCCCGCGTCGCACGCCGTATCATTGAGAGCCCACTGAT GCGCCCGGT GCT AAGCCGGGTGTAAAGAGCCAAGCTGCACGCGCTGGAGAAAATCACGGAGCCTGCCTCTCAGC TCCA

SEQ ID NO: 6:

CGGGGCGCGCCATCCGT GCGGTTGGTTTCCCCGCGGTCCCCGCTCCAGGCATGT GCCAAGTC

GTCGTGCCTCGCCGGGGCGACAAAGCGGGTGGGGGCACTCGGCCCAGTCCTGTCCAGGCAGA

CCCTCGGACTGGAGCGGGCCCCTGCGGGGAT CCGCTCAAGGAATCCCGAAGCTGAGGGCATT

GGCCGCAGTCGGCCGGT CCTCCCCATCGCACGGGGCACCCGGATT CCCCAGAACCCCCAGGC

GCGACGCGTGTAGGAGT CCACCCTGCTAGAACGTCGCCCGAGGTCGGTGCCCGGAGATCGAT

CGAGAGGCCGGT GCGGGCACAGTAGTGCCCCGGTCCCGTGAACCAACGGAGCAACTCGT GCA

TCCCGGAT GGCGGGCCCTTTT CAGACT GCTGGCCCCGCACCGACGACCCCCTGCCGCAGCAG

AGACGATT GTCT GAGCAGGAT GGCGGT CAGGGTGTTTGGACGCCGGACCGGTCGGCTGCTCG

GTCC

SEQ ID NO: 7:

GCCGCGTGTTGCATCGGCGGAAGCTATTCCGAAAGTGGTCAACGTACCTT GGGGGCGGCGTC GTGACCCCCAGGGGTCGACAGTCGCAGCGCCCGAACGGTCATGTACAACCGAGGAGCCGGGC CTGGCCGGCTAACGTCTAGCATGTGGCATGCTCGGGTCGCCCCGGCGGAGAAGCGACGCCCG AGCCCGCCAT GAGAGCGGGGTAGCATCGTTCTAGGCGTCGCGGCTTAGGCCCGGAAGAT CCG CCT GGCCCTCTCATCCCCTGTACGGCT CCCT CCCGTTACCCCCGACCCCGACACTCGAACCC GCGGTGGT GGGGCATGCCTGGAAGCGGCTAT CCGGCGGTGCCCGCCGCCCAACCTGGTGGGT GCTCGATGGCGGGGTGGGCGTGGCCGGCCCCGATCCGGAGCGGGTGCTCCGCGCCACCGAGA CGACCCACACGT TACT GGGTAGGCAAGGCCAACCCCGTCTTCGGCGCGCGCTGC CAT GGGAC GCAC

SEQ ID NO: 8:

CCGAAGGAGACCCCGTCGACGCTAGGGCGCAGCCGAT GACGGCGGGGACCCCGGCCCACTTC GGATGGAGCGCTTTTCGGATGAATGTATTACCAACTGACGCGAGGGGCCTGCGCGCAGCGTC CCGGCACGTGACCCCAGGCCGACGAAACGCGTGCTCGGCCTTACATCACCGGACCGGCCGCC GGACTCGGGGGTCCGCGCTAACGTGACCTTGCGGCGCGACTGCGCGTGTTCGGCCTGGCCGC GCGAGGGCGCGCGTAACCGTCTAGGCCAAACGGGCTAGTCGTATTGCCTGTCCAGAGTTCCC GTTCCGCTGCCCCAGCGAGCGCATCCGGGGCGACCCCGGGCCGAGGGCCGCCGTCCTCGTGA GAGGTGAGGGGTTCCCCTCGGCCCGGGGTGCTCACGCGGGATGTGGCGGGCGGGATCACCCC CAACACCATCCTCTGGTCCGTGGCAGGGGCCACTCCTACCGGCCACAGGAGGTATTACGTCC ATGC

SEQ ID NO: 9:

CGCAAAACACGTCGTTGCCGATACGGTGGGCGGGGAGCGTTGTGCGAACATGCCGCAACTGA

TCGCCCGTAGTGCGTCGCGCCTCCATCGGCTCTAGTGCGAGGCGGCCTACCTGGTCTCGGGG

AAGCTGGGGACGCCCCTCTGCTCAGCGTCCGGCCGCGGAGGCTGTCAGCGGCGGTTTCCCCG

ACACCGTCTCGCTAACGCACTCCCGTGGACTGGACCGGCATGGGTGGCCCCGGAGAGGGCTC

TCTGCAGCGTGGGAGGGAAGGGCCGCGAACTCACCGCTACCCCCACGGCGGATGAGTACAGA

CACCTACAGCGGTCTGGTCGTCCGCAGGAGACCTGCCGGGCTCCACCGAGGCGGCGCCTGTG

GCCGACGGCGGCAGTGACGCCTCTCCACGTCTAGGCCGGAGAGCGCCATCCCGATCAGAGTC

GAGACGGGGGCCCTGCTACGATCGCCCTCCTCCACCAAGTGGGGTGCCTCCGCGGCGGTGCC

CCGC

SEQ ID NO: 10:

CCGGAGGGGCCTGCGTCGGGCGGGGCAGCCACAGGTGCCCGTGTCGAGCGGTGCCCGCACGG CTCATCGCCGTGCGGGGCCGAGTACAACGCGGGTGCGCCCCCAGCAACACCGTAGCGGGGTC TGTACGGGCCCTCCGCAGCCCGCGTCCCGAGGGGCTGCGCGCCCGATAGGGCGTCTGTCCTC TCCCGGGCTAACGGAGAGCGCGTACAGGTGGGAGCCGGTGCAGGGGAAGGCTGTCCGTACTC CGTGACCCGGGTCTGCCTACACGGGCGACGATCAGCTCAGCGGCCCCGGACCCCATACTCAA GCCGAACCCACTAT GAGCTTTGTATAAGCCCACCGGTTTCACGCGAGGAGCCCTCAAGGCGG GCGAGTGGGTTCCAGGGACACGGTAGGCCGTTGCCCCGCCCAACTCGACGCCCCACCTTGGC CGTGTGTTGCACCGGGCACGTGGTTAGGTCCGTTCATAGGCGACCCGGGATCGGCACCTCCC GTCT

SEQ ID NO: 11:

ATAGGGAGCAGCGACTGTGCTTCATGGCATGCAAGGGAATCTAAGAAACGTAAGTTAGGTGC AAA GAT GAGGGGCATTCCTCGGCTTCTGTTGCGACAATTGTCACCCAAACACCGAAGACTGT GAGCAAGTCGTCAGATCAT GAACCATTGACCCCTACCATGCGGTTTGAAGGGGATGTAGTTT TAAGGACTGCCTCTCGGCCACTTCTGAGACATCTTTTACACGGGAGAGCTATTTTGAATCGG TTTGGTTTGATCCCCGTCTTCCAAATAGCCACTCCTGCCGAAGGAAGCGTGCGTCATGGGTG TTCGAGAGTGGGCACACTGTTCGTTATTTCAGCGTAAATCTGCAGCCGAGTTTCTACCAAAC GCAAAACATTTTCCGTCCGATCACGGAGCTTTTGTTCTAGGTGCGACCCACTTGCCCGACGC TATCTCCCACAAGAAACACTTCTCCTACTAGTCCAGATCACGAAAAGACTCCGCAGGTAAGT GGGG SEQ ID NO: 12:

ATAGGTGGGATGGCATCCCGAAACATCTGTATAAGGAAGGGTGAAGATCGGCACGCCATTCG TGGTAT GAGAAGGTTGCGCACAAACGCCCCTTCCGCAAGACTTTGACTTTTAAGTTGATCAG TTTGCTGTTAAGGCTAACGGCAACACACGCTGCCGTCATTTCTATCATCCTATTACGCACTC CCGAGAGGGATATGTTTCAGTAATCCGAAGATCTCAGGGGCCAGTGGGTTATCCATACGCGA ATTGTAGCAGAACCAGCCCTGTTGAACGTTCTGATTGCGGAGCCCGGGAATCATTAGTTTTG CCTTTGTATACACGAGTCACACAACGGACCAGACCGCCTATACGCGGGGTATCGCTATACAA AGTCGAGAAACCTCGAACAGGGCGTGCTACCGTTTTATACTCCGTCGGACACGGGTGCCAGT TGCAAGTATTACTGCTTGTAAGCCTGGCTACTATCTTTTAACTTCAGGGCACGCCCATCGAC ATCG

SEQ ID NO: 13:

TAGCTTTAACCGCCCTGGAGCATTGTTTCAGAATGCTCCCTCCCGTTAGAAGTCGGACCCCT GCGATCCACTATCAATGGGCGGCACTTGGATCACAGTAGATTGCAAGCAATTGCAAGCTGCC TAAAGGACGCTAACGAGTCAACCCCTGAGTGTTTGTGAATGTTGTGTTTTCCTTGCAGGACG GCGGTTCACCTTCGCTTGACTTTTATTCTACGACGGGACACAATTGCCTGCTCGGGCCAACC GTCCCATGTTGCTGTTTAGCCATCCAACAGACCCCGACATAAAAGCAGGCGAACGGGGTTAC GTTTTAAGGCTCCCCGGTTCATACATATGATACGCCGACCCATATTACCTCTAATGCCTGAC TCCCGGGTAGCAAGAAATGGATGAAAGTTGGGCGATGGTAGCCGTCAGGGCGAAGTGTAGAC TTGCATTAAGTACATTTCAGAGTACGTGAGTCCTTATTGAATAATCACGCAGGGAGAAT GAG GCAA

SEQ ID NO: 14:

AATACGCCAGTATCGGTGGACGAGTTTCGTATCGGGGAGTTTTGATAAACTACTACCCCTTG

CATTCGCATGCTGCAGCTAACCTGCTTCACGACCCGCGGAGAAGATAGCCTCGTATAGGCCT

CCTAGCTATGAATTCCGCTTTTCCGCATATCTATGCTTATCACGGAATGCAGAACGCGAGGG

TGGAGGCATGCAATTGGTACTAGTACGACCGGCTTCGACGCTAAGATACTTTTGTTCGGCAA

CCTGACATACGCTTGCCATGGTTTCAGGACGCTCTTTCAGGGGATTACCCTCATGCAAGCTG

GGGAGCATTTATACGACGCTCGGTCATCAGTATAATTGAACGCACAGGGACACAGCACAGCA

GTTGGTGTCACACTTTAGTCAAGGTAAATGCAAACGAACCGGCAGCTCGCTAGCGATGTTTC

ATGGGTCCTGACTGAAGCAGAGTGAACAGGCTCAGCTATATGTTAAAATCTCCTTCGCCACA

CTGG

SEQ ID NO: 15:

GAACTCAAATCCTAGTATAACGAAAGGTAGAAGAACTCCGCCATCTACGACAGGCCTTTAGC CTAAATAGTGACAAGGGGTCAGGAAGCCCCTGTGTTAAAACGGGTAGACTTCTTCAATGGGG AGGGTCGTAGACTGGCGAGTCCGTGGTAGCTCCTAGGTGCCCCGTCCTAGAGGGTTGGAGTC TCCTGTTAATAAGACGACAAGTATGCGAGGGCCGTACGACGCCTTTCTGTCACTTCAGTGCC TATCCACTCACTTGTGATTTTACCACACGTCGCCTAAATTGAAGTAGGTCCTAGCACTTCAC TCCAGACTTCAGTCACTCTCCGTCTTTGGCGTTATTCAACCCCGAGCCATTTCACTCCGCCA TAACTAATCGTCTGGAGGACAATGTCTCCTTTGGGGCAGACATTGGGCCACGTAGGCGCGTC CTCCTTGAAAAT GAGGTTGAGTAGGGACTATCGGCTATGGAACGAATCTGTAAAACGTAGAA TTGA

SEQ ID NO: 16: GTAATATCTACCGACAGCTCTCACCCGGTCATAAGTGCCGAAGTGACGGCCGATACACATTT CAGGCCGTGAGAGTGGAGAGATCCAAACTTGAGTCCTTAGAGTCGACTCGATAGACGCGACT CTACAATATTCCAAACTAACGTGACGAGAGCAGGGGTTGTGGGTTTCATGTACCCCGAGTGT GGTGGGGCTGTTCTTTGGATTTGACCCGGAGTTGTTAGCCAGTTTGCAGACCCGGATAGGCG GGCAGTAATCGACCTAAAT GAAGCTTACAGCTATTCTAGGAGGCAAAATCGGGGCTTTGTCT TTTCGCAGACCATACCATCTAGGTCAACGTTTTAATGGTAGGCGCCAGGCCAAACTACTACT GTCACGGAACTCTTCACAAGTCCCGTGAACTTTCTGGTGTGACCATGCCAAACCGTCTGGTA ACCGTCCACCTAAGGAACATGCCCAATCTCCGTTTACTTCCAAGTTAGAGTCTATCGTCACG TAGC

SEQ ID NO: 17:

AGTTACATTGAAAAGGTAGGTAGAAACAGGGTTGGCAAAAGTTGCCTAACTTCTCACTCTGT

GTAGGGGTGGTTGTACTTAAGTGGTCGACTATGGCGTTCCGACTTGTGGAACCGGAGTAACA

GGGGTAAACGAGCCAACAGTCTGAACCGTGCTATCGATCATTCGCAGCATTGTCATCGAGCT

TTTCGCATTAAGCTAAAGCAAAGAGAGGTGACTCACCCCTGCCGACACCCAAGACACGCTTT

TGAGCTGGTGCGCGAACCTACGATATTTAGAGTCGGCAGGGGATCTCGCAACTAGTTTGTTA

GTTCTAGGGGCATTTCCGCTAACGGCCCTCTGACGGTTGTTTAAGGGTCTGCTCTAACCTCC

CAAAAGTTTAATACGCGCATTCAGAGACCTCTCTGTCACGGGACCCGGCCAAACTCATCTTT

CGTAATGCCTTCGCGGACGTATACGTTGACCTATTAGAACAACCAAGCACCCAGTTCACCTC

GGGC

SEQ ID NO: 18:

CGTCTAATACCCCAATTTTCCGTAAACGAATTCTGGTATCGCGGACGTGTGTGTTCACGCTG GCGCGGTCTAAAACGGATCCCTCAGTGTCTGCTACAATTCGTCGAAGAGACAGTAGAAGAGG GTATCCATATTTGATGTGTGAGCGTTGGTGATCGATTAAGATACATTTGAACCAGCCGTGCC TGCATCTCACCAAAGCTTGTAACGCGTCATCTCTTCCCCTTCCGTTCCCGCTTCAGGGGCGA GCTGTTCCGGTTATACCGGAAAGAAGGCAATGCAAATCCCCGATCTCCTTGCAGACGTCGTC GACAAGTCGCGTTGGGTGTCCACTCCTAACACCCGGCGATACTAGCGTCTCACCACGTTAAG TGCTAAAGAATCTGATTGAGCTTGACTAGTAGTTGCGCTGGTAGGACTAT GAAGTTACGGCT AGTAACAAGTACGTCAGTGCGTAGACTCCGAAAACTGTAGAAAGGCAAACAAGAGTGCTGGC CACT

SEQ ID NO: 19:

CGGTATCCAGCAAACTCCGCCGACCAGAGGTGCTGCTAAGCCGGAAAACGCTATGTCGATTG TCGGAGCACCCTTTCCCGAACTATCAATTCCGTTAACTCTCACACCAAAATGGTCTCTACGC ACGTTAACGGGGCTTGTGGGGAAAGATCTAGCCCAGATTCCAAAATCATCCATTGTGATCTT ATCGGTTGAACTGCCTTTTAAGAGTTGCCCCAGAGATTGTTCACCCGCGCAATCAAGCTGCC AACCAGGGGAATACCCAATGTTTTGAGTGGTTGTAATCCTGGAGGTTCGCCTAGATAATACG CGTCTAAGCCGATCAACCTTGGGGAGGCTCCAAGATTATAGCCCTATGTCTGGGGATTGGAC TTCTTTATGCTACGATAACGGAATACCCGAAAGGCGAT GAAGTAACCTTATGGTCCTTCTCT CGATGGTACTGGTAGGCCAACTACCGCCTAAGTGCGTGAGGGCGTGTGAAGAGTGTGGGTAA CCTC

SEQ ID NO: 20:

CCGGGATTCCAATGACCCGTGCTCCGCACGACGGGTGACGACCACGGCGAATCTTTTCACCC CAAGCACATGCCCTATGCCGATATAATGCTAAGTCGCGGCTCCTCGGATGTCACAT GATCTT CGCTCGACCTTCGGATGTTGGCGCATCATGTCTGGTTGTAAATCCCCGGTACATTAATTCAT CTACTCAT CACCATTCTAAGATGTCACAGAAGTTGTCCGGGGCTT GCGTGTAGCTTATCATT

TGACAGAGTATGCATCGGGAGCAGTGCATCCGGAAGAAGATCAATTAGAT GCGCAGAAGAGC

TCCTGTAACGATTACCGAAAATACACGGTCT GCGGTCAGATACGTTGGAGAGGGGCTAGACG

CCGGGTTTTATGTTAACTCTACTTGCGACTTTTACATGTCCCTTAGTCACAATGTTTTGTCG

AACTAGCGGATGAGCAAAACGCACGAAACACTTAAAGGGGTGGGAGTATTACCCTCCCGACA

AGGG

SEQ ID NO: 21:

ATTTCCGTTAATAGAATTTTATTGGTGGTGGTCTTAATATAGGCTTAT GACGAAATGTAAGA TTT GGAAAATTCTTTGACTGATCAATGAATGCATCTTAACATTCATAATTTGCTAAAATTAA TTGAGATACCAT GTTTCGCCCTTCTTAGAACGTAAAAGAAAAATGAGAAATGAGGGGGACTA CGTATCGGTTCCATTATTATCAAAAGCAACAGAGTATACCTAATTATGTCTATT GACTGGTT TTTAAGGT GTTATAATATCGGCGTTTAATCTAATCTAATAAACTATACATTACCAAGTACGC TTTTAAGTTATACGTATATGCTCTAGTATTTTACTTCTCAAATTGATGGGTTAAAGGTTATA ATTAGATTATTAATACTTAAACCCTCGCTTTACTTACGATACACTAATACTATCTCATCAAT ACCTTGGAATAT GCTCCCCATAGTAATAAAACCTTAAAAATAGATAAAAT CATGGTAGCTAA ATAC

SEQ ID NO: 22:

AAATTGAATGCT GCAACTGTTTTCTTTATACTTAGATATTAACATAATAACATAGCGAGAAT CT T C AT T AT GT C T T T T T C AAC T GC AT AT AT AC GT AAT T C AAAAAT T GAT AT T T AT AGAC C GA GACACTTT CTAATGCGATGATTGAAAATTAT CATCAAAAT GGGTGGGCGCGATGTTAAGTTA ATT GTATGCAATAAATCGTAT CAT GAAAGACTAATTACCT GCCTAAGCCT CTAGAGATTATT ATTTGAAT GGACAATATTAAT GAACTGTAAAGTCAAATGCTTTTTAACATTGGAACTAAGCC GTTTTTCT CTTT CTATAGATATACCGGTTTAGTTAGTTAACTAAATTAGTAGATAATTTTGC TTAAT GAT CGTTATTATAAGACCGACCCAAGGTACCTAAAATTTGATTGT GTCATAGAACAA GTT CAAAACAACCATACATTT GTTCTACCTTTAACCCAAT CTGTAGTATTTAAT GGGGATAA CAAA

SEQ ID NO: 23:

CGAAACCAAAAGATAAAGTGCTTAATGATCTAATGTATTGCGTATAGACATCATAGTGT GAT AATATCTCTAAATCTACTTGAATTTCAGTTT CAGT CTAAATAGAACTTGTTACTACACTTAA TGT GGAAAACAACTCTAAAGCCATTAATGCT CTGAATTAGCTATT GGATAGCGCAAACT GGT AAT GT GT CAT AAT T C AAT T GG GAC AAAAT T T T GAAC AAAAT T AC C C AT AAAT AAAAC AT C C A ATTTGTAT CTTGCCATAAAATTCTTATTATT CATGTCTGGATCTTTTATCTTACTATTTTAT ACAT AAGGAT AT CTT AT ACGT TT GAAACT CAAATT GT AGC AAAGAACAT AAT GAT GAAC ACT GATATAAGTTCTTTACT GATTTTCGACACTATTATATTGTACTGATTTTT CAATTGCAGCAT TAT GTTTT GGTGGAAGAAAGTACCCATAGTGATAGGAGTT GGATTATAACGCGCACTTTATT AACG

SEQ ID NO: 24:

GTATAGCTAT GATACCTTTTGAAGTGCAGTATAGATTCGCTGAGACCCTTAAGACTAACAAG T CAT GAT GAGAATTTCGTAAGAGAGGAGAATAAGAT ACGT ACGT C ATT AAT AAT AT CACGTT TCCCCACACTATACTATAAGAATACTACTCTTGCACAACTAAATT GTTAGACACACAGTAAA CAGAAAAATAGATAT GAATAATTTTTGGCGCTTAATTCTCAGAGCTTTAAAACCTGCATTAT TTAGTTTT AAT AAT GCGT AT GAGTTGGCGT CAAATT GAT AAT AACTTGCGAACAACGTGGAT T AAATT AT CT AAT AAT AT AGAT AAAAT TTCT GT AT TATTTT ACAT T AAAT CTGCTAACT GTT TTCTCGAATCGTAGTTTTCTAATCCTACGAAAAGGTAGATACTGTAAGTAACGTTACCATGA TAT CTGTATTTATATTGTAGCAT GATTTTACCCTTTTCATATCTTAATTATCTATTAGATTT AGAA

SEQ ID NO : 25 :

TATATTTGAATTTATTT GGGTTGTATT CGGCATAGTATATAAGACTTACAAATAAGTGTACT ACT GGCTAAGGTTTGAAGAAGAAT GAAGTCT GTCT GATACGCGATATAAAAGGTAAATATGA TCGATATTACAATAAATTATCTTTAAAATTT CTACATAAATCAAGTTTAACACATCATACAA AGATTAACTTAGAATTCTGCCCAAATCTACTTGGGACAAT CGATCTGATTTTACAGGCTTTT ATGAGTTACGTATTCGCATTAATCTCAATTAGGAT GACGATTCATTATTTTCTACTTGTATT TAT GAAAAACACTTCTGTTCATCAATTTATCTTATAGAGCTAAGATTACCCCCTAATTGAAG GGGATATTAAAACAAAAGCCGTTTTAGTTATTGAGGCAATTAT GATAGCGTAAAGTAAGATA AAT GCTCATTATTTTCTTATGACCAACCACTACAGGAGAATAACTATTTCTCAACTTAGCCC ATTT

SEQ ID NO : 26 :

ATT TT T ATT CAGTAATT TAT C TT T CAACTT G GAT AAGTT C TT CAT AAGC G C GATT CAAATAA TTAACAACTAGGTGGGTACCT GTTGTT GGAATAAGCAATT GATATACACAGTAATAGTAAAG AACGCATTTATAGAT GACTAGATTTAGGTGACACTAAAGT CTATTAACTTTAATTTGAATTC TTTTGTGTTAATAGATGCTCCGCGTTATTACTTTATGAAACGAAATCCATTTTATTGAATAA CAACACTGTATTTAATTAGTAGTAAATATAT GGAAATCTGTCAGGAGTAATCGCAATTAGTA CCCCTTTTTCACGTTCT CAGTTTCATT GAACGAGCACAAGATAAAGTTAATTTT GGTTT GGT ATAAAAAGAATCGACATAAAACCAGCT CTTATATAACGTTTTCAATCACACGGATTTTAATA CTT CTACATAGAAAGAAAATCTAAGACTCCGGTATTATTT CACTAACACTTAGCTGTATAAT GACA

SEQ ID NO : 27 :

GTTTATTCTCAATATAATGTATTACCAATATTTAAGTATACTGACATACGAAGAATTAT GTA TCGTTCTT GTAT CATACTATTTGCTCGCGTCCTACTCTGAAAGTAACAGCATTGAGTAAGAT ATGCAAAGCTTTATAAACAACCTATGGTAAT GAGCAAAAGGATAGTCTGT GCAGTTCAAATA AGT CTAGTTGAT GTTTCTCTGAAGAGCGTAAGTGCACTTCATATGAAAAATCTAGGGCTAAT CCT CATTTAATAATCAAAACTTTTATGCCTAAGACTGTAATAATTAATCTACAATATCGTTT TCTATAAAATCTTGACTATGTAATATGTGTCTGATTTTTAATCTGAGAAGTTCGAAGTATAT GTCTTACTATACTCTATTTCAGGCTTAATGCATCTATTGAACATAAAAGCTCAT CTTTACAA ACCAAGATAACTAAACT GTAATTAGTAGAGT GAGTAAACCTAATCATTGGATAATAGTGTTA AATT

SEQ ID NO : 28 :

AAAGACATTTATTTATAGTTAGGATCTTGTAACTGCAAGCAGCAGTATTAATTGACATTAGT CAATTAATAAACACTAATCATATGTCTATAT GAT AGCGGT CCACTTCGAGCCAAGGTT AATT CTT GCTCAAGCT CAAGT GTGAATTACAGTAATGTCAGATT GTAAAATTCGATGT GTAAGTTT TAGATCATAATTTAACT CTTGTCGAAAACAATCTAACTATATTACAATTAAAAT GTTCATGT CCATTATAGATAAAGTATTTTTACGAATGGGGATGTTATAAGAATACTTAAAATAGTGCACA TTAAGCTTAAAT GTATT GAACACTTAATTATTACAATCTGTGTTT GGTATACCTAATTTTAA ACTTCCTAAACCAAT GACTAT GGCAAAACACTTTTTCTGCCAAGATATTTTATTTCCCATCT TGCGAAGT CGAGATTTAGGAAAACCTATACTTCTT GGATTAACTGTCATATCTATTGGCGGA AAAA SEQ ID NO : 29 :

CTGGCTTAAGTTAT GAGCTGAAAACAAGATTATCTTTTTCTGATATGTTTTACCTATTGCAG AACCGATCACTGAACGAAACAATCCAAACCCTAATAACAGTTAGCTCGATTACAGGATCAGT AATAAAGTTTTT GATT GTTAGATTAGCCAAACTT CAT GAATTTATATCTTTAAGATTTT CAC AAG AT GAAT T T C AT T C C T AT AAAT T T C T T AAT AAAC AAGT AT T GAATTAATAAC C AGGC GAT CTACATAGATAATTGTTTTTGCATTGAATTAGGGGTGATTATATTAGCGGTTGTACGAAAGA GTCAATTATTTACTTTT GATATGTGTCACTACCTGAAAGGGTAAGTAATACATGTTGTGTAA CAACTATCTCAT GTCTCATATATCCGACTAACATTTATACAAAATTAGTTTAGCGAATTTAC AAAAGGATTATATTCGGACCCACTCTTTAGT GAAACTATAAAATATTGAGCACGCATTTAAT TAAT

SEQ ID NO : 30 :

AGTTGCATAGGGCATACTTTCTATTTAATACATAT CTGATTCATGGTACTTTTAACCGAAAT AAAGAGAGTCAATAAGCTCGCATTTCTAACCGTTATATTGCACGACTATCGTAT GAT AT AT A GAATCTAATTAT GCGGCGTAATTTCATATAGGATAGATTGGTAATATGCTTACTTACTTAAA ATATGTCTTGCCAACGTATAAAGAACCTTAGGGACACTAT CTAAAAGATTTTTCTCCAAACT TTATTTTT CTAGGATTT CAAGATAAGTAAAT CAGATGCTT GCATGTTAGTATAT CGAAAAAC AGGTTATAATAAGGTATTTACTTGTAAAATT CCTGCCATCAGAACAGATTTCTTTCTCGCTA AAT GAGCTAT GAGGAATATAACGCCTT CATAATTAAATTCTTTAAGAACATACGGATATACA T T C AT T T T AT AAAC CT AT AAAAAGAT CAT AT T GAT T T C GA

F F F

[0092] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A reagent for storage, lysis or purification from a collected biological sample comprising a spike-in standard.

2. The reagent of claim 1, wherein spike-in standard allows tracking to reflect quality of the collected sample; the quantity of a collected sample; the detection limit within the collected sample or integrity measurements with the biological sample.

3. The reagent of claim 1, wherein the reagent comprises a biological sample.

4. The reagent of claim 1, wherein the biological sample comprises bacteria, archaea, fungi, a eukaryote, viruses, DNA, RNA, proteins, metabolites, lipids, and/or carbohydrates.

5. The reagent of claim 1, wherein the spike-in standard comprises DNA, RNA, and/or cells.

6. The reagent of claim 5, wherein the cells are microbial cells.

7. The reagent of claim 1, wherein the biological sample comprises RNA and/or DNA.

8. The reagent of claim 1, is a fecal sample, sputum sample, saliva sample, swab samples, CSF samples, or blood sample.

9. The reagent of claim 1, wherein the biological sample is a mammalian biological sample.

10. The reagent of claim 1, wherein the biological sample is an environmental sample

11. The reagent of claim 1, wherein the biological sample is a soil, water, plant, surface swab or filtrate sample.

12. The reagent of claim 5, wherein the spike-in standard is a cellular spike-in standard.

13. The reagent of claim 5, wherein the spike-standard comprises 1, 2, 3, 5, 6, 7, 8, 9, or 10 different microbes.

14. The reagent of claim 5, wherein the spike-standard comprises 20 to 100 different microbes.

15. The reagent of claim 13, wherein three different microbes are used and the organisms comprise Treupera radiovictrix, Imtechella halotolerans , and/ or Allobacillus halotolerans.

16. The reagent of claim 1, wherein the spike-in standard is further defined as a nucleic acid spike-in standard.

17. The reagent of claim 16, wherein the nucleic acid spike-in standard comprises artificial nucleic acids not matching any known organisms.

18. The reagent of claim 16, wherein the nucleic acid spike-in standard comprises one or more nucleic acid molecules comprising a sequence at least 80% or 90% identical to SEQ ID NO: 1-30.

19. The reagent of claim 16, wherein the nucleic acid spike-in standard enables collection, preservation, and/or transportation of the biological sample without nucleic acid degradation, microbial growth gene induction, and/or epigenetic change.

20. The reagent of claim 16, wherein the nucleic acid spike-in standard comprises 500, 1000, and 2000 bp RNA.

21. The reagent of claim 16, wherein the spike-in standard comprises 17, 20, and 25 base pair miRNA.

22. The reagent of claim 5, wherein the RNA is miRNA.

23. The reagent of claim 16, wherein the nucleic acid spike-in standard comprises 1000, 2000, and 3000 base pair double-stranded DNA.

24. The reagent of claim 16, wherein the nucleic acid spike-standard comprises log dilutions.

25. The reagent of claim 24, wherein the log dilutions comprise 10 ³ ng, 10 ⁴ ng, and/or 10 ⁵ ng of DNA or RNA.

26. The reagent of claim 5, wherein the DNA comprises 35, 50, or 65% GC-content.

27. The reagent of claim 16, wherein the nucleic acid spike-in standard is GC-rich.

28. The reagent of claim 16, wherein the nucleic acid spike-in standard is AT-rich.

29. The reagent of claim 5, wherein the DNA or RNA is epigenetically modified DNA or RNA.

30. The reagent of claim 29, wherein the epigenetically modified DNA or RNA comprises at least 10%, 25%, 50%, or 100% 5-methylcytosine methylation.

31. The reagent of claim 1 , wherein the biological sample has undergone storage, transport, purification, library preparation, and/or processing.

32. A container comprising the reagent of any one of claims 1-31.

33. The container of claim 32, wherein the container is further defined as a swab and collection tube, blood vacutainer, fecal scoop tube, or saliva collection device.

34. A kit comprising the reagent of any one of claims 1-31 and a primer or probe that can be used to quantify the amount or integrity of the nucleic acid molecule in a sample.

35. The kit of claim 34, wherein the reagent is provided in a swab and collection tube, blood vacutainer, fecal scoop tube, or saliva collection device.

36. A method for assessing the quality of a biological sample comprising:

(a) adding a spike-in control of known quantity to a biological sample;

(b) sequencing the biological sample comprising the spike-in control;

(c) performing bioinformatics analysis to obtain relative abundance of molecule(s) of interest; and

(d) converting the relative abundance of the molecule(s) of interest into absolute quantification of the molecule(s) of interest based on known abundances of the spike-in control.

37. The method of claim 36, further comprising extracting DNA and/or RNA from the biological sample comprising the spike-in control prior to step (b).

38. The method of claim 37, further comprising preparing a library from the extracted DNA and/or RNA.

39. The method of claim 36, wherein converting comprises generating a standard curve of the abundance of the molecule of interest versus abundance of the spike-in standard.

40. The method of claim 36, wherein converting comprises subtracting abundance of the spike-in standard from abundance of the molecule(s) of interest.

41. The method of claim 36, wherein the biological sample has undergone storage, transport, purification, library preparation, and/or processing.

42. The method of claim 36, wherein the method comprises assessing quality of the biological sample at collection, purification, and analysis stages.

43. The method of claim 42, wherein analysis comprises PCR and/or sequencing.

44. The method of claim 42, wherein assessing quality comprises measuring degradation of the molecule(s) of interest.

45. The method of claim 42, wherein the molecule(s) of interest are microbes.

46. The method of claim 45, wherein assessing quality comprises determining a measurement integrity characterization (MIQ) score.

47. The method of claim 36, wherein the method comprises using a nucleic acid spike-in standard to assess bias in sequencing.

48. The method of claim 36, wherein the molecule(s) of interest is a virus.

49. The method of claim 48, wherein the virus is human immunodeficiency virus (HIV), influenza virus, rabies virus, or herpes simplex virus (HSV).

50. The method of claim 36, further comprising adding a lysis or preservation reagent to the biological with the spike-in control.

51. A nucleic acid molecule comprising a sequence at least 80% identical to SEQ ID NO: 1-30.

52. The nucleic acid molecule of claim 51, wherein the nucleic acid molecule is a DNA.

53. The nucleic acid molecule of claim 51, wherein the nucleic acid molecule is a RNA.

54. The nucleic acid molecule of claim 51, wherein the nucleic acid molecule comprises a label.

55. A reagent for storage, lysis or purification of biological molecules comprising a nucleic acid molecule of any one of claims 1-7.

56. The reagent of claim 9, wherein the reagent comprises a known concentration of the nucleic acid molecule.

57. A kit comprising the reagent of claim 9 and a primer or probe that can be used to quantify the amount or integrity of the nucleic acid molecule in a sample.