WO2024092158A1 - Stabilization of nucleic acids in body fluids - Google Patents

Abstract

Methods of preparing stabilized cell-free nucleic acid control material in a body fluid are provided. Compositions including stabilized cell-free nucleic acid control materials and methods of their use are also provided. Kits including the compositions are also provided.

Description

STABILIZATION OF NUCLEIC ACIDS IN BODY FLUIDS

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 63/420,056, filed October 27, 2022, which is incorporated herein by reference in its entirety.

FIELD

This disclosure relates to methods to stabilize nucleic acid compositions in body fluids, particularly for use as a quality control material in assays for detection of biomarkers or to mimic infectious agents in a sample.

BACKGROUND

Human body fluids contain valuable biomarkers or infectious agents that can be used for disease diagnosis. Liquid biopsies including whole blood, plasma, serum, cerebrospinal fluid, saliva, urine, and so on, are useful for detection of important biomarkers for diagnostic, prognostic, and/or monitoring purposes. Cell-free total nucleic acids (cfTNA), which includes both cell-free DNA (cfDNA) and cell-free RNA (cfRNA), for example, from liquid biopsy, carry important genetic information from normal or diseased cells, including cancerous cells. However, due to the short half-life of naked fragmented nucleic acids, especially RNA, in human plasma or serum, it is very challenging to improve the stability of the cfTNA and ensure the sensitivity for any specific targets. cfDNA contains deoxyribose and may be protected by histones in plasma, rendering it more stable than RNA, which contains an active 2'-hydroxyl group, resulting in increased susceptibility to hydrolysis. Currently, although there are a few cfDNA control products commercially available either in storage buffer, synthetic plasma matrix, or real human plasma, there are very limited options for cfRNA controls, which are available only in preservative buffer format.

SUMMARY

The present disclosure provides methods to stabilize both cfDNA and cfRNA components in body fluids to create quality control materials applicable to both oncology and infectious disease controls. The disclosed methods can be applied to creation of any synthetic spike-in quality control materials in body fluids that mimic patient samples. Compositions including stabilized cell-free nucleic acids are also provided. In some examples, the disclosed methods of increasing stability of cell-free nucleic acid control material in a body fluid include adding one or more stabilizing agents and cell-free nucleic acid control material to a body fluid sample, In other examples, the disclosed methods of increasing stability of cell-free nucleic acid control material in a body fluid include adding a preparation of encapsulated cell-free nucleic acid control material to a body fluid sample. In further examples, the disclosed methods of increasing stability of cell-free nucleic acid control material in a body fluid include adding one or more stabilizing agents and a preparation of encapsulated cell-free nucleic acid control material to a body fluid sample. In particular examples, the body fluid is blood, plasma, synthetic plasma, serum, cerebrospinal fluid, saliva, or urine. In some examples, the body fluid is a human body fluid.

In some examples, the stabilizing agent includes ammonium sulfate, EDTA, or both. In additional examples, the stabilizing agent further includes bovine serum albumin, trehalose, or both.

In some examples, the encapsulated cell-free nucleic acid control material is encapsulated in oil droplets, liposomes, or lipid nanoparticles. In some examples, the oil droplets are monodisperse droplets (for example, prepared by microfluidics-based partitioning). In other examples, the oil droplets are particle-templated emulsification droplets, for example, including monodisperse polyacrylamide. In some examples, the particle-templated emulsification droplets are produced by microfluidics-free methods, such as vortexing the cell- free nucleic acids with an oil and monodisperse polyacrylamide beads at about 500 rpm to about 4000 rpm for about 15 seconds to about 15 minutes.

The cell-free nucleic acid control material may include cell-free DNA control material, cell- free RNA control material, or both. In some examples, the cell-free DNA control material comprises genomic DNA, DNA molecules including one or more variants, or both. The DNA molecules including one or more variants may include variants selected from the group consisting of one or more single nucleotide polymorphisms (SNP), single nucleotide variants, substitution of one or more nucleic acids (e.g., point mutations), insertion (INS), deletion (DEL), premature stop codons, trinucleotide repeats, translocations, inversions, somatic rearrangements, allelomorphs, splice variants, regulatory variants, gene fusions, copy number variation (CNV), and any combination of two or more thereof. In some examples, the cell-free RNA control material includes genomic RNA, one or more RNA molecules including one or more variants selected from fusions, isoforms, splice variants, and any combination thereof.

Also provided are cell-free nucleic acid control compositions. In one example, the cell-free nucleic acid control composition includes a body fluid, one or more stabilizing agents, and a cell- free nucleic acid control material. In another example, the cell-free nucleic acid control composition includes a body fluid and an encapsulated cell-free nucleic acid control material. In a further example, the cell-free nucleic acid control composition includes a body fluid, one or more stabilizing agents, and an encapsulated cell-free nucleic acid control material. In particular examples, the body fluid is blood, plasma, synthetic plasma, serum, cerebrospinal fluid, saliva, or urine. In some examples, the body fluid is a human body fluid.

In some examples, the stabilizing agent includes ammonium sulfate, EDTA, or both. In additional examples, the stabilizing agent further includes bovine serum albumin, trehalose, or both.

Also provided are kits including a cell-free nucleic acid composition described herein. In some examples, the kit further includes one or more enzymes, buffers, oligonucleotide primers, oligonucleotide adaptors, oligonucleotide probes, or any combination of two or more thereof.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an exemplary workflow for creating cfTNA in human plasma matrix. DNA (e.g., synthetic or DNA from a cell line) is mixed with RNA (e.g., IVT or cRNA from a cell line) at a 10: 1 ratio in a nucleic acid stabilization buffer (nucleic acid dilution solution, NADS) and encapsulated in oil emulsion or lipid nanoparticle. The copy number of DNA and RNA are determined and quantified before spiking into human plasma matrix.

FIGS. 2A and 2B show fragmentation results on the TapeStation for DNA (FIG. 2A) and RNA (FIG. 2B). A2 is DNA ladder. B2 and C2 are replicate samples from the same DNA fragmentation condition. Al is RNA ladder. Bl, Cl, and DI are the fragmentation condition used to fragment the total RNA.

FIG. 3 is a graph showing DNA recovery for different formulations after accelerated stability test. DNA was extracted from cfTNA controls after incubation at 4°C for 51 hours (equivalent to 2 years stored at -80°C) or at 37°C for 7 days (equivalent to 1 year stored at -20°C). The concentration was determined as set forth in Example 3 and the recovery rate was calculated by comparing to the total input. N=2, two replicate extraction for each sample.

FIG. 4 is a graph showing DNA mutation detection with NGS test for cfTNA extracted from Formulations C, E, and F after 51 hours at 4°C or 7 days at 37°C.

FIG. 5 is a graph showing RT-qPCR results for RNA using the indicated conditions. FIG. 6 is a graph showing RNA yield from Formulations C, E, and F after accelerated stability test. RT-qPCR of the extracted samples and RNA standards were conducted using ABI 7500 Fast Real Time PCR system and ACTB ml assay. RNA recovery yield was calculated by comparing the sample Ct to the Standard RNA calibration curve and its formula. Note: 0 h samples were left at room temperature for -2 hours before storing at -80°C.

FIG. 7 is a graph showing IVT RNA recovery yield based on ddPCR data with IVT RNA- specific assays from Formulations C, E, and F after accelerated stability test. The extracted cfTNA from Formulations C, E, and F and a control sample was tested with QX200™ AutoDG™ Droplet Digital™ PCR System (Bio-Rad Laboratories). Briefly, extracted cfTNA was diluted 10 times and then 3 pL of the diluted cfTNA was used together with one-Step RT-ddPCR Advanced Kit for the reaction. QuantaSoft (version of 1.7.4.0917) was used for the data analysis to quantify the copy number of IVT RNA.

FIG. 8 is a graph showing IVT RNA recovery yield based on NGS data from Formulations C, E, and F after accelerated stability test. The extracted cfTNA from Formulations C, E, and F and a control sample was tested with Next Generation Sequencing platform using Genexus™ Integrated Sequencer (Thermo Fisher Scientific). Preparation of the NGS libraries and analysis of data was as set forth in Example 6. Molecular counts are sequencing reads per one million total reads.

FIGS. 9A and 9B show testing of Formulation F with different conditions for preparing the encapsulated nucleic acids. The cfTNA extracted from Formulation F was tested either by using RT-qPCR with ABI 7500 Fast Real Time PCR system and ACTB ml assay (FIG. 9A), or by using Bio-Rad QX200 AutoDG Droplet Digital PCR System and IVT KIF5B(24)-RET(8) assay (FIG. 9B). Condition 1: Vortexing at -3000 rpm (speed level 10) for 1 minute; Condition 2: Vortexing at -2500 rpm (speed level 8) for 3 minutes; Condition 3: Vortexing at -2500 rpm (speed level 8) for 6 minutes. N=2, two replicate extraction for each sample.

DETAILED DESCRIPTION

I. Terms

Section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc., discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. It is noted that, as used in this specification, singular forms “a,” “an,” and “the,” and any singular use of a word, include plural referents unless expressly and unequivocally limited to one referent. Also, the use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” are not intended to be limiting. It is to be understood that both the general description is exemplary and explanatory only and not restrictive of the invention.

Unless otherwise defined, scientific and technical terms used in connection with the invention described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization used herein are those well-known and commonly used in the art. The practice of the present subject matter may employ, unless otherwise indicated, techniques and descriptions used in organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art. Such techniques include, but are not limited to, preparation of synthetic polynucleotides, polymerization techniques, chemical and physical analysis of polymer particles, preparation of nucleic acid libraries, nucleic acid sequencing and analysis, and the like. Specific illustrations of suitable techniques can be used by reference to the examples provided herein. Other equivalent procedures can also be used. Such techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Hermanson, Bioconjugate Techniques, Second Edition (Academic Press, 2008); Merkus, Particle Size Measurements (Springer, 2009); Rubinstein and Colby, Polymer Physics (Oxford University Press, 2003); and the like. As utilized in accordance with teachings provided herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

As used herein, the term “nucleic acid” refers to natural nucleic acids, artificial nucleic acids, analogs thereof, or combinations thereof, including polynucleotides and oligonucleotides. As used herein, the terms “polynucleotide” and “oligonucleotide” are used interchangeably and mean single-stranded and double-stranded polymers of nucleotides including, but not limited to, 2’- deoxyribonucleotides (nucleic acid) and ribonucleotides (RNA) linked by intemucleotide phosphodiester bond linkages, e.g. 3’-5’ and 2’-5’, inverted linkages, e.g. 3’ -3’ and 5’-5’ , branched structures, or analog nucleic acids. Polynucleotides have associated counter ions, such as H⁺, NH₄ ⁺, trialkylammonium, Mg²⁺, Na⁺, and the like. An oligonucleotide can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. Oligonucleotides can be comprised of nucleobase and sugar analogs. Polynucleotides typically range in size from a few monomeric units, e.g. 5-40, when they are more commonly frequently referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units, when they are more commonly referred to in the art as polynucleotides; for purposes of this disclosure, however, both oligonucleotides and polynucleotides may be of any suitable length. Unless denoted otherwise, whenever a oligonucleotide sequence is represented, it will be understood that the nucleotides are in 5’ to 3’ order from left to right and that “A” denotes deoxy adenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, “T” denotes thymidine, and “U’ denotes deoxyuridine. As discussed herein and known in the art, oligonucleotides and polynucleotides are said to have “5’ ends” and “3’ ends” because mononucleotides are typically reacted to form oligonucleotides via attachment of the 5’ phosphate or equivalent group of one nucleotide to the 3’ hydroxyl or equivalent group of its neighboring nucleotide, optionally via a phosphodiester or other suitable linkage.

As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification. This process for amplifying the polynucleotide of interest consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired polynucleotide of interest, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded polynucleotide of interest. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the polynucleotide of interest molecule. Following annealing, the primers are extended with a polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (e.g., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired polynucleotide of interest. The length of the amplified segment of the desired polynucleotide of interest (amplicon) is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of repeating the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the polynucleotide of interest become the predominant nucleic acid sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.” As defined herein, target nucleic acid molecules within a sample including a plurality of target nucleic acid molecules are amplified via PCR. In a modification to the method discussed above, the target nucleic acid molecules can be PCR amplified using a plurality of different primer pairs, in some cases, one or more primer pairs per target nucleic acid molecule of interest, thereby forming a multiplex PCR reaction. Using multiplex PCR, it is possible to simultaneously amplify multiple nucleic acid molecules of interest from a sample to form amplified target sequences. It is also possible to detect the amplified target sequences by several different methodologies (e.g., quantitation with a bioanalyzer or qPCR, hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified target sequence). Any oligonucleotide sequence can be amplified with the appropriate set of primers, thereby allowing for the amplification of target nucleic acid molecules from genomic DNA, cDNA, formalin-fixed paraffin-embedded DNA, fine-needle biopsies and various other sources. In particular, the amplified target sequences created by a multiplex PCR process are themselves efficient substrates for subsequent PCR amplification or various downstream assays or manipulations.

II. Methods of Stabilizing Nucleic Acids in Body Fluids

Provided herein are methods of stabilizing nucleic acids (such as cell-free nucleic acids) in a body fluid (such as a human body fluid matrix). It is particularly challenging to stabilize cell-free nucleic acids (e.g., nucleic acids that are not in a cellular microenvironment) due to RNA active 2'- hydroxyl group, which increases the susceptibility to hydrolysis in an aqueous environment, as well as active enzymatic degradation for both RNA and DNA in body fluid matrix. One exemplary method of stabilizing cell-free nucleic acids (such as a cell-free nucleic acid control material) is illustrated in FIG. 1. The illustrated method is only one example of the methods provided herein, and should not be considered to be limiting.

In some examples, stabilizing cell-free nucleic acids in a body fluid relates to decreasing or inhibiting degradation of nucleic acids in a body fluid (e.g. , a test sample) compared to a control. In other examples, stabilizing cell-free nucleic acids in a body fluid includes increasing or maintaining (for example, not substantially or significantly increasing or decreasing) DNA and/or RNA recovery from nucleic acids in a body fluid compared to a control. Exemplary methods for determining DNA or RNA recovery are described in the Examples below. One of ordinary skill in the art can identify other suitable methods of determining DNA or RNA recovery. In some examples, the control is a mixture of the body fluid and nucleic acids, where the body fluid does not include a stabilizing agent and/or the nucleic acids are not encapsulated. In other examples, the control is a mixture of body fluid and nucleic acids with the same composition (e.g., including the same stabilizing agent(s) and/or including encapsulated nucleic acids), but prepared under different conditions or treated with different conditions after preparation. The methods described herein may stabilize nucleic acids in a body fluid when assessed by one method, but may not stabilize nucleic acids in a body fluid when assessed by a different method.

In some examples, the methods provided herein include adding (or contacting) one or more stabilizing agents and a cell-free nucleic acid control material to a body fluid sample. In some examples, the one or more stabilizing agents is added to the body fluid sample prior to adding the cell-free nucleic acid control material. In other examples, the cell-free nucleic acid control material is added to the body fluid sample prior to adding the one or more stabilizing agents. In further examples, the one or more stabilizing agents and the cell-free control material are added to the body fluid sample simultaneously or substantially simultaneously.

In other examples, the methods provided herein include adding (or contacting) a preparation of encapsulated cell-free nucleic acid control material to a body fluid sample.

In further examples, the provided methods include adding (or contacting) one or more stabilizing agents and a preparation of encapsulated cell-free nucleic acid control material to a body fluid sample. In some examples, the one or more stabilizing agents is added to the body fluid sample prior to adding the preparation of encapsulated cell-free nucleic acid control material. In other examples, the preparation of encapsulated cell-free nucleic acid control material is added to the body fluid sample prior to adding the one or more stabilizing agents. In further examples, the one or more stabilizing agents and the preparation of encapsulated cell-free nucleic acid control material are added to the body fluid sample simultaneously or substantially simultaneously.

In some examples, the body fluid is plasma, such as human plasma. In some examples, the plasma is DNA-depleted plasma. In other examples, the body fluid is a synthetic plasma or a plasma-based diluent. Exemplary commercially available synthetic plasma include artificial plasma fluid (Biochemazone, Leduc, Canada) and commercially available plasma-based diluents include Basematrix diluent, Seracon diluent, and Matribase diluent (all from Seracare, Milford, MA). In other examples, the body fluid is blood, serum, cerebrospinal fluid, saliva, or urine. In some examples, the body fluid is a human body fluid.

In some examples, the methods include adding one or more nucleic acid stabilizing agents to a body fluid sample. In some examples, the one or more stabilizing agents include ammonium sulfate ((NH4)2SO4), ethylenediaminetetraacetic acid (EDTA), or both. In some examples, the one or more stabilizing agents include about 3 mM to 200 mM (NI-UESC (such as about 3-5 mM, about 5-10 mM, about 10-20 mM, about 20-50 mM, about 50-100 mM, about 100-150 mM, or about 150-200 mM), about 10 mM to 100 mM (such as about 10-15 mM, about 15-20 mM, about 20-30 mM, about 30-50 mM, about 50-75 mM, or about 75-100 mM) EDTA, or both. In specific examples, the one or more stabilizing agents include about 10 mM (NH^SCh, about 10 mM EDTA, or about 10 mM (NFLrhSC and about 10 mM EDTA.

In other examples, the one or more stabilizing agents include (NH4)2SO4 and EDTA and further include bovine serum albumin (BSA), trehalose, or both. In some examples, the one or more stabilizing agents include about 2-10% (such as about 2-4%, about 3-5%, about 5-8%, about 6-9%, or about 7-10%) BSA or about 20 mM to 200 mM (such as about 20-30 mM, about 30-40 mM, about 40-50 mM, about 50-60 mM, about 60-80 mM, about 80-100 mM, about 100-150 mM, or about 150-200 mM) trehalose. In one specific example, the one or more stabilizing agents include about 10 mM (NID SC , about 10 mM EDTA, and about 5% BSA. In another specific example, the one or more stabilizing agents include about 10 mM (NFU SC , about 10 mM EDTA, and about 50 mM trehalose.

In another example, the one or more stabilizing agents includes tetradecyltrimethylammonium bromide (TTAB). In one example, the stabilizing agent includes about 4% TTAB.

In some examples, the one or more stabilizing agents may be a commercially available RNA stabilizing solution, such as RNALater® stabilization solution (Thermo Fisher Scientific, Waltham, MA), RNAProtect® reagent (Qiagen, Germantown, MD), NucleoProtect RNA (Takara Bio, Kusatsu, Shiga, Japan), or DNA/RNA Shield reagent (Zymo Research Corporation, Irvine, CA).

In some examples, the one or more stabilizing agents are included in a buffer solution, for example prior to adding to the body fluid. Exemplary buffers include phosphate buffered saline (PBS) and sodium citrate.

In some examples, the methods include adding a preparation of encapsulated nucleic acids (such as DNA, RNA, or both) to the body fluid. Without being bound by theory, it is believed that encapsulation prevents contact between the nucleic acid and human plasma matrix components or aqueous environment which may lead to rapid degradation of DNA/RNA.

In some examples, the preparation of encapsulated cell-free nucleic acid control material is encapsulated in an oil droplet, a liposome, or a lipid nanoparticle.

In some examples, the cell-free nucleic acid control material is encapsulated in an oil droplet or a lipid-oil mixture. In some examples, the encapsulated cell-free nucleic acid control material is in an emulsion, such as a monodisperse emulsion. In some examples, the cell-free nucleic acid control material is encapsulated in a composition including an oil. In some examples, the oil includes a silicone oil. Exemplary oils are described in U.S. Pat. No. 9,427,737 and Wu et al., Advanced Functional Materials 28: 1803559, 2018. In one specific example, the oil is Droplet Generation Oil for Probes (Bio-Rad Laboratories, Hercules, CA). One of ordinary skill in the art can select appropriate oils for use in the methods described herein.

In some examples, the encapsulated cell-free nucleic acid control material is prepared by a microfluidics-based method, such as using Bio-Rad Digital Droplet technology. In other examples, the encapsulated cell-free nucleic acid control material is prepared by agitating (for example, vortexing) the nucleic acid with oil. For example, the encapsulated cell-free nucleic acid control material may be prepared by agitating the nucleic acid with oil for about 15 seconds to about 15 minutes (such as about 15 seconds to about 1 minute, about 30 seconds to about 90 seconds, about 1 minute to about 3 minutes, about 2 minutes to about 5 minutes, about 3 minutes to about 6 minutes, about 4 minutes to about 7 minutes, about 5 minutes to about 8 minutes, about 6 minutes to about 9 minutes, about 7 minutes to about 10 minutes, about 8 minutes to about 11 minutes, about 9 minutes to about 12 minutes, about 10 minutes to about 13 minutes, about 11 minutes to about 14 minutes, or about 12 minutes to about 15 minutes). In some examples, the nucleic acid is agitated with the oil for about 15 seconds, about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, or about 15 minutes. The agitating is carried out at a speed of about 500 rpm to about 4000 rpm (such as about 500 rpm to about 1500 rpm, about 1000 rpm to about 2500 rpm, about 2000 rpm to about 3500 rpm, about 3000 rpm to about 4500 rpm, or about 4000 rpm to about 5000 rpm). In some examples, the nucleic acid is agitated with the oil at about 500 rpm, about 1000 rpm, about 1500 rpm, about 2000 rpm, about 2500 rpm, about 3000 rpm, about 3500 rpm, about 4000 rpm, about 4500 rpm, or about 5000 rpm. In particular examples, the cell-free nucleic acid control material is agitated with the oil for about 30 seconds, about 1 minute, or about 2 minutes at about 2300 rpm, about 1 minute at about 3000 rpm, about 3 minutes at about 2500 rpm, about 6 minutes at about 2500 rpm, or about 10 minutes at about 3000 rpm.

In other examples, the encapsulated cell-free nucleic acid control material is encapsulated in particle-templated emulsification (PTE) droplets. Exemplary methods for preparing PTE droplets are described in WO 2019/139650. In some examples, the encapsulated cell-free nucleic acid control material is prepared microfluidics -free methods, such as by agitating (for example, vortexing) the nucleic acid with oil and a population of monodisperse template particles. In some examples, the monodisperse template particles is a hydrogel, for example, collagen, hyaluronan, chitosan, fibrin, gelatin, alginate, agarose, chondroitin sulfate, polyacrylamide, polyethylene glycol, polyvinyl alcohol, polyacrylamide/poly(acrylic acid), hydroxyethyl methacrylate, poly-N-isopropyl acrylamide, polyanhydrides, polypropylene fumarate, or a combination of two or more thereof. In one example, the monodisperse template particle includes monodisperse polyacrylamide.

In some examples, the encapsulated cell-free nucleic acid control material may be prepared by a microfluidics-free methods, such as agitating the nucleic acid with oil (for example, Droplet Generation Oil for Probes (Bio-Rad Laboratories, Hercules, CA)) and monodisperse template particles for about 15 seconds to about 15 minutes (such as about 15 seconds to about 1 minute, about 30 seconds to about 90 seconds, about 1 minute to about 3 minutes, about 2 minutes to about 5 minutes, about 3 minutes to about 6 minutes, about 4 minutes to about 7 minutes, about 5 minutes to about 8 minutes, about 6 minutes to about 9 minutes, about 7 minutes to about 10 minutes, about 8 minutes to about 11 minutes, about 9 minutes to about 12 minutes, about 10 minutes to about 13 minutes, about 11 minutes to about 14 minutes or about 12 minutes to about 15 minutes). In some examples, the nucleic acid is agitated with the oil and monodisperse template particles for about 15 seconds, about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, or about 15 minutes. The agitating is carried out at a speed of about 500 rpm to about 4000 rpm (such as about 500 rpm to about 1500 rpm, about 1000 rpm to about 2500 rpm, about 2000 rpm to about 3500 rpm, about 3000 rpm to about 4500 rpm, or about 4000 rpm to about 5000 rpm). In some examples, the nucleic acid is agitated with the oil at about 500 rpm, about 1000 rpm, about 1500 rpm, about 2000 rpm, about 2500 rpm, about 3000 rpm, about 3500 rpm, about 4000 rpm, about 4500 rpm, or about 5000 rpm. In particular examples, the cell-free nucleic acid control material is agitated with the oil and monodisperse template particles for about 30 seconds to 2 minutes at 2300 rpm, about 1 minute at 3000 rpm, about 3 minutes at 2500 rpm, about 6 minutes at 2500 rpm, or about 10 minutes at 3000 rpm.

In some examples, at least about 2000 droplets (such as at least about 2000, at least about 4000, about least about 6000, at least about 8000, at least about 10,000, at least about 12,000, at least about 14,000, at least about 16,000, at least about 18,000, at least about 20,000, or more droplets) per 40 pL sample including the nucleic acid and monodisperse template particle.

Alternatively, lipid nanoparticles can also be used for encapsulation of the cell-free nucleic acid control material. In some examples, the nucleic acids are encapsulated in one or more cationic lipids. Exemplary cationic lipids include dioleoyl-trimethylammoniumpropane (DOTAP), polyethyleneimine (PEI), dimethyldioctadecylammonium bromide (DDA), l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), 2,3-dioleyloxy-N-[2-sperminecarboxamido)ethyl]-N,N- dimethyl-l-propanaminium trifluoracetate (DOSPA), and ethylphosphaidylcholine (ePC). In some examples, the one or more cationic lipids are mixed with one or more neutral or zwitterionic lipids, for example, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoylphosphocholine (DOPC), or monoolein (MO). In other examples, the nanoparticle includes cholesterol or polylysine. In further examples, the nucleic acids may be encapsulated in a lipidoid, for example an ionizable lipidoid (such as C 12-200).

The cell-free nucleic acid control material includes cell-free DNA, cell-free RNA, or both. The cell-free nucleic acid control material includes nucleic acids that are not present in a cell or cellular microenvironment. In some examples, the cell-free nucleic acid control material includes isolated or purified DNA, RNA, or both. In some examples, the cell-free RNA includes in vitro transcribed (IVT) RNA. In some examples, the cell-free nucleic acid control material is a non- naturally occurring mixture of cell-free DNAs, cell-free RNAs, or both. In some examples, the cell-free nucleic acid control material includes one or more target nucleic acids (such as one or more nucleic acids including any nucleic acid sequence suspected or expected to be present in a sample) corresponding to target nucleic acids for which the material is serving as a control. In particular examples, cell-free nucleic acid control material is disclosed in U.S. Provisional Patent Application No. 63/402,027, filed August 29, 2022 and International Patent Application No. PCT/US2023/072998, filed August 28, 2023.

In some examples, the cell-free nucleic acid control material includes one or more DNA variant nucleic acids (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more DNA variants). In other examples, the cell-free nucleic acid control material includes 20 or more, 100 or more, 200 or more, or even 500 or more DNA variants. In some examples, the cell- free nucleic acid control material includes one or more DNAs, including one or more plasmids, artificial chromosomes, and/or double-stranded DNA fragments (such as gBlocks) including one or more DNA variants. The variants may be variants from a single gene or more than one gene (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 500, or more genes).

The DNA variants may be selected from one or more of a single nucleotide polymorphism (SNP), single nucleotide variants, substitution of one or more nucleic acids (e.g., point mutations), insertion (INS), deletion (DEL), premature stop codons, trinucleotide repeats, translocations, inversions, somatic rearrangements, allelomorphs, splice variants, regulatory variants, gene fusions, copy number variation (CNV), and any combination thereof. In some examples, the DNA variants may be selected from one or more of a single nucleotide polymorphism (SNP), insertion (INS), deletion (DEL), inversions, duplication, substitution, copy number variation (CNV), translocation, gene rearrangement, alternative splicing, gene fusion, and any combination of two or more thereof. In some examples, the cell-free nucleic acid control material includes at least one SNP, at least one insertion, at least one deletion, at least one splice variant, and at least one copy number variant fragment.

In some examples, the DNA molecules including one or more variants are included in the form of plasmid DNA or artificial chromosome DNA (such as human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), Pl-derived artificial chromosome (PAC)), cosmid, or fosmid DNA including the DNA variant(s)). In some examples, the DNA including the variant(s) (such as the plasmid or artificial chromosome) is fragmented, purified, and included in the cell-free nucleic acid control material. In some examples, the fragmented DNA is of a selected length or range of lengths. Thus, in some examples, the composition includes a collection of DNA fragments (such as plasmid or artificial chromosome DNA fragments) of a set range of length, such as about 100 bp to about 500 bp (for example, about 100 bp to about 200 bp, about 150 bp to about 250 bp, about 200 bp to about 300 bp, about 250 bp to about 350 bp, about 300 bp to about 400 bp, about 350 bp to about 450 bp, or about 400 to 500 bp). In one specific example, the collection of DNA fragments ranges from about 100 bp to about 500 bp, having a peak at about 240 bp. In other examples, the DNA variant fragments are from gBlock DNA fragments including one or more DNA variants. In some examples, the gBlock DNA fragments are about 100 bp to about 500 bp, for example, about 100 bp to about 200 bp, about 150 bp to about 250 bp, about 200 bp to about 300 bp, about 250 bp to about 350 bp, about 300 bp to about 400 bp, about 350 bp to about 450 bp, or about 400 to 500 bp, for example, about 200 bp.

In some examples, the cell-free nucleic acid control material includes one or more (such as I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) RNA molecules, such as RNA variant nucleic acids. In other examples, the cell-free nucleic acid control material includes 20 or more, 100 or more, 200 or more, or even 500 or more RNA variants. The RNA variants may be selected from RNA fusions, isoforms (e.g., splice variants), and any combination thereof. In some examples, the cell-free nucleic acid control material includes at least one RNA fusion and at least one isoform. In some examples, the one or more RNA variants are generated in vitro, for example by in vitro transcription from a DNA, such as plasmid DNA.

In other examples, the cell-free nucleic acid control material includes a combination of fragmented DNA and RNA variants in a background of genomic DNA (such as genomic DNA extracted from human cells). DNA variants (or mutations) include but are not limited to one or more of single nucleotide polymorphism (SNP), single nucleotide variants, substitution of one or more nucleic acids (e.g., point mutations), insertion (INS), deletion (DEL), premature stop codons, trinucleotide repeats, translocations, inversions, somatic rearrangements, allelomorphs, splice variants, regulatory variants, gene fusions, and copy number variation (CNV), for example, across several different cancer hotspot genes, while RNA variants (or mutations) include one or more of RNA fusions and isoforms (such as alternative splice forms).

In some examples, the cell-free nucleic acid control material includes one or more DNA variants and/or one or more RNA variants with sequences including mutations or variants associated with cancer or other diseases or disorders (including genetic disorders). In some examples, the cell-free nucleic acid control material includes one or more DNA variant and/or one or more RNA variant having mutations or variants associated with one or more solid tumor cancers selected from the group consisting of head and neck cancers (e.g., HNSCC, nasopharyngeal, salivary gland), brain cancer (e.g., glioblastoma, glioma, gliosarcoma, glioblastoma multiforme, neuroblastoma), breast cancer (e.g., TNBC, trastuzumab resistant HER2+ breast cancer, ER+/HER- breast cancer), gynecological (e.g., uterine, ovarian cancer, cervical cancer, endometrial cancer, fallopian cancer), colorectal cancer, gallbladder cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, bladder cancer, prostate cancer, testicular cancer, urothelial cancer, liver cancer (e.g., hepatocellular carcinoma, HCC), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), kidney (renal cell) cancer, pancreatic cancer (e.g., adenocarcinoma, ductal), thyroid cancer, bile duct cancer, pituitary tumor, Wilms’ tumor, Kaposi sarcoma, hairy cell carcinoma, osteosarcoma, thymus cancer, skin cancer, melanoma, heart cancer, oral and larynx cancer, neuroblastoma, mesothelioma, and other solid tumors (e.g., thymic, bone, soft tissue, oral SCC, myelofibrosis, synovial sarcoma). In other examples, the cell-free nucleic acid control material includes one or more DNA variant and/or one or more RNA variant having mutations or variants associated with one or more blood/hematologic cancers selected from the group consisting of multiple myeloma, diffuse large B cell lymphoma (DLBCL), lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, follicular lymphoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and myelodysplastic syndrome. In some examples, the cell-free nucleic acid control material includes one or more DNA variants and one or more RNA variants including sequences having mutations or variants in targets selected from those listed in Table 1.

Table 1. Exemplary targets by variant class

In some examples, the cell-free nucleic acid control material includes one or more DNA fragments including variants (such as one or more SNPs, insertions, and/or deletions) in one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, or all) of EGFR, KRAS, KEAP1, BRAE, KIT, STK11, RET, ERBB2, and MET. In other examples, the cell-free nucleic acid control material includes one or more DNA fragments including CNVs in ERBB2, MET, or both. In additional examples, the cell- free nucleic acid control material includes one or more RNAs including a fusion selected from one or more (such as 1, 2, 3, 4, 5, or all) of EML4-ALK, TPM3-NTRK1, NACC2-NTRK2, ETV6- NTRK3, CCDC6-RET, and SLC34A2-ROS1. In another example, the cell-free nucleic acid control material includes an RNA including an alternative splice form of MET.

In other examples, the cell-free nucleic acid control material includes one or more nucleic acids (such as DNA or RNA) from an infectious disease agent. In some examples, the nucleic acid is a viral nucleic acid, a bacterial nucleic acid, or a fungal nucleic acid. In some examples, the cell- free nucleic acid control material includes one or more of a SARS-CoV-2 nucleic acid, an influenza virus nucleic acid, a Hepatitis B virus nucleic acid, a Hepatitis C virus nucleic acid, a Human Immunodeficiency virus nucleic acid, a Human Papilloma virus nucleic acid, an Epstein-Barr virus nucleic acid, a Neisseria gonorrhoeae bacteria nucleic acid, a Chlamydia bacteria nucleic acid, a Staphylococcus aureus bacteria nucleic acid, a V ancomycin-resistant Enterococci bacteria nucleic acid, a Candida albicans fungus nucleic acid, or an Aspergillus fungus nucleic acid.

III. Compositions

Also provided are compositions that included stabilized cell-free nucleic acid control material. In some examples, the composition includes a body fluid, one or more stabilizing agents, and a cell-free nucleic acid control material. In other examples, the composition includes a body fluid and an encapsulated cell-free nucleic acid control material. In further examples, the composition includes a body fluid, one or more stabilizing agents, and an encapsulated cell-free nucleic acid control material.

In some examples, the body fluid is plasma, such as human plasma. In some examples, the plasma is DNA-depleted plasma. In other examples, the body fluid is a synthetic plasma or a plasma-based diluent. Exemplary commercially available synthetic plasma include artificial plasma fluid (Biochemazone, Leduc, Canada) and commercially available plasma-based diluents include Basematrix diluent, Seracon diluent, and Matribase diluent (all from Seracare, Milford, MA). In other examples, the body fluid is blood, serum, cerebrospinal fluid, saliva, or urine. In some examples, the body fluid is a human body fluid.

In some examples, the methods include adding one or more nucleic acid stabilizing agents to a body fluid sample. In some examples, the one or more stabilizing agents include ammonium sulfate ((NH4)2SO4), ethylenediaminetetraacetic acid (EDTA), or both. In some examples, the one or more stabilizing agents include about 3 mM to 200 mM (NHihSC (such as about 3-5 mM, about 5-10 mM, about 10-20 mM, about 20-50 mM, about 50-100 mM, about 100-150 mM, or about 150-200 mM), about 10 mM to 100 mM (such as about 10-15 mM, about 15-20 mM, about 20-30 mM, about 30-50 mM, about 50-75 mM, or about 75-100 mM) EDTA, or both. In specific examples, the one or more stabilizing agents include about 10 mM (NH4)2SO4, about 10 mM EDTA, or about 10 mM (NH4)2SO4 and about 10 mM EDTA.

In other examples, the one or more stabilizing agents include (NH4)2SO4 and EDTA and further include bovine serum albumin (BSA), trehalose, or both. In some examples, the one or more stabilizing agents include about 2-10% (such as about 2-4%, about 3-5%, about 5-8%, about 6-9%, or about 7-10%) BSA or about 20 mM to 200 mM (such as about 20-30 mM, about 30-40 mM, about 40-50 mM, about 50-60 mM, about 60-80 mM, about 80-100 mM, about 100-150 mM, or about 150-200 mM) trehalose. In one specific example, the one or more stabilizing agents include about 10 mM (NFU SCU, about 10 mM EDTA, and about 5% BSA. In another specific example, the one or more stabilizing agents include about 10 mM (NFU SC , about 10 mM EDTA, and about 50 mM trehalose.

In another example, the one or more stabilizing agents includes tetradecyltrimethylammonium bromide (TTAB). In one example, the stabilizing agent includes about 4% TTAB.

In some examples, the one or more stabilizing agents may be a commercially available RNA stabilizing solution, such as RNALater® stabilization solution (Thermo Fisher Scientific, Waltham, MA), RNAProtect® reagent (Qiagen, Germantown, MD), NucleoProtect RNA (Takara Bio, Kusatsu, Shiga, Japan), or DNA/RNA Shield reagent (Zymo Research Corporation, Irvine, CA).

In some examples, the composition includes one or more additional agents, such as a buffer. Exemplary buffers include phosphate buffered saline (PBS) and sodium citrate.

In some examples, the encapsulated cell-free nucleic acid control material is encapsulated in an oil droplet, a liposome, or a lipid nanoparticle. In some examples, the cell-free nucleic acid control material is encapsulated in a composition including an oil. In some examples, the oil includes a silicone oil. Exemplary oils are described in U.S. Pat. No. 9,427,737 and Wu et al., Advanced Functional Materials 28:1803559, 2018. In one specific example, the oil is Droplet Generation Oil for Probes (Bio-Rad Laboratories, Hercules, CA). One of ordinary skill in the art can select appropriate oils for use in the methods described herein.

In other examples, the composition includes cell-free nucleic acid control material encapsulated in a lipid nanoparticle. In some examples, the nucleic acids are encapsulated in one or more cationic lipids. Exemplary cationic lipids include dioleoyl-trimethylammoniumpropane (DOTAP), polyethyleneimine (PEI), dimethyldioctadecylammonium bromide (DDA), 1,2-di-O- octadecenyl-3 -trimethylammonium propane (DOTMA), 2,3-dioleyloxy-N-[2- sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoracetate (DOSPA), and ethylphosphaidylcholine (ePC). In some examples, the one or more cationic lipids are mixed with one or more neutral or zwitterionic lipids, for example, l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), dioleoylphosphocholine (DOPC), or monoolein (MO). In other examples, the nanoparticle includes cholesterol or poly lysine. In further examples, the nucleic acids may be encapsulated in a lipidoid, for example an ionizable lipidoid (such as C12-200). The cell-free nucleic acid control material includes cell-free DNA, cell-free RNA, or both. The cell-free nucleic acid control material includes nucleic acids that are not present in a cell or cellular microenvironment. In some examples, the cell-free nucleic acid control material includes isolated or purified DNA, RNA, or both. In some examples, the cell-free RNA includes in vitro transcribed (IVT) RNA. In some examples, the cell-free nucleic acid control material is a non- naturally occurring mixture of cell-free DNAs, cell-free RNAs, or both. In some examples, the cell-free nucleic acid control material includes one or more target nucleic acids (such as one or more nucleic acids including any nucleic acid sequence suspected or expected to be present in a sample) corresponding to target nucleic acids for which the material is serving as a control. In particular examples, cell-free nucleic acid control material is disclosed in U.S. Provisional Patent Application No. 63/402,027, filed August 29, 2022 and International Patent Application No. PCT/US2023/072998, filed August 28, 2023.

In some examples, the cell-free nucleic acid control material includes one or more DNA variant nucleic acids (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more DNA variants). In other examples, the cell-free nucleic acid control material includes 20 or more, 100 or more, 200 or more, or even 500 or more DNA variants. In some examples, the cell- free nucleic acid control material includes one or more DNAs, including one or more plasmids, artificial chromosomes, and/or double-stranded DNA fragments (such as gBlocks) including one or more DNA variants. The variants may be variants from a single gene or more than one gene (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 500, or more genes).

The DNA variants may be selected from one or more of single nucleotide polymorphism (SNP), single nucleotide variants, substitution of one or more nucleic acids (e.g., point mutations), insertion (INS), deletion (DEL), premature stop codons, trinucleotide repeats, translocations, inversions, somatic rearrangements, allelomorphs, splice variants, regulatory variants, gene fusions, copy number variation (CNV), and any combination thereof. In some examples, the DNA variants may be selected from one or more of single nucleotide polymorphism (SNP), insertion (INS), deletion (DEL), inversions, duplication, substitution, copy number variation (CNV), translocation, gene rearrangement, alternative splicing, gene fusion, and any combination of two or more thereof. In some examples, the cell-free nucleic acid control material includes at least one SNP, at least one insertion, at least one deletion, at least one splice variant, and at least one copy number variant fragment.

In some examples, the DNA molecules including one or more variants are included in the form of plasmid DNA or artificial chromosome DNA (such as human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), Pl-derived artificial chromosome (PAC)), cosmid, or fosmid DNA including the DNA variant(s)). In some examples, the DNA including the variant(s) (such as the plasmid or artificial chromosome) is fragmented, purified, and included in the cell-free nucleic acid control material. In some examples, the fragmented DNA is of a selected length or range of lengths. Thus, in some examples, the composition includes a collection of DNA fragments (such as plasmid or artificial chromosome DNA fragments) of a set range of length, such as about 100 bp to about 500 bp, for example, about 100 bp to about 200 bp, about 150 bp to about 250 bp, about 200 bp to about 300 bp, about 250 bp to about 350 bp, about 300 bp to about 400 bp, about 350 bp to about 450 bp, or about 400 to 500 bp. In one specific example, the collection of DNA fragments ranges from about 100 bp to about 500 bp, having a peak at about 240 bp. In other examples, the DNA variant fragments are from gBlock DNA fragments including one or more DNA variants. In some examples, the gBlock DNA fragments are about 100 bp to about 500 bp, for example, about 100 bp to about 200 bp, about 150 bp to about 250 bp, about 200 bp to about 300 bp, about 250 bp to about 350 bp, about 300 bp to about 400 bp, about 350 bp to about 450 bp, or about 400 to 500 bp, for example, about 200 bp.

In some examples, the cell-free nucleic acid control material includes one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) RNA molecules, such as RNA variant nucleic acids. In other examples, the cell-free nucleic acid control material includes 20 or more, 100 or more, 200 or more, or even 500 or more RNA variants. The RNA variants may be selected from RNA fusions, isoforms (e.g., splice variants), and any combination thereof. In some examples, the cell-free nucleic acid control material includes at least one RNA fusion and at least one isoform. In some examples, the one or more RNA variants are generated in vitro, for example by in vitro transcription from a DNA, such as plasmid DNA.

In other examples, the cell-free nucleic acid control material includes a combination of fragmented DNA and RNA variants in a background of genomic DNA (such as genomic DNA extracted from human cells). DNA variants (or mutations) include but are not limited to one or more of single nucleotide polymorphism (SNP), single nucleotide variants, substitution of one or more nucleic acids (e.g., point mutations), insertion (INS), deletion (DEL), premature stop codons, trinucleotide repeats, translocations, inversions, somatic rearrangements, allelomorphs, splice variants, regulatory variants, gene fusions, and copy number variation (CNV), for example, across several different cancer hotspot genes, while RNA variants (or mutations) include one or more of RNA fusions and isoforms (such as alternative splice forms). IV. Kits

Also provided are kits including one or more of the compositions disclosed herein. The kit may also include a container (e.g., a vial, test tube, flask, bottle, syringe, or other packaging system) into which the one or more of the compositions may be placed/contained. Where more than one component is included in the kit, it will generally include at least one second, third, or other additional container into which the additional components can be separately placed. Various combinations of components may also be packaged in a single container. The kits may also include reagent containers in close confinement for commercial sale. The kit may include the composition in a liquid solution (such as an aqueous solution).

In some examples, the kit is for use in methods of detecting one or more biomarkers, such as one or more biomarkers relating to a disease or disorder (e.g., cancer, genetic disorders, or infectious disease) using next-generation sequencing or other methods (such as digital PCR or qPCR). In one example, the kit is for use in methods of detecting one or more biomarkers associated with cancer (including one or more SNPs, mutations, CNVs, and fusions). In some examples, the kit includes a composition that includes nucleic acids including one or more (or each) of the biomarkers detected in the method.

The kit may also include instructions for employing the kit components as well as the use of any other reagent not included in the kit. Instructions may include variations that may optionally be implemented. The instructions may be provided as a separate part of the kit (e.g. , a paper or plastic insert or attachment) or as an internet-based application. Kits may further include one or more of a polymerase, one or more oligonucleotide primers, one or more buffers, other reaction mixtures, or any combination thereof. Other variations and arrangements for the kits of this disclosure are contemplated as would be understood by those of ordinary skill in the art.

V. Methods of Use

This disclosure also provides methods for confirming the validity of an assay (such as a next-generation sequencing assay) by including a composition disclosed herein in a sample. In some examples, the composition includes a known number of representative sequences and/or variants thereof and detection of all of the representative sequences and/or variants in the mixture indicates the sequencing reaction was accurate.

The disclosed compositions can be used during NGS method development, method validation, operator training and routine QC monitoring. During assay development, the NGS assay may be developed using the composition to align the VAF of specific variants. For validation purposes, the composition can be used to test the assay accuracy, precision, repeatability and reproducibility. The composition can also be used for new operator training, lab proficiency testing, and routine testing to monitor any systematic errors.

EXAMPLES

The following examples are provided to illustrate features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

Example 1. Stabilization

Stabilization reagents were added to human plasma and thoroughly mixed before spiking in the DNA/RNA mixture (60 ng DNA and 6 ng RNA per 1 mL plasma). Several different formulations were tested.

Formulation A: A solution comprising 25% Invitrogen™ RNAlater™ stabilization solution (Thermo Fisher Scientific) and 75% human plasma was made. After thoroughly mixing, DNA/RNA mixtures were spiked into this solution, referred to as Formulation A. Invitrogen™ RNAlater™ stabilization solution comprises ammonium sulfate and EDTA in a sodium citrate buffer at pH 5.2.

Formulation B: Human plasma was pre-mixed with stabilization reagents to have the final concentration of 10 mM (NFL SCL + 10 mM EDTA + 5% BSA in RNase-free phosphate buffered saline at pH 7.4. Then DNA/RNA mixtures were spiked into this solution and referred to as Formulation B.

Formulation C: Human plasma was pre-mixed with stabilization reagents to have the final concentration of 10 mM (NFL SCL + 10 mM EDTA + 50 mM trehalose in RNase-free phosphate buffered saline at pH 7.4. Then DNA/RNA mixtures were spiked into this solution and referred to as Formulation C.

Example 2. Encapsulation

Encapsulated DNA/RNA mixture was added to human plasma (60 ng DNA and 6 ng RNA per 1 mL plasma). Several encapsulation methods were tested:

Formulation D: Invitrogen™ Lipofectamine MessengerMAX™ mRNA transfection reagent (Thermo Fisher Scientific) was diluted using IX PBS and mixed with DNA/RNA mixtures at 25 pg/mL concentration. The reaction was incubated for 10 mins at 22°C before spiking into human plasma.

Formulation E: The Bio-Rad™ QX200™ droplet generator (Bio-Rad Laboratories, catalog #1864002) was used to encapsulate cfTNA mixture. In an exemplary method, 20 pL DNA/RNA mixture was loaded into the sample cartridge, and 70 pL of the droplet generation oil for probes was used to generate an emulsion. The emulsion was then spiked into human plasma.

Formulation F: Another method for encapsulation is microfluidics-free Particle-Templated Emulsification (PTE). Aqueous particles containing nucleic acids emulsify with oil particles under vortexing to form droplets of similar size. As a general description of one example, particles of Bio-Gel™ P-60 gel (Bio-Rad Laboratories) were mixed with DNA/RNA mixture to a final concentration of approximately 250 mM ammonium sulfate and then incubated at room temperature for 5 min under gentle agitation before adding 60 pL of droplet generation oil for probes per 40 pL sample. Then the aqueous solution and oil mixture was mixed using a vortex mixer at high speed (e.g. 2500-3000 rpm) for approximately 1-8 minutes. The collected emulsion was then spiked into the plasma.

Example 3. Extraction and Nucleic Acid Quantification

The nucleic acids from each of Formulations A through F were extracted using Applied Biosystems™ MagMAX™ Cell-Free Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific), and 2 pL of the extracted materials were used to quantify the DNA concentration using Invitrogen™ Qubit™ dsDNA HS assay kit (Thermo Fisher Scientific) on Qubit 3.0 fluorometer (Thermo Fisher Scientific). To determine the RNA concentration, reverse transcription of the extracted samples was conducted using Applied Biosystems™ TaqPath™ 1-Step RT-qPCR Master Mix, CG (Thermo Fisher Scientific) and ACTB ml assay (assay ID: PN Hs99999903_ml). The RNA calibration curve was generated using Applied Biosystems™ total RNA control (Thermo Fisher Scientific). The RNA control was serially diluted from 50 ng/pL down to 0.0005 ng/pL, and 5 pL of the diluted RNA controls (final concentrations at 5.0, 0.5, 0.05, 0.005, 0.0005 ng/pL) combined with extracted RNA was used for reverse transcription and real time PCR reaction on Applied Biosystems™ 7500 Fast Real Time PCR system using the following program: 1 cycle of 2 mins at 25°C, 30 mins at 50°C, 2 mins at 95°C, followed by 40 cycles of 15 secs at 95°C, 1 min at 60°C and then hold at 10°C.

Example 4. Stability Studies

The preliminary shelf-life of the product was determined by accelerated stability studies. In brief, samples were incubated at 4°C for 51 hours (equivalent to 2 years stored at -80°C), or 37°C for 7 days (equivalent to 1 year stored at -20°C) before extraction and quantification. The DNA and RNA yield from the elevated stressed samples were compared to the yield of samples that were not temperature-stressed. Example 5. Nucleic Acid Quantification with ddPCR

The cfTNA extracted from each of Formulations C, E, and F was tested with QX200™ AutoDG™ Droplet Digital™ PCR System (Bio-Rad Laboratories). For in vitro transcribed (IVT) RNA ddPCR method, extracted cfTNA was diluted 10 times and then 3 p L of the diluted cfTNA was used together with One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad Laboratories) for the reaction. QuantaSoft software (version of 1.7.4.0917; Bio-Rad Laboratories) was used for the data analysis to quantify the copy number of IVT RNA.

Example 6 . Nucleic Acid Quantification with NGS

The cfTNA extracted from each of Formulations C, E, and F was also tested on Next Generation Sequencing (NGS) platform using Ion Torrent™ Genexus™ Integrated Sequencer (Thermo Fisher Scientific). Up to 10 ng total purified cfTNA was used to generate library using the Oncomine™ Dx Express Test (Thermo Fisher Scientific) and sequenced on Genexus Integrated Sequencer. The data was analyzed using Ion Torrent™ Genexus™ software version 6.6.2.1 (Thermo Fisher Scientific).

Example 7. DNA Recovery Yield from each Formulation

Nucleic acid was extracted and the DNA concentration was determined in accordance with Example 3. DNA was spiked in at 240 ng/4 mL, and the recovery rates ranged from 15 %-85%. Formulation A showed the worst yield and Formulation E showed the best yield (Table 2). Formulation F was not tested in this experiment. Formulations C, E, and F were further tested in DNA-depleted EDTA plasma and, again, Formulation E showed the best yield (FIG. 3). After stress testing at 4°C for 51 hours and at 37°C for 7 days as set forth in Example 4, the DNA yield remained similar to the one before stress testing (FIG. 3). This suggests that DNA is stable in almost all the formulations, except Formulation A (data not shown). The RNAlater™ stabilization solution may reduce DNA extraction yield or perhaps results in less stable DNA in the formulation. Mutation specific targets from Formulation C, E, and F also showed stable variant allelic frequency (AF%) on NGS testing (FIG. 4), which indicates that the formulations did not affect variant detection. The accelerated stability test results indicate DNA is stable at -80°C for 24 months or - 20°C for 12 months using the disclosed stabilization methods. Table 2. DNA yield after extraction from different formulations

Example 8. RNA Recovery from each Formulation

RNA extracted from each formulation was quantified using RT-qPCR. Formulation E showed the best yield as compared to the RNA only control, while Formulation A showed the worst yield (FIG. 5). This result is consistent with the recovery results from DNA, indicating that Formulation E showed the best efficiency to stabilize both RNA and DNA in human plasma. Formulations C, E, and F were further used to perform the accelerated stability tests described in Example 4, and the results suggested that the cfTNA control material can be stably stored at -80°C for 24 months or -20°C for 12 months (FIG. 6).

Example 9. RNA Fusion Genes

To evaluate the efficacy and efficiency of Formulations C, E, and F to stabilize RNA fusion genes, in addition to the wild type background RNA, multiple IVT RNA fusions were spiked into the cfTNA mix. Similar to the high stabilization efficiency observed with Formulation E for background RNA, Formulation E also efficiently stabilized RNA fusion genes under accelerated stability testing based on ddPCR™ quantification. Formulation F was less effective and efficient in stabilizing nucleic acids but still performed better than the control (FIG. 7). An NGS platform, Genexus™ Integrated Sequencer platform, was used to perform sequencing from the extracted cfTNA and confirmed a similar pattern with that in ddPCR™ platform results (FIG. 8). These data indicate that the cfTNA control in human plasma can be stable stored at -80°C for 24 months or -20°C for 12 months. Example 10. Formulation F Testing

Preparation of Formulation F with different conditions was tested. Condition 1: Vortexing at -3000 rpm (speed level 10) for 1 minute; Condition 2: Vortexing at -2500 rpm (speed level 8) for 3 minutes; Condition 3: Vortexing at -2500 rpm (speed level 8) for 6 minutes. The cfTNA extracted from Formulation F was tested either by using RT-qPCR with ABI 7500 Fast Real Time PCR system and ACTB ml assay (FIG. 9A), or by using Bio-Rad QX200 AutoDG Droplet Digital PCR System and 1VT KIF5B(24)-RET(8) assay (FIG. 9B). The encapsulation efficiency of Condition 2 was relatively lower than that of Condition 1 by comparing the RNA recovery at time point zero, but its encapsulated emulsion was more stable during the storage by comparing the RNA recovery for 51 hours at 4°C.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim:

1. A method of increasing stability of cell-free nucleic acid control material in a body fluid, comprising: adding one or more stabilizing agents and cell-free nucleic acid control material to a body fluid sample; adding a preparation of encapsulated cell-free nucleic acid control material to a body fluid sample; or adding one or more stabilizing agents and a preparation of encapsulated cell-free nucleic acid control material to a body fluid sample.

2. The method of claim 1, wherein the body fluid is blood, plasma, synthetic plasma, serum, cerebrospinal fluid, saliva, or urine.

3. The method of claim 1 or claim 2, wherein the stabilizing agent comprises ammonium sulfate, EDTA, or both.

4. The method of claim 3, wherein the stabilizing agent further comprises bovine serum albumin, trehalose, or both.

5. The method of any one of claims 1 to 4, wherein the encapsulated cell-free nucleic acid control material is encapsulated in oil droplets, liposomes, or lipid nanoparticles.

6. The method of claim 5, wherein the oil droplets are monodisperse droplets.

7. The method of claim 5, wherein the oil droplets are particle- templated emulsification droplets.

8. The method of claim 7, wherein the particle-templated emulsification droplets comprise monodisperse polyacrylamide beads.

9. The method of claim 7 or claim 8, wherein the particle-templated emulsification droplets are produced by vortexing the cell-free nucleic acids with an oil and the monodisperse polyacrylamide beads at about 500 rpm to about 4000 rpm for about 15 seconds to about 15 minutes.

10. The method of any one of claims 1 to 9, wherein the cell-free nucleic acid control material comprises cell-free DNA control material, cell-free RNA control material, or both.

11. The method of claim 10, wherein the cell-free DNA control material comprises genomic DNA, DNA molecules comprising one or more variants, or both.

12. The method of claim 11, wherein the DNA molecules comprising one or more variants comprise variants selected from the group consisting of one or more single nucleotide polymorphisms (SNP), single nucleotide variants, substitution of one or more nucleic acids (e.g., point mutations), insertion (INS), deletion (DEL), premature stop codons, trinucleotide repeats, translocations, inversions, somatic rearrangements, allelomorphs, splice variants, regulatory variants, gene fusions, copy number variation (CNV), and any combination of two or more thereof.

13. The method of claim 10, wherein the cell-free RNA control material comprises genomic RNA, one or more RNA molecules comprising one or more variants selected from fusions, isoforms, splice variants, and any combination thereof.

14. A cell-free nucleic acid control composition comprising: a body fluid; one or more stabilizing agents; and a cell-free nucleic acid control material.

15. A cell-free nucleic acid control composition comprising: a body fluid; and an encapsulated cell-free nucleic acid control material.

16. A cell-free nucleic acid control composition comprising; a body fluid; one or more stabilizing agents; and an encapsulated cell-free nucleic acid control material.

17. The cell-free nucleic acid control composition of claim 14 or claim 16, wherein the stabilizing agent comprises ammonium sulfate, EDTA, or both.

18. The cell-free nucleic acid control composition of claim 17, wherein the stabilizing agent further comprises bovine serum albumin, trehalose, or both.

19. The cell-free nucleic acid control composition of claim 15 or claim 16, wherein the encapsulated cell-free nucleic acid control material is encapsulated in oil droplets, liposomes, or lipid nanoparticles.

20. The cell-free nucleic acid control composition of claim 19, wherein the oil droplets are monodisperse droplets.

21. The cell- free nucleic acid control composition of claim 19, wherein the oil droplets are particle-templated emulsification droplets.

22. The cell-free nucleic acid control composition of claim 21, wherein the particle- templated emulsification droplets comprise monodisperse polyacrylamide beads.

23. The cell-free nucleic acid control composition of any one of claims 14 to 22, wherein the body fluid is blood, plasma, synthetic plasma, serum, cerebrospinal fluid, saliva, or urine.

24. The cell-free nucleic acid control composition of any one of claims 14 to 23, wherein the cell-free nucleic acid control material comprises cell-free DNA, cell-free RNA, or both.

25. The cell-free nucleic acid control composition of claim 24, wherein the cell-free DNA comprises genomic DNA, DNA molecules comprising one or more variants, or both.

26. The cell-free nucleic acid control composition of claim 25, wherein the DNA molecules comprising one or more variants comprise variants selected from the group consisting of one or more single nucleotide polymorphisms (SNP), single nucleotide variants, substitution of one or more nucleic acids (e.g., point mutations), insertion (INS), deletion (DEL), premature stop codons, trinucleotide repeats, translocations, inversions, somatic rearrangements, allelomorphs, splice variants, regulatory variants, gene fusions, copy number variation (CNV), and any combination of two or more thereof.

27. The cell-free nucleic acid control composition of claim 24, wherein the cell-free RNA control material comprises genomic RNA, one or more RNA molecules comprising one or more variants selected from fusions, isoforms, splice variants, and any combination thereof.

28. A kit comprising the cell-free nucleic acid control composition of any one of claims 14 to 27.

29. The kit of claim 28, further comprising one or more enzymes, buffers, oligonucleotide primers, oligonucleotide adaptors, oligonucleotide probes, or any combination of two or more thereof.