Strategies to minimize microbial contamination during the development and deployment of pathogen agnostic targets and metagenomic diagnostic methods

Abstract

The present disclosure provides methods for minimizing microbial contamination during pathogen agnostic targeted and metagenomic diagnostic workflows. The methods include: performing one or more workflow steps on one or more samples in a space with an isolated airflow; adding one or more DNA crosslinking reagents to the one or more samples; obtaining and storing one or more reaction reagents from single use-sized aliquots; generating one or more polymerase chain reaction (PCR) products from the one or more samples by amplifying the one or more samples using one or more capture probes and primers, wherein the capture probes encode a binding region sequence that does not overlap with one or more binding site sequences of the primers; performing a first and a second polymerase chain reaction (PCR) step, wherein the first PCR step is performed using deoxyuridine triphosphate (dUTP) nucleotides and uracil-N-glycosylases / uracil-DNA glycosylases (UNG / UDG), and the second PCR step is performed using dUTP nucleotides; and analyzing the formation of the one or more PCR products using one or more bioinformatics techniques, wherein bioinformatics technique is performed using a stringent threshold level.

Description

STRATEGIES TO MINIMIZE MICROBIAL CONTAMINATION DURING THE DEVELOPMENT AND DEPLOYMENT OF PATHOGEN AGNOSTIC TARGETS AND METAGENOMIC DIAGNOSTIC METHODSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 777,843, filed March 26, 2025, which is incorporated by reference herein in its entirety for any and all purposes.TECHNICAL FIELD

[0002] The present disclosure addresses the problem of false positives in the pathogen detection workflows due to contaminations.BACKGROUND

[0003] Accurate species identification and quantitation are critical for interpretation of universal and pathogen-agnostic pathogen detection workflows. These pathogen-detection workflows could be targeted or metagenomic in design. In targeted workflows, the steps like universal polymerase chain reactions (PCRs) are agnostic to any microbe or microbial DNA present in the sample. This is because these universal PCRs use generic primers that target conserved 16s rDNA, 18s rDNA and 23s rDNA regions in the bacterial and fungal genomic DNA. These generic primers do not differentiate between target versus a contaminant present in the starting sample. Hence, when such amplified DNA is detected, either by sequencing or probe-based hybridization methods, it can result in false positives being reported. Similarly, in metagenomic diagnostic workflows which include whole genome amplification, the random primers can amplify targets and contaminants present in the sample.

[0004] These misdetections can confound researchers during a diagnostic workflow development. The contaminants in samples often compete with the targets during the universal amplification steps. This effect is more pronounced when the targets are present at extremely low levels, such as less than 10 colony-forming units (CFUs) / mL. Such competition for PCR reagents results in contaminant amplification superseding the target amplification. This affects the analytical and clinical sensitivity of the assay.104402.010225

[0005] In addition, in such a clinically deployed diagnostic workflow, these misdetections can prevent accurate interpretation of the diagnostic report with negative implications for clinical therapeutic decisions. False positive results impact patient outcomes. Accordingly, there is a need for improved methods for minimizing microbial contamination during the development of and during the deployment of pathogen agnostic targeted and metagenomic diagnostic methods.SUMMARY

[0006] The present disclosure provides systems and methods for minimizing microbial contamination during the development and deployment of pathogen agnostic targeted and metagenomic diagnostic methods.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In the drawings, which are not necessarily drawn to scale, like numerals can describe similar components in different views. Like numerals having different letter suffixes can represent different instances of similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. In the drawings:

[0008] FIG. 1 depicts a table summarizing the strategies to minimize the contaminants seen in targeted and metagenomic pathogen diagnostic tests.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0009] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.104402.010225The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0011] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0012] As used in the specification and in the claims, the term "comprising" can include the embodiments "consisting of and "consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as "consisting of' and "consisting essentially of the enumerated ingredients / steps, which allows the presence of only the named ingredients / steps, along with any impurities that might result therefrom, and excludes other ingredients / steps.

[0013] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated, some other value approximately, or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0014] Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0015] All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints.

[0016] False positive assay results or misdetections can result by sample contamination, experimental contamination, and computational factors. The methods as contemplated herein provide strategies to minimize sources of false positive results.

[0017] The technical features described herein are a combination of multiple individual practices that are followed during the diagnostic development. Each of the individual practices can be employed independently to decrease the occurrence of false104402.010225positive results due to contamination. However, the combination of these practices gives the best results for controlling contamination in metagenomics workflows.

[0018] The present disclosure provides methods for minimizing microbial contamination of an analytical sample during the development and deployment of pathogenagnostic pathogen diagnostic methods. The pathogen-agnostic diagnostic methods include targeted and metagenomic diagnostic methods. Advantageously, the methods address the problem of false positive detection of pathogens during pathogen detection workflows that are susceptible to contamination.

[0019] Embodiments of the methods include performing one or more workflow steps on one or more samples in a space with an isolated airflow. The design or layout of the space, such as a laboratory space in which the pathogen detection workflow is performed, is important for minimizing contamination. The space includes a space with an isolated airflow that can include an isolated biosafety cabinet, a room with an isolated air handling unit, or a combination of an isolated biosafety cabinet in a room with an isolated air handling unit.

[0020] In some embodiments, the isolated biosafety cabinet includes a high-efficiency particulate air (HEPA)-filtered inflow ventilation. This minimizes the risk of contaminants from the environment (i.e., the air outside the biosafety cabinet) from compromising any procedure or experiment performed within the biosafety cabinet or space. In some embodiments, the space with an isolated airflow (e.g., biosafety cabinet) includes a HEPA-filtered exhaust ventilation. This ensures that air leaving the space is not contaminated with reagents or target DNA or RNA from one experiment so as to minimize the risk of contaminating subsequent experiments. In some embodiments, the one or more workflow steps are performed in a space with an isolated airflow include one or more workflow steps including, for example, performing DNA extraction, sample preparation, amplification reaction, PCR amplicon preparation, sequencing procedures, or one or more combinations thereof. In some embodiments, the one or more PCR amplicons prepared in the space with an isolated airflow for post-PCR clean-up are accessed within the biosafety cabinet, which minimizes cross contamination of target DNA or RNA across experiments. Advantageously, performing the DNA extraction in a space with a separate air handling unit minimizes the DNA-containing aerosols from contaminating subsequent experiments at amplification and sequencing which is performed in different rooms with a different air handling unit.104402.010225

[0021] The sample can include a DNA sample. The sample can include an RNA sample. The sample can include DNA or RNA isolated from one or more biological sample including for example, a blood sample, a tissue sample, a cellular sample, a urine sample, a saliva sample, a mucous sample, a biological fluid sample, a homogenized tissue sample, or any other biological sample suitable for analysis by one or more diagnostic methods.

[0022] Embodiments of the pathogen-agnostic diagnostic methods can include one or more targeted genomics techniques such as targeted next generation sequencing. The one or more pathogen-agnostic diagnostic methods can include one or more metagenomic techniques making use of PCR-based sequencing, shotgun sequencing or a combination thereof. The one or more targeted genomics techniques can include one or more targetenrichment techniques including, for example, hybridization capture, amplicon sequencing, primer-extension, and / or a combination thereof. In some embodiments, the hybridization capture uses one or more capture probes such as biotinylated oligonucleotide probes for capturing a target sequence in a DNA sample. In some embodiments, the amplicon sequencing uses PCR amplification in order to enrich a target region of DNA or RNA before a sequencing step is performed. The PCR amplification uses one or more use as primers or primer pairs in order to target the region of interest. In some embodiments, the primerextension uses a primer pair to capture and release a target region of DNA or RNA. In some embodiments the primer-extension uses a biotinylated capture primer and a release primer in order to capture and release the target region of DNA or RNA. Embodiments of the pathogen-agnostic diagnostic methods include metagenomics analysis.

[0023] Embodiments of the methods include designing the one or more capture probes used in the one or more pathogen-agnostic diagnostic methods such that the binding region of the capture probe is outside of the primer binding sites. In some embodiments, the methods include generating one or more (PCR) products or PCR amplicons from the one or more samples by amplifying the one or more samples using one or more targeted genomics techniques using the capture probes and primers, wherein the capture probes encode a target region or binding region having a sequence that does not overlap with one or more target region sequences or binding region sequences of the primers. Advantageously, this prevents any amplicons from a previous experiment or amplification step from being captured in a subsequent experiments or amplification step.104402.010225

[0024] Embodiments of the methods include removing double-stranded DNA contaminants from the sample by adding one or more DNA crosslinking dyes to the sample. The one or more DNA crosslinking dyes can include, for example, ethidium monoazide (EMA). EMA is a DNA crosslinking dye that binds any double stranded DNA that is present and renders it unamplifiable. EMA can also penetrate compromised cells and bind to them. These cells could be lysed blood cells or lysed microbial cells. When blood sample containing microbial target is treated with EMA, the EMA can bind the contaminating DNA / DNA from compromised cells or any circulating DNA present in the blood sample, and render it unamplifiable. This ensures that, during the universal PCR, only target DNA gets amplified and not contaminating DNA.

[0025] Embodiments of the methods include preparing and storing all reagents in single use-sized aliquots. The one or more reagents in single use aliquots can include any reagents for use in pathogen-agnostic diagnostic methods as understood in the art, including for example, probes, hybridization enzymes, amplification enzymes, nucleotides, master mixes, or a combination thereof. The one or more reagents are prepared into volumes that are adequate for single use to ensure minimal contamination of targets into the reagents which can hinder subsequent experiments with different targets. The methods can include not reusing any detergents in the development of diagnostic workflows or methods as contemplated herein.

[0026] Embodiments of the methods include generating one or more polymerase chain reaction (PCR) products or PCR amplicons from the one or more samples by amplifying the one or more samples using one or more capture probes and primers, wherein the capture probe(s) encode a binding region sequence that does not overlap with one or more binding site sequences of the primer(s). In certain aspects, this prevents any PCR products or PCR amplicons from a previous experiment from being captured in one or more subsequent experiments, which improves the fidelity of the procedure.

[0027] Embodiments of the methods include performing a first and a second polymerase chain reaction (PCR) step, wherein the first PCR step is performed using deoxyuridine triphosphate (dUTP) nucleotides and uracil-N-glycosylases / uracil-DNA glycosylases (UNG / UDG), and the second PCR step is performed using dUTP nucleotides. Because PCR can amplify very small amounts of DNA, preventing amplicon contamination prevents false positive detection of pathogens or targets of interest. Contamination can104402.010225include cross-contamination from other samples, DNA contamination from elsewhere in the laboratory, and carryover contamination from amplification products and primers used in prior PCR experiments. UNG is active on single- and double-stranded dU-containing DNA, but dUTP is not a substrate for UNG. Accordingly, the methods include using dUTP in PCR procedures, so that dU-containing PCR products or PCR amplicons can then be degraded using UNG in the subsequent experiments. However, UNG is not used in the second round of PCR, including for example, library preparation.

[0028] Embodiments of the methods include analyzing the formation of the one or more PCR products using one or more bioinformatics techniques, wherein the bioinformatics technique is performed using a stringent threshold level. As used herein, the threshold level or threshold setting refers to the minimum number of multiple primers to amplify any genomic DNA including microbial DNA before it is identified. The stringent threshold level can include a level configured for allowing a minimum number of PCR product reads for a particular target in order to reduce amplifying any potential contaminant primer pairs or closely related contaminating primer pairs. The one or more bioinformatics techniques can include a fluorescence-based technique and wherein the stringent threshold level comprises a fluorescence threshold value. The stringent threshold level can include a level allowing a minimum number of primer pairs to amplify. The stringent threshold level can include a threshold level that is set above a level for one or more known contaminants for a particular experiment. The known contaminants can include one or more DNA extraction inhibitors, one or more microbial DNA, one or more fungal DNA, or a combination thereof.

[0029] The methods as presented herein are a combination of good laboratory practices (GLP) in addition to other multiple practices as outlined herein. A summary of the strategies for minimizing contaminated described herein are shown in FIG. 1. Each of the individual practices can be employed independently to decrease the occurrence of false positive results due to contamination. However, the combination of these technical practices used in the development or deployment of targeted or metagenomic pathogen detection workflows gives the best results for controlling contamination in such metagenomics workflows. The biggest advantage from this approach is that both cellular and DNA contaminants that can interfere with the target pathogen detection in the conventional pathogen detection tests can be minimized.

Claims

104402.010225What is claimed:

1. A method for minimizing microbial contamination during a pathogen agnostic targeted or metagenomic detection workflow, the method comprising:i. performing one or more workflow steps on one or more samples in a space with an isolated airflow;ii. adding one or more DNA crosslinking reagents to the one or more samples;iii. obtaining and storing one or more reaction reagents from single usesized aliquots;iv. generating one or more polymerase chain reaction (PCR) products from the one or more samples by amplifying the one or more samples using one or more capture probes and primers, wherein the capture probes encode a binding region sequence that does not overlap with one or more binding site sequences of the primers;v. performing a first and a second polymerase chain reaction (PCR) step, wherein the first PCR step is performed using deoxyuridine triphosphate (dUTP) nucleotides and uracil-N-glycosylases / uracil- DNA glycosylases (UNG / UDG), and the second PCR step is performed using dUTP nucleotides; andvi. analyzing the formation of the one or more PCR products using one or more bioinformatics techniques, wherein bioinformatics technique is performed using a stringent threshold level.

2. The method of claim 1, wherein the one or more samples comprises one or more DNA samples.

3. The method of claim 1, wherein the one or more sample comprises one or more RNA samples.104402.0102254. The method of claim 1, wherein the space with isolated airflow comprises an isolated biosafety cabinet, a room with an isolated air handing unit, or both in an isolated biosafety cabinet and in a room with an isolated air handling unit5. The method of claim 1, wherein the one or more workflow steps performed in a space with an isolated airflow comprises one or more of: sample preparation, amplification reaction, PCR amplicon preparation, sequencing run, or a combination thereof.

6. The method of claim 1, wherein the isolated biosafety cabinet comprises a high- efficiency particulate air (HEPA)-filtered inflow ventilation.

7. The method of claim 1, wherein the space with an isolated airflow comprises a HEPA-filtered exhaust ventilation.

8. The method of claim 1, wherein one or more PCR amplicons prepared in the space with an isolated airflow for post-PCR clean-up are accessed within the biosafety cabinet.

9. The method of claim 1, wherein the one or more DNA crosslinking reagents comprises a DNA crosslinking dye.

10. The method of claim 9, wherein the DNA crosslinking dye comprises ethidium monoazide bromide (EMA).

11. The method of claim 1, wherein the one or more reaction reagents comprise one or more of: probes, hybridization enzymes, amplification enzymes, nucleotides, or a combination thereof.

12. The method of claim 1, wherein the one or more bioinformatics techniques comprises a fluorescence-based technique and wherein the stringent threshold level comprises a fluorescence threshold value.104402.01022513. The method of claim 1, wherein the stringent threshold level comprises a level allowing a minimum number of primer pairs to amplify.

14. The method of claim 1, wherein the stringent threshold level comprises a level configured for allowing a minimum number of PCR product reads.

15. The method of claim 1, wherein the stringent threshold level comprises a level above a level of known contaminants for a particular experiment.

16. The method of claim 14, wherein the known contaminants comprise one or more DNA extraction inhibitors, one or more microbial DNA, one or more fungal DNA, or a combination thereof.

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