WO2021055495A1 - Procédés et systèmes d'appauvrissement sélectif d'acide nucléique - Google Patents

Procédés et systèmes d'appauvrissement sélectif d'acide nucléique Download PDF

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WO2021055495A1
WO2021055495A1 PCT/US2020/051097 US2020051097W WO2021055495A1 WO 2021055495 A1 WO2021055495 A1 WO 2021055495A1 US 2020051097 W US2020051097 W US 2020051097W WO 2021055495 A1 WO2021055495 A1 WO 2021055495A1
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cells
population
dna
amount
intercalating agent
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PCT/US2020/051097
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Patrick BROWNE
Leighton TURNER
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Juno Bio Limited
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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • Microbiome studies often focus on samples that are rich in microbial deoxycibonucleic acid (DNA) (e.g., bacterial DNA).
  • DNA microbial deoxycibonucleic acid
  • a large percentage of human microbiome studies may be focused on intestinal microbiome, which may be analyzed through fecal samples, which are rich in microbial DNA.
  • certain biological samples such as vaginal samples, oral samples, and nasal samples, may contain far more human DNA than microbial DNA, thereby necessitating methods of de-hosting in order to selectively deplete human DNA in a biological sample in order to detect low-abundance microbial DNA with high sensitivity.
  • certain biological samples may contain bacterial DNA corresponding to a plurality of different species of bacteria, for which species-specific depletion of bacterial DNA may be performed.
  • the present disclosure provides methods and systems for performing selective deoxyribonucleic acid (DNA) depletion (e.g., via selective cell lysis).
  • such systems and methods may be used to perform analysis of biological samples comprising mixtures of cell populations, such as a first population of cells originating from a subject (e.g., a human subject) and a second population of cells not originating from the subject (e.g., microbial cells).
  • such systems and methods may be used to perform analysis of biological samples comprising mixtures of bacterial populations, such as a first population of cells originating from a particular species of bacteria and a second population of cells not originating from the particular species of bacteria.
  • the present disclosure provides a method for performing selective cell lysis, comprising: (a) obtaining a biological sample comprising a plurality of cells, wherein thethe plurality of cells comprises a first population of cells originating from a subject of a first species and a second population of cells not originating from the subject; (b) isolating the plurality of cells from the biological sample; (c) contacting the plurality of cells with an amount of a permeabilization agent to selectively permeabilize the plurality of cells, wherein the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 90% of the first population of cells to produce a population of permeabilized cells and (ii) permeabilize no more than about 10% of the second population of cells, thereby producing a cell suspension comprising (1) the population of permeabilized cells originating from the subject of the first species and (2) the second population of cells not originating from the subject; (d) contacting the cell suspension with a first amount of a deoxyribonucleic acid
  • the first species is a mammal. In some embodiments, the mammal is a human.
  • the second population of cells comprises a second species. In some embodiments, the second species comprises one or more microbial species.
  • the biological sample is a vaginal sample, a fecal sample, an oral sample, a nasal sample, a throat sample, a respiratory tract sample, a skin sample, an endometrial sample (e.g. aspirate or biopsy), a brain sample, an ear sample, a sperm sample, a derivative thereof, or a combination thereof of the subject. In some embodiments, the biological sample is the vaginal sample or the derivative thereof.
  • the vaginal sample or the derivative thereof is a vaginal swab.
  • (b) comprises performing centrifugation of the biological sample. In some embodiments, the centrifugation is performed at about 10,000 x the gravitational acceleration vector (g).
  • the amount of the permeabilization agent is sufficient to permeabilize at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% of the first population of cells to produce the population of permeabilized cells.
  • the amount of the permeabilization agent is sufficient to permeabilize no more than about 40%, no more than about 35%, no more than about 30%, no more than about 25%, no more than about 20%, no more than about 15%, no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, no more than about 0.5%, or no more than about 0.1% of the second population of cells .
  • the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 95% of the first population of cells to produce the population of permeabilized cells and (ii) permeabilize no more than about 5% of the second population of cells. In some embodiments, the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 99% of the first population of cells to produce the population of permeabilized cells and (ii) permeabilize no more than about 1% of the second population of cells. In some embodiments, the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 99.9% of the first population of cells to produce the population of permeabilized cells and (ii) permeabilize no more than about 0.1% of the second population of cells. In some embodiments, the amount of the permeabilization agent is sufficient to (i) permeabilize substantially all of the first population of cells to produce the population of permeabilized cells and (ii) permeabilize substantially none of the second population of cells.
  • the permeabilization reagent comprises an organic solvent, a surfactant, hypotonic water, a lysin, a cytolysin, or an endolysin.
  • the organic solvent comprises one or more of: methanol, acetone, phenol, benzene, and toluene.
  • the surfactant comprises one or more of: saponin, Triton X-100, Tween-20, 3-([3-cholamidopropyl] dimethylammonio)-l-propanesulfonate hydrate (CHAPS) cell extract buffer, NP-40, sodium dodecyl sulfate (SDS), Span 80, and Digitonin.
  • the surfactant comprises saponin.
  • the amount of the permeabilization agent e.g., saponin is sufficient to produce a weight-to-volume percentage concentration of the permeabilization agent in the cell suspension of about 0.025%.
  • the hypotonic water is ultrapure nuclease-free water.
  • the cytolysin is obtained or derived from a bacterial species.
  • the cytolysin lyses human cells.
  • the cytolysin is Listeriolysin O (e.g., obtained or derived from Listeria bacteria).
  • the amount of the Listeriolysin O is sufficient to produce a concentration of the Listeriolysin O in the cell suspension of about 0.5 mM.
  • the endolysin is obtained or derived from a phage that disrupts bacterial cell membranes. In some embodiments, the endolysin lyses bacterial cells. In some embodiments, the endolysin is specific to a species of bacteria species or a clade of bacteria. [008]
  • (c) further comprises incubating the plurality of cells with the amount of the permeabilization agent for at least about 5 minutes. In some embodiments, (c) further comprises incubating the plurality of cells with the amount of the permeabilization agent for at least about 10 minutes. In some embodiments, (c) further comprising vortexing the cell suspension. In some embodiments, (c) further comprising continually vortexing the cell suspension.
  • the DNA-intercalating agent comprises a photoreactive DNA- intercalating agent.
  • the photoreactive DNA-intercalating agent comprises propidium monoazide (PMA), propidium iodide (PI), ethidium bromide (EtBr), or ethidium monoazide (EMA).
  • the photoreactive DNA-intercalating agent comprises the PMA, and the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 10 micromolar (mM), at least about 20 mM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 120 pM, at least about 140 pM, at least about 160 pM, at least about 180 pM, at least about 200 pM, or more than about 200 pM.
  • mM micromolar
  • the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 10 micromolar (mM), at least about 20 mM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about
  • the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 30 micromolar (pM). In some embodiments, the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 50 micromolar (pM). In some embodiments, the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 100 micromolar (pM).
  • the first amount of the DNA-intercalating agent is sufficient to produce at least about a 3 -fold, at least about a 4-fold, at least about a 5-fold, at least about a 6-fold, at least about a 7-fold, at least about an 8-fold, at least about a 9-fold, at least about a 10-fold, at least about a 12-fold, at least about a 14-fold, at least about a 16-fold, at least about an 18-fold, at least about a 20-fold, or more than about a 20-fold molar excess of the DNA-intercalating agent as compared to the second amount of the DNA-intercalating agent.
  • the first amount of the DNA-intercalating agent is sufficient to produce at least about a 5 -fold molar excess of the DNA-intercalating agent as compared to the second amount of the DNA-intercalating agent. In some embodiments, the first amount of the DNA-intercalating agent is sufficient to produce at least about a 10-fold molar excess of the DNA-intercalating agent as compared to the second amount of the DNA-intercalating agent.
  • (d) further comprises incubating the cell suspension with the first amount of the DNA-intercalating agent for at least about 5 minutes. In some embodiments, (d) further comprises incubating the cell suspension with the first amount of the DNA-intercalating agent for at least about 15 minutes. In some embodiments, (d) further comprises incubating the cell suspension with the first amount of the DNA-intercalating agent for at least about 30 minutes.
  • (d) further comprises incubating the cell suspension with the first amount of the DNA-intercalating agent in an absence of illumination. In some embodiments, (d) further comprises incubating the cell suspension with the first amount of the DNA-intercalating agent in a presence of illumination. In some embodiments, (d) further comprises vortexing the cell suspension. In some embodiments, (d) further comprises continually vortexing the cell suspension.
  • (e) comprises performing centrifugation of the biological sample.
  • the centrifugation is performed at about 10,000 x g.
  • the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40- fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80- fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, or more than about 200-fold more than the second amount of DNA.
  • the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 10-fold more than the second amount of DNA. In some embodiments, the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 10-fold more than the second amount of DNA. In some embodiments, the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 30-fold more than the second amount of DNA. In some embodiments, the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 100-fold more than the second amount of DNA.
  • the method further comprises contacting the cell suspension with DNase to remove DNA molecules of the population of permeabilized cells.
  • the DNase is Turbo DNase.
  • the method further comprises extracting a plurality of DNA molecules from the second population of cells originating from the second species.
  • the method further comprises sequencing the plurality of DNA molecules to produce a plurality of sequence reads corresponding to the second population of cells.
  • the sequencing comprises next-generation sequencing (NGS).
  • the sequencing comprises whole genome sequencing (WGS).
  • the method further comprises amplifying the plurality of DNA molecules.
  • the amplifying comprises polymerase chain reaction (PCR) amplification.
  • the PCR amplification comprises quantitative PCR (qPCR) amplification.
  • the first population of cells comprises a larger number of cells as compared to the second population of cells.
  • the first population of cells comprises at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold, at least about a 6-fold, at least about a 7-fold, at least about an 8-fold, at least about a 9-fold, at least about a 10-fold, at least about a 12-fold, at least about a 14-fold, at least about a 16-fold, at least about an 18-fold, at least about a 20-fold, at least about a 25- fold, at least about a 30-fold, at least about a 40-fold, at least about a 50-fold, at least about a 60-fold, at least about a 70-fold, at least about an 80-fold, at least about a 90-fold, at least about a 100-fold, at least about a 200-fold, or more than about a 200-
  • the first population of cells comprises at least about a 5-fold greater number of cells as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 10-fold greater number of cells as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 30-fold greater number of cells as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 100-fold greater number of cells as compared to the second population of cells.
  • the first population of cells comprises a larger number of DNA molecules as compared to the second population of cells.
  • the first population of cells comprises at least about a 2-fold, at least about a 3-fold, at least about a 4- fold, at least about a 5-fold, at least about a 6-fold, at least about a 7-fold, at least about an 8- fold, at least about a 9-fold, at least about a 10-fold, at least about a 12-fold, at least about a 14-fold, at least about a 16-fold, at least about an 18-fold, at least about a 20-fold, at least about a 25-fold, at least about a 30-fold, at least about a 40-fold, at least about a 50-fold, at least about a 60-fold, at least about a 70-fold, at least about an 80-fold, at least about a 90- fold, at least about a 100-fold, at least about a 200-fold, or more than about a 200
  • the first population of cells comprises at least about a 5-fold greater number of DNA molecules as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 10-fold greater number of DNA molecules as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 30-fold greater number of DNA molecules as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 100-fold greater number of DNA molecules as compared to the second population of cells.
  • the method further comprises depleting from among the plurality of cells at least about 90% of DNA molecules of first population of cells. In some embodiments, the method further comprises depleting from among the plurality of cells at least about 95% of DNA molecules of first population of cells. In some embodiments, the method further comprises depleting from among the plurality of cells at least about 99% of DNA molecules of first population of cells. In some embodiments, the method further comprises depleting from among the plurality of cells at least about 99.9% of DNA molecules of first population of cells.
  • the present disclosure provides a method for performing selective extraction or amplification of DNA (e.g., via selective cell lysis), comprising: (a) obtaining a plurality of cells, wherein the plurality of cells comprises a first population of cells and a second population of cells; (b) contacting the plurality of cells with a permeabilization agent to selectively permeabilize the first population of cells but not the second population of cells, thereby producing a cell suspension comprising permeabilized cells and the second population of cells; (c) contacting the cell suspension with at least about a 10-fold molar excess of a DNA-intercalating agent as compared to a concentration of the DNA-intercalating agent that is sufficient to inhibit extraction or amplification of DNA molecules of the permeabilized cells; and extracting or amplifying DNA molecules of the second population of cells.
  • the present disclosure provides a method for performing selective extraction or amplification of a nucleic acid molecule (e.g., via selective cell lysis), comprising: (a) obtaining a plurality of cells, wherein the plurality of cells comprises a first population of cells and a second population of cells; (b) contacting the plurality of cells with a permeabilization agent to selectively permeabilize the first population of cells and no more than 10% of the second population of cells, thereby producing a cell suspension comprising (i) permeabilized cells derived from the first population of cells and (ii) cells of the second population of cells; (c) contacting the cell suspension or a derivative thereof with a molar excess of a nucleic acid intercalating agent as compared to a concentration of the nucleic acid intercalating agent that is sufficient to inhibit extraction or amplification of nucleic acid molecules of the permeabilized cells; and extracting or amplifying nucleic acid molecules of the cells of the second population of cells.
  • the nucleic acid intercalating agent is a deoxyribonucleic acid intercalating agent.
  • the molar excess is at least a 2 fold molar excess.
  • the present disclosure provides a system comprising one or more computer processors that are individually or collectively programmed to implement a method for performing selective cell lysis, comprising: (a) obtaining a biological sample comprising a plurality of cells, wherein the plurality of cells comprises a first population of cells originating from a subject of a first species and a second population of cells not originating from the subject; (b) isolating the plurality of cells from the biological sample; (c) contacting the plurality of cells with an amount of a permeabilization agent to selectively permeabilize the plurality of cells, wherein the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 90% of the first population of cells to produce a population of permeabilized cells and (ii) permeabilize no more than about 10% of the second population of cells,
  • the present disclosure provides a system comprising one or more computer processors that are individually or collectively programmed to implement a method for performing selective extraction or amplification of a nucleic acid molecule, comprising:
  • the present disclosure provides a method comprising: (a) contacting a mixture comprising a first cell from a subject and a second cell not from the subject with an agent that renders the first cell accessible while the second cell is maintained substantially inaccessible; and (b) contacting the mixture or derivative thereof with an intercalating agent to thereby extract or isolate the second cell from the mixture.
  • the agent comprises an organic solvent, a surfactant, hypotonic water, a lysin, a cytolysin, or an endolysin.
  • the intercalating agent is a nucleic acid intercalating agent.
  • the nucleic acid intercalating agent is a deoxyribonucleic acid intercalating agent.
  • (b) comprises contacting the mixture or derivative thereof with a molar excess of the intercalating agent as compared to a concentration of the intercalating agent that is sufficient to inhibit extraction or amplification of nucleic acid molecules of the first cell. In some embodiments, the molar excess is at least a 2 fold molar excess.
  • Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine-executable code that, upon execution by one or more computer processors, implements any method of the present disclosure (e.g., by directing one or more processing operations).
  • Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto.
  • the computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.
  • FIG. 1 shows a non-limiting example of a workflow of a method for performing selective deoxyribonucleic acid (DNA) depletion (e.g., via selective cell lysis) of a biological sample containing a plurality of cell populations, in accordance with disclosed embodiments.
  • FIG. 2 illustrates a computer system that is programmed or otherwise configured to implement methods provided herein.
  • sample generally refers to a biological sample obtained from or derived from one or more subjects. Biological samples may be processed or fractionated before further analysis.
  • a biological sample may include a fecal sample, a urine sample, a vaginal sample, an oral sample, a nasal sample, a throat sample, a respiratory tract sample, a skin sample, an endometrial sample (e.g.
  • a biological sample may be purified to obtain a cell sample.
  • the sample can be a tissue sample (e.g., aspirate or biopsy).
  • the sample can be a cell-free sample.
  • the term “derived from,” as used herein, generally refers to an origin or source, and may include naturally occurring, recombinant, unpurified, or purified molecules. [037] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.
  • the term “subject” refers to an entity or a medium that has, may have, or is suspected of having testable or detectable genetic information.
  • the subject can be a person ( or an individual).
  • the subject can be a patient.
  • the subject can be a vertebrate, such as, for example, a mammal.
  • Non-limiting examples of mammals include humans, simians, farm animals, sport animals, rodents, and pets.
  • the subject may be displaying a symptom(s) indicative of a health or physiological state or condition of the subject, such as a disease or disorder of the subject.
  • the subject can be asymptomatic with respect to such health or physiological state or condition.
  • Microbiome studies may focus on samples that are rich in microbial deoxyribonucleic acid (DNA). For example, a large percentage of human microbiome studies may be focused on intestinal microbiome, which may be analyzed through fecal samples, which are rich in microbial DNA. However, certain biological samples, such as vaginal samples, oral samples, and nasal samples, may contain far more human DNA than microbial DNA, thereby necessitating methods of de-hosting in order to selectively deplete human DNA in a biological sample.
  • DNA microbial deoxyribonucleic acid
  • Such depletion may result in a decrease of a concentration or relative amount of nucleic acid molecules (e.g., DNA) from one source (e.g., a human) as compared to nucleic acid molecules from another source (e.g., a microbe).
  • nucleic acid molecules e.g., DNA
  • the present disclosure provides methods and systems for performing selective DNA depletion (e.g., via selective cell lysis).
  • such systems and methods may be used to perform analysis of biological samples comprising mixtures of cell populations, such as a first population of cells originating from a subject (e.g., a human subject) and a second population of cells not originating from the subject (e.g., microbial cells).
  • the present disclosure provides a method for performing selective DNA depletion (e.g., via selective cell lysis), comprising: (a) obtaining a biological sample comprising a plurality of cells, wherein the plurality of cells comprises a first population of cells originating from a subject of a first species and a second population of cells not originating from the subject; (b) isolating the plurality of cells from the biological sample; (c) contacting the plurality of cells with an amount of a permeabilization agent to selectively permeabilize the plurality of cells, wherein the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 90% of the first population of cells to produce a population of permeabilized cells and (ii) permeabilize no more than about 10% of the second population of cells, thereby producing a cell suspension comprising (1) the population of permeabilized cells originating from the subject of the first species and (2) the second population of cells not originating from the subject; (d) contacting the cell suspension with a first amount
  • the first species is a mammal. In some embodiments, the mammal is a human.
  • the second population of cells comprises a second species. In some embodiments, the second species comprises one or more microbial species.
  • the biological sample is a vaginal sample, a fecal sample, an oral sample, a nasal sample, a throat sample, a respiratory tract sample, a skin sample, an endometrial sample (e.g. aspirate or biopsy), a brain sample, an ear sample, a sperm sample, a derivative thereof, or a combination thereof of the subject. In some embodiments, the biological sample is the vaginal sample or the derivative thereof.
  • the vaginal sample or the derivative thereof is a vaginal swab.
  • (b) comprises performing centrifugation of the biological sample. In some embodiments, the centrifugation is performed at about 10,000 x the gravitational acceleration vector (g).
  • the amount of the permeabilization agent is sufficient to permeabilize at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% of the first population of cells to produce the population of permeabilized cells.
  • the amount of the permeabilization agent is sufficient to permeabilize no more than about 40%, no more than about 35%, no more than about 30%, no more than about 25%, no more than about 20%, no more than about 15%, no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, no more than about 0.5%, or no more than about 0.1% of the second population of cells .
  • the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 95% of the first population of cells to produce the population of permeabilized cells and (ii) permeabilize no more than about 5% of the second population of cells. In some embodiments, the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 99% of the first population of cells to produce the population of permeabilized cells and (ii) permeabilize no more than about 1% of the second population of cells. In some embodiments, the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 99.9% of the first population of cells to produce the population of permeabilized cells and (ii) permeabilize no more than about 0.1% of the second population of cells. In some embodiments, the amount of the permeabilization agent is sufficient to (i) permeabilize substantially all of the first population of cells to produce the population of permeabilized cells and (ii) permeabilize substantially none of the second population of cells.
  • the permeabilization reagent or permeabilization agent comprises an organic solvent, a surfactant, hypotonic water, a lysin, a cytolysin, or an endolysin.
  • the organic solvent comprises one or more of: methanol, acetone, phenol, benzene, and toluene.
  • the surfactant comprises one or more of: saponin, Triton X-100, Tween-20, 3-([3-cholamidopropyl] dimethylammonio)-l- propanesulfonate hydrate (CHAPS) cell extract buffer, NP-40, sodium dodecyl sulfate (SDS), Span 80, and Digitonin.
  • the surfactant comprises saponin.
  • the amount of the permeabilization agent e.g., saponin
  • the hypotonic water is ultrapure nuclease-free water.
  • the cytolysin is obtained or derived from a bacterial species. In some embodiments, the cytolysin lyses human cells. In some embodiments, the cytolysin is Listeriolysin O (e.g., obtained or derived from Listeria bacteria). In some embodiments, the amount of the Listeriolysin O is sufficient to produce a concentration of the Listeriolysin O in the cell suspension of about 0.5 mM. In some embodiments, the endolysin is obtained or derived from a phage that disrupts bacterial cell membranes. In some embodiments, the endolysin lyses bacterial cells.
  • the endolysin is specific to a species of bacteria species or a clade of bacteria. [045] In some embodiments, (c) further comprises incubating the plurality of cells with the amount of the permeabilization agent for at least about 5 minutes. In some embodiments, (c) further comprises incubating the plurality of cells with the amount of the permeabilization agent for at least about 10 minutes. In some embodiments, (c) further comprising vortexing the cell suspension. In some embodiments, (c) further comprising continually vortexing the cell suspension.
  • the nucleic acid intercalating agent may be an agent or moiety capable of insertion (e.g., non-covalent insertion) between two molecular moieties, such as nucleotide base units of double-stranded nucleic acid.
  • the DNA-intercalating agent may be an agent or moiety capable of insertion (e.g., non-covalent insertion) between stacked base pairs in a nucleic acid double helix.
  • the nucleic acid intercalating agent e.g., DNA-intercalating agent
  • comprises a photoreactive nucleic acid intercalating agent e.g., photoreactive DNA-intercalating agent.
  • the nucleic acid intercalating agent (e.g., photoreactive DNA- intercalating agent) comprises propidium monoazide (PMA), propidium iodide (PI), ethidium bromide (EtBr), or ethidium monoazide (EMA).
  • PMA propidium monoazide
  • PI propidium iodide
  • EtBr ethidium bromide
  • EMA ethidium monoazide
  • the nucleic acid intercalating agent (e.g., photoreactive DNA- intercalating agent) comprises the PMA, and the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 10 micromolar (mM), at least about 20 mM, at least about 30 pM, at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 120 pM, at least about 140 pM, at least about 160 pM, at least about 180 pM, at least about 200 pM, or more than about 200 pM.
  • mM micromolar
  • the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 10 micromolar (mM), at least about 20 mM, at least about 30 pM, at least about 40
  • the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 30 micromolar (pM). In some embodiments, the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 50 micromolar (mM). In some embodiments, the amount of the PMA is sufficient to produce a molar concentration of the PMA in the cell suspension of at least about 100 micromolar (pM).
  • the first amount of the nucleic acid intercalating agent is sufficient to produce at least about a 3-fold, at least about a 4- fold, at least about a 5-fold, at least about a 6-fold, at least about a 7-fold, at least about an 8- fold, at least about a 9-fold, at least about a 10-fold, at least about a 12-fold, at least about a 14-fold, at least about a 16-fold, at least about an 18-fold, at least about a 20-fold, or more than about a 20-fold molar excess of the nucleic acid intercalating agent (e.g., DNA- intercalating agent) as compared to the second amount of the nucleic acid intercalating agent (e.g., DNA-intercalating agent).
  • the nucleic acid intercalating agent e.g., DNA-intercalating agent
  • the first amount of the nucleic acid intercalating agent is sufficient to produce at least about a 5- fold molar excess of the nucleic acid intercalating agent (e.g., DNA-intercalating agent) as compared to the second amount of the nucleic acid intercalating agent (e.g., DNA- intercalating agent).
  • the first amount of the nucleic acid intercalating agent is sufficient to produce at least about a 10-fold molar excess of the nucleic acid intercalating agent (e.g., DNA-intercalating agent) as compared to the second amount of the nucleic acid intercalating agent (e.g., DNA-intercalating agent).
  • (d) further comprises incubating the cell suspension with the first amount of the nucleic acid intercalating agent (DNA-intercalating agent) for at least about 5 minutes.
  • (d) further comprises incubating the cell suspension with the first amount of the nucleic acid intercalating agent (e.g., DNA-intercalating agent) for at least about 15 minutes. In some embodiments, (d) further comprises incubating the cell suspension with the first amount of the nucleic acid intercalating agent (e.g., DNA- intercalating agent) for at least about 30 minutes.
  • the nucleic acid intercalating agent e.g., DNA-intercalating agent
  • (d) further comprises incubating the cell suspension with the first amount of the nucleic acid intercalating agent (e.g., DNA-intercalating agent) in an absence of illumination. In some embodiments, (d) further comprises incubating the cell suspension with the first amount of the nucleic acid intercalating agent (e.g., DNA- intercalating agent) in a presence of illumination. In some embodiments, (d) further comprises vortexing the cell suspension. In some embodiments, (d) further comprises continually vortexing the cell suspension.
  • the nucleic acid intercalating agent e.g., DNA-intercalating agent
  • (e) comprises performing centrifugation of the biological sample.
  • the centrifugation is performed at about 10,000 x g.
  • the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40- fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80- fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, or more than about 200-fold more than the second amount of DNA.
  • the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 10-fold more than the second amount of DNA. In some embodiments, the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 10-fold more than the second amount of DNA. In some embodiments, the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 30-fold more than the second amount of DNA. In some embodiments, the first population of cells comprises a first amount of DNA and the second population of cells comprises a second amount of DNA, wherein the first amount of DNA is at least about 100-fold more than the second amount of DNA.
  • the method further comprises contacting the cell suspension with deoxyribonuclease (DNase) to remove DNA molecules of the population of permeabilized cells.
  • DNase deoxyribonuclease
  • the DNase is Turbo DNase.
  • the method further comprises extracting a plurality of DNA molecules from the second population of cells originating from the second species.
  • the method further comprises sequencing the plurality of DNA molecules to produce a plurality of sequence reads corresponding to the second population of cells.
  • the sequencing comprises next-generation sequencing (NGS).
  • the sequencing comprises whole genome sequencing (WGS).
  • the method further comprises amplifying the plurality of DNA molecules.
  • the amplifying comprises polymerase chain reaction (PCR) amplification.
  • the PCR amplification comprises quantitative PCR (qPCR) amplification.
  • the first population of cells comprises a larger number of cells as compared to the second population of cells.
  • the first population of cells comprises at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold, at least about a 6-fold, at least about a 7-fold, at least about an 8-fold, at least about a 9-fold, at least about a 10-fold, at least about a 12-fold, at least about a 14-fold, at least about a 16-fold, at least about an 18-fold, at least about a 20-fold, at least about a 25- fold, at least about a 30-fold, at least about a 40-fold, at least about a 50-fold, at least about a 60-fold, at least about a 70-fold, at least about an 80-fold, at least about a 90-fold, at least about a 100-fold, at least about a 200-fold, or more than about a 200-
  • the first population of cells comprises at least about a 5-fold greater number of cells as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 10-fold greater number of cells as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 30-fold greater number of cells as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 100-fold greater number of cells as compared to the second population of cells.
  • the first population of cells comprises a larger number of DNA molecules as compared to the second population of cells.
  • the first population of cells comprises at least about a 2-fold, at least about a 3-fold, at least about a 4- fold, at least about a 5-fold, at least about a 6-fold, at least about a 7-fold, at least about an 8- fold, at least about a 9-fold, at least about a 10-fold, at least about a 12-fold, at least about a 14-fold, at least about a 16-fold, at least about an 18-fold, at least about a 20-fold, at least about a 25-fold, at least about a 30-fold, at least about a 40-fold, at least about a 50-fold, at least about a 60-fold, at least about a 70-fold, at least about an 80-fold, at least about a 90- fold, at least about a 100-fold, at least about a 200-fold, or more than about a 200
  • the first population of cells comprises at least about a 5-fold greater number of DNA molecules as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 10-fold greater number of DNA molecules as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 30-fold greater number of DNA molecules as compared to the second population of cells. In some embodiments, the first population of cells comprises at least about a 100-fold greater number of DNA molecules as compared to the second population of cells.
  • the method further comprises depleting from among the plurality of cells at least about 90% of DNA molecules of first population of cells. In some embodiments, the method further comprises depleting from among the plurality of cells at least about 95% of DNA molecules of first population of cells. In some embodiments, the method further comprises depleting from among the plurality of cells at least about 99% of DNA molecules of first population of cells. In some embodiments, the method further comprises depleting from among the plurality of cells at least about 99.9% of DNA molecules of first population of cells.
  • the present disclosure provides a method for performing selective extraction or amplification of a nucleic acid molecule (e.g., via selective cell lysis), comprising: (a) obtaining a plurality of cells, wherein the plurality of cells comprises a first population of cells and a second population of cells; (b) contacting the plurality of cells with a permeabilization agent to selectively permeabilize the first population of cells and no more than 10% of the second population of cells, thereby producing a cell suspension comprising (i) permeabilized cells derived from the first population of cells and (ii) cells of the second population of cells; (c) contacting the cell suspension or a derivative thereof with a molar excess of a nucleic acid intercalating agent as compared to a concentration of the nucleic acid intercalating agent that is sufficient to inhibit extraction or amplification of nucleic acid molecules of the permeabilized cells; and extracting or amplifying nucleic acid molecules of the cells of the second population of cells.
  • the nucleic acid intercalating agent is a deoxyribonucleic acid intercalating agent.
  • the molar excess is at least a 2 fold molar excess. In some embodiments, the molar excess is at least a 5 fold molar excess. In some embodiments, the molar excess is at least a 10 fold molar excess.
  • the present disclosure provides a system comprising one or more computer processors that are individually or collectively programmed to implement a method for performing selective cell lysis, comprising: (a) obtaining a biological sample comprising a plurality of cells, wherein the plurality of cells comprises a first population of cells originating from a subject of a first species and a second population of cells not originating from the subject; (b) isolating the plurality of cells from the biological sample; (c) contacting the plurality of cells with an amount of a permeabilization agent to selectively permeabilize the plurality of cells, wherein the amount of the permeabilization agent is sufficient to (i) permeabilize at least about 90% of the first population of cells to produce a population of permeabilized cells and (ii) permeabilize no more than about 10% of the second population of cells, thereby producing a cell suspension comprising (1) the population of permeabilized cells originating from the subject of the first species and (2) the second population of cells not originating from the subject; (d)
  • the present disclosure provides a system comprising one or more computer processors that are individually or collectively programmed to implement a method for performing selective extraction or amplification of a nucleic acid molecule, comprising:
  • the present disclosure provides a method comprising: (a) contacting a mixture comprising a first cell from a subject and a second cell not from the subject with an agent that renders the first cell accessible while the second cell is maintained substantially inaccessible; and (b) contacting the mixture or derivative thereof with an intercalating agent to thereby extract or isolate the second cell from the mixture.
  • the agent comprises an organic solvent, a surfactant, hypotonic water, a lysin, a cytolysin, or an endolysin.
  • the intercalating agent is a nucleic acid intercalating agent.
  • the nucleic acid intercalating agent is a deoxyribonucleic acid intercalating agent.
  • (b) comprises contacting the mixture or derivative thereof with a molar excess of the intercalating agent as compared to a concentration of the intercalating agent that is sufficient to inhibit extraction or amplification of nucleic acid molecules of the first cell. In some embodiments, the molar excess is at least a 2 fold molar excess.
  • FIG. 1 shows a non-limiting example of a workflow of a method for performing selective DNA depletion (e.g., via selective cell lysis) of a biological sample containing a plurality of cell populations, in accordance with disclosed embodiments.
  • a biological sample comprising a plurality of cells is obtained, wherein the plurality of cells comprises a first population of cells (e.g., originating from a subject of a first species) and a second population of cells (e.g., not originating from the subject).
  • the plurality of cells is isolated from the biological sample (e.g., by centrifugation).
  • the plurality of cells is contacted with an amount of a permeabilization agent to selectively permeabilize the plurality of cells.
  • the amount of the permeabilization agent may be sufficient to (i) permeabilize at least about 90% of the first population of cells to produce a population of permeabilized cells and (ii) permeabilize no more than about 10% of the second population of cells, thereby producing a cell suspension.
  • the cell suspension may comprise
  • the cell suspension is contacted with a first amount of a deoxyribonucleic acid (DNA)-intercalating agent.
  • the first amount of the DNA-intercalating agent may be sufficient to produce at least about a 3 -fold molar excess of the DNA-intercalating agent as compared to a second amount of the DNA-intercalating agent that is sufficient to inhibit extraction or amplification of DNA molecules of the population of permeabilized cells.
  • the second population of cells not originating from the subject of the first species is isolated from the cell suspension. The second population of cells is then ready for further processing or analysis operations, such as DNA extraction, DNA amplification, DNA sequencing to produce sequence reads, analysis of sequence reads, or a combination thereof.
  • a biological sample can be optionally pre treated, preserved (e.g., by cryopreservation such as with glycerol), frozen and/or thawed, or processed prior to use.
  • a biological sample may be analyzed under any of the methods and systems herein within about 4 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hr, 6 hr, 3 hr, 2 hr, or 1 hr from the time the biological sample is obtained, or longer if frozen or preserved.
  • the amount can vary depending upon subject size and the condition being screened. In some embodiments, at least 10 mL, 5 mL, 1 mL, 0.5 mL, 250, 200, 150, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pL of a sample is obtained. In some embodiments, 1-50, 2-40, 3-30, or 4-20 pL of sample is obtained.
  • the biological sample may be suspended in an agent (e.g., buffer solution) for ease of further processing.
  • the sample may be taken before and/or after treatment of a subject with a disease or disorder.
  • Samples may be obtained from a subject during a treatment or a treatment regime. Multiple samples may be obtained from a subject to monitor the effects of the treatment over time.
  • the sample may be taken from a subject having or suspected of having a disease or disorder for which a definitive positive or negative diagnosis is not available via clinical tests.
  • the sample may be taken from a subject suspected of having a disease or disorder.
  • the sample may be taken from a subject experiencing unexplained symptoms, such as fatigue, nausea, weight loss, aches and pains, weakness, or bleeding.
  • the sample may be taken from a subject having explained symptoms.
  • the sample may be taken from a subject at risk of developing a disease or disorder due to factors such as familial history, age, hypertension or pre-hypertension, diabetes or pre-diabetes, overweight or obesity, environmental exposure, lifestyle risk factors (e.g., smoking, alcohol consumption, or drug use), or presence of other risk factors.
  • factors such as familial history, age, hypertension or pre-hypertension, diabetes or pre-diabetes, overweight or obesity, environmental exposure, lifestyle risk factors (e.g., smoking, alcohol consumption, or drug use), or presence of other risk factors.
  • a sample can be taken at a first time point and assayed, and then another sample can be taken at a subsequent time point and assayed.
  • a method as described herein can be used for longitudinal monitoring purposes to track the development or progression of a disease or disorder.
  • the progression of a disease can be tracked before treatment, after treatment, or during the course of treatment, to determine the treatment’s effectiveness.
  • the sample may be processed to isolate a plurality of cells from the biological sample.
  • processing the sample obtained from the subject may comprise subjecting the sample to conditions that are sufficient to isolate, enrich, extract, or amplify a plurality of cells or nucleic acid molecules.
  • processing the sample obtained from the subject may comprise selectively lysing, isolating, enriching, extracting, or amplifying one or more selected populations of cells or nucleic acid molecules but not other non-selected populations of cells or nucleic acid molecules.
  • the cells or nucleic acid molecules may be analyzed using one or more assays.
  • methods of assaying may include any suitable assay, for example, a cytology assay, a microarray assay, a sequencing assay (e.g., DNA sequencing or ribonucleic acid (RNA) sequencing), or a quantitative polymerase chain reaction (qPCR) assay.
  • a plurality of nucleic acid molecules is extracted from the plurality of cells and subjected to sequencing to generate a plurality of sequence reads.
  • the nucleic acid molecules may comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
  • the extraction method may extract all RNA or DNA molecules from a plurality of cells of a sample. Alternatively, the extraction method may selectively extract a portion of RNA or DNA molecules from a plurality of cells of a sample. Extracted RNA molecules from a sample may be converted to complementary DNA (cDNA) molecules by reverse transcription (RT).
  • cDNA complementary DNA
  • FIG. 2 shows a computer system 201 that is programmed or otherwise configured to, for example, direct the obtaining of a biological sample comprising a plurality of cells, direct the isolation of cells from biological samples, direct the contacting of cells with a permabilization agent to selectively permeabilize the cells, direct the contacting of cell suspensions with a DNA-intercalating agent, direct the isolation of cells from cell suspensions, direct the performing of DNA depletion, direct the performing of selective cell lysis, and direct the performing of selective extraction or amplification of DNA.
  • the computer system 201 can regulate various aspects of analysis, calculation, and generation of the present disclosure, such as, for example, directing the obtaining of a biological sample comprising a plurality of cells, directing the isolation of cells from biological samples, directing the contacting of cells with a permabilization agent to selectively permeabilize the cells, directing the contacting of cell suspensions with a DNA- intercalating agent, directing the isolation of cells from cell suspensions, directing the performing of DNA depletion, directing the performing of selective cell lysis, and directing the performing of selective extraction or amplification of DNA.
  • the computer system 201 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the computer system 201 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 205, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 201 also includes memory or memory location 210 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 215 (e.g., hard disk), communication interface 220 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 225, such as cache, other memory, data storage and/or electronic display adapters.
  • the memory 210, storage unit 215, interface 220 and peripheral devices 225 are in communication with the CPU 205 through a communication bus (solid lines), such as a motherboard.
  • the storage unit 215 can be a data storage unit (or data repository) for storing data.
  • the computer system 201 can be operatively coupled to a computer network (“network”) 230 with the aid of the communication interface 220.
  • the network 230 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network 230 in some cases is a telecommunication and/or data network.
  • the network 230 can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • one or more computer servers may enable cloud computing over the network 230 (“the cloud”) to perform various aspects of analysis, calculation, and generation of the present disclosure, such as, for example, directing the isolation of cells from biological samples, directing the contacting of cells with a permabilization agent to selectively permeabilize the cells, directing the contacting of cell suspensions with a DNA-intercalating agent, directing the isolation of cells from cell suspensions, directing the performing of DNA depletion, directing the performing of selective cell lysis, and directing the performing of selective extraction or amplification of DNA.
  • cloud computing may be provided by cloud computing platforms such as, for example, Amazon Web Services (AWS), Microsoft Azure, Google Cloud Platform, and IBM cloud.
  • the network 230 in some cases with the aid of the computer system 201, can implement a peer-to-peer network, which may enable devices coupled to the computer system 201 to behave as a client or a server.
  • the CPU 205 may comprise one or more computer processors and/or one or more graphics processing units (GPUs).
  • the CPU 205 can execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory 210.
  • the instructions can be directed to the CPU 205, which can subsequently program or otherwise configure the CPU 205 to implement methods of the present disclosure. Examples of operations performed by the CPU 205 can include fetch, decode, execute, and writeback.
  • the CPU 205 can be part of a circuit, such as an integrated circuit.
  • One or more other components of the system 201 can be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC).
  • the storage unit 215 can store files, such as drivers, libraries and saved programs.
  • the storage unit 215 can store user data, e.g., user preferences and user programs.
  • the computer system 201 in some cases can include one or more additional data storage units that are external to the computer system 201, such as located on a remote server that is in communication with the computer system 201 through an intranet or the Internet.
  • the computer system 201 can communicate with one or more remote computer systems through the network 230.
  • the computer system 201 can communicate with a remote computer system of a user.
  • remote computer systems include personal computers (PCs) (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants.
  • PCs personal computers
  • slate or tablet PC’s e.g., Apple® iPad, Samsung® Galaxy Tab
  • telephones e.g., Apple® iPhone, Android-enabled device, Blackberry®
  • the user can access the computer system 201 via the network 230.
  • Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 201, such as, for example, on the memory 210 or electronic storage unit 215.
  • the machine executable or machine readable code can be provided in the form of software.
  • the code can be executed by the processor 205.
  • the code can be retrieved from the storage unit 215 and stored on the memory 210 for ready access by the processor 205.
  • the electronic storage unit 215 can be precluded, and machine-executable instructions are stored on memory 210.
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.
  • “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
  • Volatile storage media include dynamic memory, such as main memory of such a computer platform.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system.
  • Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications.
  • Computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a compact disc read-only memory (CD-ROM), digital versatile disc (DVD) or digital versatile disc-read only memory (DVD-ROM), any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a random access memory (RAM), a read-only memory (ROM), a programmable read-only memory (PROM) and erasable programmable read-only memory (EPROM), a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • the computer system 201 can include or be in communication with an electronic display 235 that comprises a user interface (E ⁇ ) 240 for providing, for example, (i) a visual display indicative of a workflow for performing selective DNA depletion, selective cell lysis of a plurality of cells, or selective extraction or amplification of DNA.
  • UIs include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • GUI graphical user interface
  • the algorithm can, for example, direct the obtaining of a biological sample comprising a plurality of cells, direct the isolation of cells from biological samples, direct the contacting of cells with a permabilization agent to selectively permeabilize the cells, direct the contacting of cell suspensions with a DNA-intercalating agent, direct the isolation of cells from cell suspensions, direct the performing of DNA depletion, direct the performing of selective cell lysis, and direct the performing of selective extraction or amplification of DNA.
  • Example 1 Method for De-Hosting a Biological Sample Using Saponin and Propidium Monoazide
  • a large percentage of human microbiome work may be focused on the intestinal microbiome, which is analyzed primarily through fecal samples. Fecal samples tend to be very rich in microbial DNA. For example, when fecal samples are processed by whole genome sequencing (WGS) sequencing, typically 90% or more of the resulting sequencing reads are aligned or mapped to a bacteria genome.
  • WGS whole genome sequencing
  • NGS next-generation sequencing
  • WGS whole genome sequencing
  • De-hosting refers to the process of removing human DNA from a mixed sample in order to improve results (e.g., increase signal-to-noise ratio by increasing the “signal” and/or decreasing the “noise”).
  • de-hosting may be performed on a “live” or viable biological sample (e.g., containing whole cells from two different popultations) or a biological sample containing cells with a membrane potential, which process is generally referred to as differential lysis. Differential lysis of cells may generally encounter two challenges.
  • the differential cell lysis may need to efficiently lyse as high a percentage of human cells as possible, while lysing as low a percentage of microbial cells as possible (ideally, substantially none of the microbial cells, so as not to bias the results).
  • the differential cell lysis may need to effectively deplete (e.g., remove all or substantially all of) the extracellular DNA in the cell suspension resulting from the lysed population of cells (e.g., human cells), so that the DNA of the desired population of cells (e.g., microbial cells) can be isolated, extracted, or amplified for further downstream analyses.
  • a biological sample is de-hosted using saponin and propidium monoazide (PMA) as follows.
  • the method for de-hosting a biological sample may comprise processing a plurality of cells of the biological sample using various surfactants, detergents (e.g., as described by Hasan et al., “Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next- Generation Sequencing,” J. Clin. Microbiol ., 54(4):919-27, Jan. 13, 2016; which is incorporated herein by reference in its entirety), or lysins.
  • detergents e.g., as described by Hasan et al., “Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next- Generation Sequencing,” J. Clin. Microbiol ., 54(4):919-27, Jan. 13, 2016; which is incorporated herein by reference in its entirety
  • the method for de-hosting a biological sample may comprise processing a plurality of cells of the biological sample using a DNA-intercalating agent, such as propidium monoazide (PMA) (e.g., as described by Marotz et al., “Improving saliva shotgun metagenomics by chemical host DNA depletion,” Microbiome , (2016) 6:42; which is incorporated herein by reference in its entirety), propidium iodide (PI), ethidium bromide (EtBr), or ethidium monoazide (EMA).
  • PMA propidium monoazide
  • PI propidium iodide
  • EtBr ethidium bromide
  • EMA ethidium monoazide
  • a biological sample may be de-hosted as follows. First, a sample collection vial containing a biological sample with a plurality of cells is obtained from a subject, where the plurality of cells comprises a first population of cells originating from the subject and a second population of cells not originating from the subject. Next, the sample collection vial is vortexed, and a 250-pL aliquot of the contents therein is transferred to a new tube. Next, the sample aliquot is harvested by centrifugation at 10,000 x g for 8 minutes at room temperature to produce a pellet comprising the plurality of cells. Next, the supernatant is carefully discarded, leaving the cell pellet.
  • the cell pellet is suspended in 250 pL of 0.025% saponin to produce a cell suspension.
  • the suspension is incubated at room temperature for 10 minutes, while vortexing every 2 minutes, thereby permeabilizing the first population of cells while not permeabilizing the second population of cells.
  • a suitable amount of a stock of propidium monoazide (PMA) is added to achieve a concentration of 100 pM.
  • This final concentration of PMA may be at least about 3X, about 4X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 12X, about 14X, about 16X, about 18X, about 20X, or more than about 20X greater than an amount of PMA needed to inhibit DNA extraction or amplification from the permeablized cells.
  • the sample is incubated in dark conditions for 5 minutes.
  • the sample is arranged on ice and exposed to light (e.g., from a flashlight) to cause the photoactivation of the PMA.
  • the sample is incubated on ice for 40 minutes, while vortexing and rearranging every 8 minutes.
  • the sample is harvested at 10,000 x g for 8 minutes at room temperature to produce a cell pellet.
  • the supernatant is carefully discarded.
  • the cell pellet is re-suspended in 35 pL of lysis coc.
  • the suspension is incubated at room temperature for 10 minutes, while vortexing every 2 minutes.
  • the sample is subjected to DNA purification to obtain DNA molecules originating from the second population of cells.
  • the combination of saponin and PMA produces a synergistic effect to yield more efficient selective cell lysis of the mixed sample, while also optimally preserving microbial diversity.
  • a biological sample is de-hosted using lysins as follows.
  • the method for de-hosting a biological sample may comprise processing a plurality of cells of the biological sample using various protein-based disruptors (e.g., lysins).
  • the various protein-based disruptors e.g., lysins
  • lysins used to selectively permeabilize cell membranes are selective for cells of a certain species (e.g., human cell-specific, bacteria species-specific, or bacteria clade-specific).
  • a human cell-specific (host) cell lysis may be performed using a cytolysin (e.g., a lysin obtained or derived from a bacteria that lyses human cells).
  • cytolysins may selectively disrupt human cell membranes.
  • the use of such cytolysins may be performed alternatively to, or in combination with, the use of surfactants and detergents (e.g., saponin, hypotonic water, etc.), as described elsewhere herein.
  • the cytolysin Listeriolysin O e.g., a lysin derived from Listeria bacteria
  • the host-depletion method may comprise adding about 7.5 microliters (pL) of 50 pg/mL Listeriolysin O to produce a final weight-by-volume concentration in the cell suspension of about 1.5 pg/mL (e.g., a molar concentration of about 25 nM). Further, the host-depletion method may comprise adding an amount of dithiothreitol (DTT) sufficient to produce a final molar concentration in the cell suspension of about 0.5 mM.
  • the lysin may be a recombinant lysin (e.g., a heat-stable lysin).
  • a bacteria species-specific or clade-specific cell lysis may be performed using an endolysin (e.g., a lysin obtained or derived from a phage).
  • an endolysin e.g., a lysin obtained or derived from a phage.
  • Such endolysins may selectively disrupt cell membranes of certain bacterial species or clades.
  • any bacterial species or clade may be selectively lysed given a cognate endolysin, thereby enabling the editing of the microbiome of the specific bacterial species or clade.
  • the method for de-hosting a biological sample may comprise processing a plurality of cells of the biological sample using a DNA-intercalating agent, such as propidium monoazide (PMA), propidium iodide (PI), ethidium bromide (EtBr), or ethidium monoazide (EMA).
  • a DNA-intercalating agent such as propidium monoazide (PMA), propidium iodide (PI), ethidium bromide (EtBr), or ethidium monoazide (EMA).
  • a biological sample may be de-hosted as follows. First, a sample collection vial containing a biological sample with a plurality of cells is obtained from a subject, where the plurality of cells comprises a first population of cells originating from the subject and a second population of cells not originating from the subject. Next, the sample collection vial is vortexed, and a 250-pL aliquot of the contents therein is transferred to a new tube. Next, the sample aliquot is harvested by centrifugation at 10,000 x g for 8 minutes at room temperature to produce a pellet comprising the plurality of cells. Next, the supernatant is carefully discarded, leaving the cell pellet.
  • the cell pellet is suspended a lysin (e.g., a cytolysin or an endolysin) to produce a cell suspension.
  • a lysin e.g., a cytolysin or an endolysin
  • the suspension is incubated at room temperature for 10 minutes, while vortexing every 2 minutes, thereby permeabilizing the first population of cells while not permeabilizing the second population of cells.
  • a suitable amount of a stock of propidium monoazide (PMA) is added to achieve a concentration of 100 mM.
  • This final concentration of PMA may be at least about 3X, about 4X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 12X, about 14X, about 16X, about 18X, about 20X, or more than about 20X greater than an amount of PMA needed to inhibit DNA extraction or amplification from the permeablized cells.
  • the sample is incubated in dark conditions for 5 minutes.
  • the sample is arranged on ice and exposed to light (e.g., from a flashlight) to cause the photoactivation of the PMA.
  • the sample is incubated on ice for 40 minutes, while vortexing and rearranging every 8 minutes.
  • the sample is harvested at 10,000 x g for 8 minutes at room temperature to produce a cell pellet.
  • the supernatant is carefully discarded.
  • the cell pellet is re-suspended in 35 pL of lysis coc.
  • the suspension is incubated at room temperature for 10 minutes, while vortexing every 2 minutes.
  • the sample is subjected to DNA purification to obtain DNA molecules originating from the second population of cells.
  • the combination of lysin and PMA produces a synergistic effect to yield more efficient selective cell lysis of the mixed sample, while also optimally preserving microbial diversity.

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Abstract

La présente invention concerne un procédé de réalisation d'une lyse cellulaire sélective, comprenant les étapes suivantes : (a) obtention d'un échantillon biologique comprenant une pluralité de cellules comprenant une première population de cellules provenant d'un sujet d'une première espèce et une seconde population de cellules ne provenant pas du sujet ; (b) isolation des cellules de l'échantillon biologique ; (c) mise en contact des cellules avec un agent de perméabilisation afin de (i) perméabiliser au moins environ 90 % de la première population de cellules pour produire des cellules perméabilisées et (ii) perméabiliser pas plus d'environ 10 % de la seconde population de cellules, ce qui permet de produire une suspension cellulaire ; (d) mise en contact de la suspension cellulaire avec une quantité d'un agent d'intercalation d'acide désoxyribonucléique (ADN) suffisant pour produire au moins environ un excès molaire 3 fois supérieur par rapport à une quantité suffisante pour inhiber l'extraction ou l'amplification d'ADN des cellules perméabilisées ; et (e) isolement de la seconde population de cellules de la suspension cellulaire.
PCT/US2020/051097 2019-09-17 2020-09-16 Procédés et systèmes d'appauvrissement sélectif d'acide nucléique WO2021055495A1 (fr)

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CN113684252A (zh) * 2021-09-03 2021-11-23 中国人民解放军军事科学院军事医学研究院 一种血液样本中病原菌dna的制备方法和基于此方法的临床病原菌感染诊断试剂盒

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US20050064598A1 (en) * 2003-09-19 2005-03-24 Microfluidic Systems, Inc. Microfluidic differential extraction cartridge
US20130171615A1 (en) * 2009-12-08 2013-07-04 Biocartis Sa Selective lysis of cells
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US6221596B1 (en) * 1999-05-17 2001-04-24 Motobit Ltd. System and method for identifying and isolating rare cells from a mixed population of cells
US20050064598A1 (en) * 2003-09-19 2005-03-24 Microfluidic Systems, Inc. Microfluidic differential extraction cartridge
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US20170292146A1 (en) * 2014-09-17 2017-10-12 Helixbind, Inc. Methods and devices for detecting and identifying microorganisms
US20180142231A1 (en) * 2015-04-20 2018-05-24 Qiagen Gmbh Method, Lysis Solution and Kit for Selectively Depleting Animal Nucleic Acids in a Sample

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113684252A (zh) * 2021-09-03 2021-11-23 中国人民解放军军事科学院军事医学研究院 一种血液样本中病原菌dna的制备方法和基于此方法的临床病原菌感染诊断试剂盒

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