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WO2020041449A1 - Procédés et compositions pour le suivi de la qualité d'un échantillon - Google Patents

Procédés et compositions pour le suivi de la qualité d'un échantillon Download PDF

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Publication number
WO2020041449A1
WO2020041449A1 PCT/US2019/047474 US2019047474W WO2020041449A1 WO 2020041449 A1 WO2020041449 A1 WO 2020041449A1 US 2019047474 W US2019047474 W US 2019047474W WO 2020041449 A1 WO2020041449 A1 WO 2020041449A1
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WIPO (PCT)
Prior art keywords
reagent
spike
standard
sample
nucleic acid
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PCT/US2019/047474
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English (en)
Inventor
Ryan KEMP
Stanislav Forman
Shuiquan TANG
Michael Weinstein
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Zymo Research Corp
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Zymo Research Corp
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Publication of WO2020041449A1 publication Critical patent/WO2020041449A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • 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.
  • the invention provides a reagent for storage, lysis or purification of a biological sample comprising a spike-in standard.
  • the biological sample comprises bacteria, archaea, fungi, eurkaryote, viruses, DNA, RNA, proteins, metabolites, lipids, and/or carbohydrates.
  • 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).
  • 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.
  • the biological sample is a fecal sample, sputum sample, saliva sample, or blood sample.
  • the biological sample is a human biological sample.
  • the spike-in standard is a cellular spike-in standard.
  • the spike-standard comprises 2 or 3 microbes.
  • the microbes may be Treupera radiovictrix, Imtechella halotolerans , and/ or Allobacillus halotolerans .
  • the spike-in standard is further defined as a nucleic acid spike-in standard.
  • the nucleic acid spike-in standard comprises artificial nucleic acids any known organism.
  • 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.
  • 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.
  • 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.
  • the nucleic acid spike-in standard comprises at least 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 2000 bp RNA.
  • the nucleic acid spike-in standard comprises between about 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 2000 bp RNA.
  • the RNA is miRNA.
  • the spike-in standard may comprise a 17, 18, 19, 20, 21, 22, 23, 24 or 25 base pair miRNA.
  • the nucleic acid spike-in standard comprises a double-stranded DNA (dsDNA).
  • dsDNA can comprise at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 or 3000 bp dsDNA.
  • the dsDNA comprises between about 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 2000 bp of dsDNA.
  • the nucleic acid spike-standard comprises log dilutions.
  • 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.
  • 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.
  • the DNA or RNA is epigenetically modified DNA or RNA.
  • 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.
  • the biological sample has undergone storage, transport, purification, library preparation, and/or processing.
  • a container comprising the reagent of the embodiment and aspects described above.
  • the container is further defined as a swab and collection tube, blood vacutainer, fecal scoop tube, or saliva collection device.
  • 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.
  • the reagent is provided in a swab and collection tube, blood vacutainer, fecal scoop tube, or saliva collection device.
  • a kit comprising a sealed tube comprising a stabilization reagent and at least a first spike-in standard.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method further comprises extracting DNA from the biological sample comprising the spike-in control prior to step (b).
  • the method further comprises preparing a library from the extracted DNA.
  • the sequencing is DNA sequencing.
  • 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.
  • the method comprises assessing quality changes in the biological sample at collection, purification, and analysis stages.
  • analysis may comprise PCR and/or sequencing.
  • assessing quality may comprise measuring degradation of the molecule(s) of interest.
  • the molecule(s) of interest are derived from microbes, cells, or any biological material containing DNA or RNA.
  • assessing measurement quality comprises determining a measurement integrity quotient (MIQ) score.
  • the method comprises using a nucleic acid spike-in standard to assess bias in sequencing.
  • the molecule(s) of interest is a virus.
  • the virus may be human immunodeficiency virus (HIV) or herpes simplex virus (HSV).
  • 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.
  • the nucleic acid molecule may be a DNA or a RNA molecule.
  • the nucleic acid molecule comprises a label.
  • 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.
  • the reagent comprises a known concentration of the nucleic acid molecule (e.g the spike-in standard).
  • the reagent inactivates organisms preventing growth (e.g., to allow for safe transport).
  • 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.
  • the reagent can be defined as providing less than 5% degradation of spike-in standard over a period of 1 year.
  • 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.
  • 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.
  • the method allows for purification of nucleic acid with a G/C bias.
  • 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.
  • the method allows for sequencing of quantitation of nucleic acids from microbes without a bias for one microbe other another.
  • the lysis and purification involves the additional of a spike-in standard of a known microbe or a plurality of known microbes.
  • 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.
  • the biological sample is a liquid sample, a tissue sample, or a blood sample.
  • the blood sample is whole blood, plasma, serum, or huffy coat.
  • obtaining the blood sample comprises collecting blood in a tube comprising the lysis reagent.
  • the lysis reagent inactivates one or more microbes and nucleases in the blood sample.
  • the one or more microbes comprise a virus, bacteria, and/or yeast.
  • the virus is influenza, ebola, HIV, influenza or HSV.
  • the bacteria is E. coli, B. subtilis, L. fermentum, E. faecalis, L. monocytogenes, P. aeruginosa, S. enterica, or S. aureus.
  • the yeast is C. neoformans and/or S. cerevisiae.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the lysis agent and the sample are mixed at 1: 1 vokvol ratio.
  • 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.
  • the lysis agent comprises a chaotropic salt.
  • the chaotropic salt is a guanidinium salt.
  • the lysing step further comprises proteinase K digestion.
  • the lysing step further comprises agitation of the sample with one or more bead.
  • the one or more bead is a plurality of beads.
  • the plurality of beads are comprised of beads of different materials, sizes, or different shapes or the combination thereof.
  • 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.
  • 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.
  • the bead is substantially spherical.
  • the bead is composed of a substantially non reactive material.
  • the bead is composed of a ceramic.
  • 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.
  • the purifying step comprises applying the mixture to a silica spin column to bind the RNA to said column.
  • the mixture is diluted in an equal volume of isopropanol prior to applying said sample to the column.
  • 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.
  • analyzing micro RNAs comprises performing microarray analysis, single cell assays, northern blotting, or qRT-PCR.
  • analyzing micro RNAs comprises constructing a library for miRNA sequencing and performing next generation miRNA sequencing on said library.
  • constructing a library comprises ligating adaptors to each end of the micro RNAs.
  • the adaptors comprise barcodes.
  • the method further comprises performing Nanostring nCounter analysis on the sequencing results.
  • the method further comprises performing unbiased miRNA functional enrichment analysis.
  • the analysis comprises using a target prediction program, gene annotation data, and applying statistical analysis.
  • 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.
  • “a” or“an” may mean one or more.
  • the words“a” or“an” when used in conjunction with the word “comprising”, the words“a” or“an” may mean one or more than one.
  • “another” or“a further” may mean at least a second or more.
  • 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).
  • FIG. 2 The microbial composition profiles of a 10 mg fecal sample before and after subtracting the taxa of the spike-in microbes.
  • FIG. 3 Microbial composition profile of a sputum sample with ZymoBIOMICSTM Spike-in Control II.
  • FIG. 4 The standard curve that plots the defined DNA abundance (ng) against the percentage abundance (%) for the three spike-in microbes.
  • FIG. 5 HIV RNA spike-in control with saliva shield mixture.
  • FIG. 6 HSV DNA spike-in tracking stability in DNA/RNA shield with stool at
  • 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.
  • 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.
  • 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).
  • a purification or processing protocol from microorganisms or liquid or solid tissues or cells from essentially any organism.
  • 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.
  • 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.
  • 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.
  • the present methods may be used to track sample quality in library preparation, such as bias during preparation.
  • 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.
  • fidelity to a known biological input sample 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 Measurement Integrity Quotient
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 sample refers to any sample that comprises a organic molecules produced by biological systems.
  • the biological sample comprises DNA and/or RNA.
  • 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.
  • 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.
  • 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.
  • a tissue sample can be removed from a subject by conventional biopsy techniques.
  • 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.
  • 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.
  • assessing the quality of RNA and/or DNA can comprise assessing the level of partial degradation of RNA and/or DNA polymers.
  • the assessing quality comprises assessing partial degradation of RNA and/or DNA from a spike-in standard.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the DNA/RNA shieldTM 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.
  • 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 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the microbes may be Treupera radiovictrix, Imtechella halotolerans, and Allobacillus halotolerans .
  • the microbes may be Imtechella halotolerans and Allobacillus halotolerans, such as in Table 1.
  • the spike-in standard may include may include microbes from the Table 2 below.
  • 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, Act
  • Corynebacterium equi Corynebacterium flavescens, Corynebacterium glutamicum, Corynebacterium haemolyticum, Corynebacterium jeikeiun (corynebacteria of group JK), Corynebacterium minutissimum (Erythrasma) , Corynebacterium parvum (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 cloaca
  • 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.
  • 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., parahaemo
  • 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.
  • 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 m ⁇ of saliva or 200 m ⁇ of blood). For a specific sample type, the defined amount can be customized and optimized.
  • the sample is then mixed, such as by inversion or pipetting.
  • 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.
  • the library is prepared/
  • sequencing platforms e.g., Illumina, Ion Torrent, PacBio and Nanopore
  • 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.
  • the 16S library is then sequenced on Illumina MiSeq using the 600-cycle kit and the shotgun library is then sequenced using Illumina HiSeq.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • next-generation sequencing it can be determined if a specific detection limit is achieved by absolute quantification systems built-in.
  • 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.
  • 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).
  • each of the microbes are present in a known amount in the sealed container.
  • each microbe is present in a different amount (such as each differing from one another 10 fold in a log distribution).
  • 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).
  • a preservation reagent e.g., comprising a chaotropic salt.
  • each of the microbes are present in a known amount in the sealed container.
  • 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).
  • a preservation reagent e.g., comprising a chaotropic salt.
  • each of the microbes are present in a known amount in the sealed container (e.g., 10 3 -10 5 log distribution).
  • 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).
  • each of the microbes are present in a known amount in the sealed container.
  • each microbe is present in a different amount (such as each differing from one another 10 fold in a log distribution).
  • the seal container further comprises a biological sample, optionally wherein the biological sample is comprises on a collection matrix (e.g., a swab).
  • 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.
  • 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.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being preferred.
  • the components of the kit may be provided as dried powder(s).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • this example can further comprise a blood sample and an anti-coagulant.
  • 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).
  • a preservation reagent e.g., comprising a chaotropic salt
  • 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.
  • 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.
  • a preservation reagent e.g., comprising a chaotropic salt
  • 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.
  • ZymoBIOMICSTM Spike-in Standard I High Microbial Load
  • 100 pL of 10% (w/v) stool suspended in DNA/RNA Shield which is equivalent to a 10 mg stool sample.
  • One prep of ZymoBIOMICSTM Spike-in Standard I has 5x10 7 cells for each of the two microbes, Allobacillus halotolerans and Imtechella halotolerans , which are alien to the human microbiome.
  • Table 2 Microbial Composition of ZymoBIOMICSTM Spike-in Control II.
  • Table 3 The abundance table of some selected microbes found in the sputum sample.
  • the following describes a method for the generation of spike-in sequences with minimal collision risk against known, sequenced genomes (see FIG. 1).
  • 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.
  • 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.
  • 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.
  • 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).

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Abstract

L'invention concerne des réactifs permettant le stockage, la lyse ou la purification d'un échantillon biologique comprenant une norme "spike-in". L'invention concerne également des procédés d'utilisation de telles normes pour évaluer et surveiller des échantillons.
PCT/US2019/047474 2018-08-21 2019-08-21 Procédés et compositions pour le suivi de la qualité d'un échantillon Ceased WO2020041449A1 (fr)

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