DNA-methylation detection in animal-derived products

Abstract

The present invention relates to a method for detecting DNA methylation of a genomic material contained in a biological sample obtained from a product derived from a test animal and / or for determining a test methylation profile of a genomic material contained in a biological sample obtained from a product derived from a test animal, the method comprises the steps of:-contacting a sample of genomic material from a product derived from the test animal with an array of DNA methylation specific for the species of the test animal, wherein the test animal is a monogastric livestock.

Description

Field of the Invention

[0001] The present invention relates to a method for detecting DNA-methylation in products derived from farm animals. In particular, the method is a DNA array-based method for detecting DNA methylation in products derived from monogastric livestock. The DNA array-based method can be used to determine the methylation profile of animal-derived products, which can then be used for several applications that determine the origin and welfare of the animal, and also determine whether the animal has been exposed to, inter alia, antibiotics and / or the method of slaughtering the animal. Background of the Invention With increasing income and education levels, consumers have shown a greater interest in the quality of the food they eat and are increasingly willing to pay a premium for meat products with guarantees of quality, safety, sustainability and high animal welfare conditions (Wu et al. Foods 2021, 10, 2490). Many of these qualities cannot be visually evaluated and thus food control by regulatory bodies such as the European Food and Drug Safety Administration and the US Food and Drug Administration (FDA) has increased consumer trust. Clear, simple food labelling from independent verification bodies adds an additional level of assurance for consumers. Among the possible food labelling qualities on meat products, geographical origin has been shown to be valued by all consumers, regardless of their culture (Wu 2021). In addition, a study of 10,000 consumers in multiple countries (Japan, the United States, Germany, China and Thailand) has shown that labels are more trusted when verified by scientific experts compared to claims made by producers, retailers or even regulatory bodies (Rupprecht et al. Food and Chemical Toxicology 137 (2020) 111170).

[0003] Origin traceability via barcodes printed on packaging, quick response matrix codes (QR codes) or online links has also been shown to further increase consumer trust and may increase the premium consumers are willing to pay to ensure the origin of their meat products. Currently, traceability technologies include data recording and reporting, which can be supported by computing, artificial intelligence and / or decentralized blockchain. Since current systems mainly rely on a human reporting chain from farm to table, there are many possibilities for introducing false messages due to errors or intentional fraud.

[0004] A single food fraud incident can incur a significant loss of consumer trust in a brand, recalls, litigation, revenue loss, and even criminal charges in severe cases (Esteki et al. Comprehensive Reviews in Food Science and Food Safety Vol. 18, 2019). Many countries, such as the FDA in the United States, have specific criteria for determining when food fraud is considered a criminal offense (Jurica et al. Foods 2021, 10, 2570). However, despite the potential profit loss and the legal regime making food fraud criminally liable, the deliberate economically motivated adulteration or mislabeling of food products continues to be a problem worldwide. To better support consumer trust in high-value meat products, there is a need for scientific, evidence-based testing that reliably and consistently provides assurances such as how and where farm animals such as livestock and poultry are raised.

[0005] To date, there are several methods available for analyzing meat products. For example, origin traceability techniques have been established by measuring isotope ratios via mass spectrometry (Zhao et al. Food Chemistry 145 (2014) 300 - 305) or determining trace element fingerprints in samples. Additionally, species confirmation of meat products can be achieved using PCR-based techniques. However, the need to utilize multiple techniques on a single sample to obtain sufficient information about the origin and species of a meat sample adds unnecessary cost and time to the verification process of these food products. To our knowledge, there are currently no reliable techniques for meat detection that can determine the health status of livestock nor welfare standards for animals raised prior to slaughter. Therefore, there is a search for reliable, evidence-based techniques for determining multiple factors of meat quality, for ensuring the origin, quality, welfare, and sustainability of animal-derived food products.

[0006] Epigenetic techniques can provide solutions for the definitive, multi-target analysis of meat. DNA methylation is one of the best-understood mechanisms in epigenetics and is known to be altered by various aspects of the environmental conditions to which the animal is exposed. Thus, methylation patterns on the genome can be used to distinguish healthy animals from inflamed animals, e.g., (Raddatz et al. Communications Biology, 4:76 (2021)). WO2022 / 023208 also discloses that DNA methylation is used at least to determine the geographical origin of an animal. Traditionally, global methylation patterns, especially for non-human species, have been evaluated by using whole-genome bisulfite sequencing (WGBS) or reduced representation bisulfite sequencing (RRBS) on DNA extracted from different tissues and / or cells. Both methods first use a bisulfite treatment step to convert all unmethylated cytosine nucleotides in the genome into uracil, leaving methylated and hemimethylated cytosine nucleotides unchanged (Stevens et al. Genome Res. 2013. 23:1541-1553). Next-generation sequencing is performed, and the generated sequences are processed (aligned to a reference genome) and analyzed to show methylation differences at individual CpG sites. WGBS covers CpG sites across the entire genome, while RRBS only covers 3-4% of all methylated sites in the genome but represents 85% of the CpG sites in regions of dynamic methylation (Illumina Field Guide to Methylation Methods, 2016). Although these techniques are highly informative, they are expensive, time-consuming, and computationally intensive, thus not allowing for a rapid turnaround time.

[0007] Accordingly, there is a need in the art for improved high-throughput, cost-effective, reliable, efficient, and robust methods for determining the origin and welfare standards of farmed animals from small DNA samples.

[0008] Description of the Invention The present invention solves the above problems by providing an array-based method to determine the origin, welfare, and several other rearing conditions of an organism by using DNA-methylation profiling. DNA-methylation-based arrays allow for high-throughput and robust methods to determine semi-quantitative / quantitative DNA-methylation information from small samples of DNA of interest. These custom designed arrays can use Illumina iScan and Infinium platform technologies or their equivalents, which allow for, for example, 100,000 different bead types covalently bound to DNA-methylation probes on each chip. Each probe represents a CpG methylation site at the end of the probe sequence. The DNA sample undergoes bisulfite conversion, amplification, fragmentation, precipitation, and resuspension steps before hybridization on the array chip. Once on the chip, the DNA hybridizes to the beads at each CpG site, allowing for the specific detection of methylation changes at each site by single nucleotide extension. This is particularly advantageous because the array-based method is simple and the results of methylation-based arrays are accurate and reproducible.

[0009] Furthermore, compared to traditional sequencing which can take weeks to generate data, array technology has a much shorter turnaround time. Compared to sequencing, the amount and complexity of data generated are smaller, making it less computationally intensive. This allows for faster computations to obtain interpretable results from experimental groups. Overall, microarray technology is about 10x faster and about 1 / 10 the price of traditional sequencing, while still being able to quantify the methylation level of specific CpG sites. Thus, methylation array technology provides a fast and flexible system that can be used for many applications, allowing for the scalability of epigenetics research and the commercialization of DNA-methylation-based solutions along the food value chain.

[0010] Since methylation changes of CpG sites can occur due to different environmental conditions, the methylation patterns in the genome can be used to determine aspects of how and where an animal is reared, such as geographical location, health, and certain rearing standards, which can determine the classification of meat, such as high animal welfare, organic, kosher, or halal. The DNA array-based method according to any aspect of the present invention includes probes that bind to specific CpG sites that have known methylation changes from different locations and rearing standards, and also from promoters, from candidate genes focused on immune system genes, feed-related genes, antibiotic-related genes, pectoralis muscle development genes (myopathy), etc. The method according to any aspect of the present invention can be used to scientifically and unambiguously determine the origin, welfare, and other meat quality aspects.

[0011] As used herein, the term 'epigenetic change' refers to chemical (e.g., methylation) or protein (e.g., histone) changes that occur to the gene body or its promoter. Through epigenetic changes, environmental factors such as diet, stress, and prenatal nutrition can imprint genes that are passed from one generation to the next.

[0012] According to one aspect of the invention, there is provided a method for detecting and / or quantifying DNA methylation of genomic material contained in a biological sample obtained from a product derived from a test animal, the method comprising the steps of: - contacting a sample of genomic material from a product derived from the test animal with a DNA methylation array specific for the test animal species, wherein the test animal is a monogastric livestock.

[0013] As used herein, the term "array" refers to an intentionally created collection of probe molecules, which can be synthesized or biosynthetically prepared. The probe molecules in the array can be identical or different from each other. The array can take various forms, e.g., a library of soluble molecules; a library of compounds bound to resin beads, silicon chips, or other solid supports.

[0014] In particular, DNA methylation-based arrays, also known as DNA methylation arrays or chips, provide a convenient platform for simultaneously analyzing a large number of CpG sites, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000, 10,000, 100,000 or more sites or loci. In particular, the array comprises a plurality of different probe molecules, which can be attached to a substrate or otherwise spatially distinguishable in the array. Examples of arrays that can be used according to any aspect of the invention include slide arrays, silicon wafer arrays, liquid arrays, bead-based arrays, etc. In one example, the array technology used according to any aspect of the invention combines a miniaturized array platform, a high level of assay multiplexing, and scalable automation for sample handling and data processing.

[0015] In particular, an array according to any aspect of the present invention can be an array of arrays, also known as a compound array, which has a plurality of individual arrays configured to allow simultaneous processing of multiple samples. Examples of compound arrays and the technology behind them are disclosed at least in US 6,429,027 and US2002 / 0102578. The substrate of the compound array can include a plurality of individual array positions, each having a plurality of probes and each physically separated from other assay positions on the same substrate, such that fluid contacting one array position is prevented from contacting another array position. Each array position can have a plurality of different probe molecules, which are directly attached to the substrate or attached to the substrate via rigid particles (also referred to herein as beads in wells).

[0016] In one example, the array substrate can be an optical fiber bundle or an array of bundles, as described in US6,023,540, US6,200,737, and / or US6,327,410. The optical fiber bundle or array of bundles can have probes directly attached to the fibers or via beads. Those skilled in the art will be able to readily determine which substrate will be most suitable for an array according to any aspect of the present invention. WO2004110246 further discloses other substrates useful for an array according to any aspect of the present invention and methods for attaching beads to the substrate.

[0017] In one example, the surface of the substrate can have a physical alteration to enable attachment of probes or generation of array positions. For example, the surface of the substrate can be modified to contain chemically modified sites for covalently or non-covalently attaching probe molecules or particles having attached probe molecules. The probes can be attached using any of a variety of methods known in the art, including inkjet printing methods, spotting techniques, photolithographic synthesis methods, or printing methods utilizing a mask. WO2004110246 discloses these techniques in more detail.

[0018] In one example, a DNA methylation-based array according to any aspect of the present invention can be a bead-based array, where the beads are associated with a solid support such as those commercially available from Illumina, Inc. (San Diego, Calif.). Bead arrays useful according to any aspect of the present invention can also be in fluid form, such as the fluid stream of a flow cytometer or similar device. Commercially available fluid forms for differentiating beads include, for example, those used in the XMAP(TM) technology from Luminex or the MPSS(TM) method from Lynx Therapeutics.

[0019] In another example, a DNA methylation-based array according to any aspect of the present invention may further comprise - at least one probe molecule specific for at least one single nucleotide polymorphism (SNP) of a first animal species; and - at least one probe molecule specific for at least one SNP of a second animal species.

[0020] These probes specific for SNPs can be used for SNP genotyping, which is a measure of the genetic variation of SNPs among members of a species. In particular, an SNP is a single base pair mutation at a specific locus, typically consisting of two alleles that are conserved during evolution (where the rare allele frequency > 1%). These probes enable the identification of species, particularly breeds of a species. In particular, when a DNA sample is introduced into an array according to any aspect of the present invention, these probes specific for SNPs can be used to determine whether the sample is from the first and / or second animal species found on the array, and whether there is DNA from another species other than the first and second animal species that has contaminated the DNA sample.

[0021] As used herein, the terms "solid support", "support", and "substrate" are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many examples, at least one surface of the solid support will be substantially flat, although in some examples it may be desirable to physically separate the synthesis regions of different compounds using, for example, pores, raised areas, pins, etched trenches, etc.

[0022] A DNA methylation array according to any aspect of the present invention can be a very high-density array, e.g., having about 10,000,000 probes / cm 2 to about 2,000,000,000 probes / cm 2 or about 100,000,000 probes / cm 2 to about 1,000,000,000 probes / cm 2 of those. High-density arrays are particularly useful according to any aspect of the present invention for including a large number of CpG sites from different species on the array.

[0023] A DNA methylation array according to any aspect of the present invention can be used to simultaneously or sequentially analyze or evaluate such multiple loci as desired. In one example, multiple different probe molecules can be attached to the substrate or otherwise spatially differentiated in the array. Each probe is generally specific for a particular locus and can be used to distinguish the methylation status of that locus.

[0024] As used herein, the term "probe molecule" refers to a surface - immobilized molecule that can be recognized by a specific target. The probes used in the array can be specific for the methylated allele of a CpG site, the unmethylated allele of a CpG site, or both.

[0025] As used herein, the term "target" refers to a molecule that has an affinity for a given probe molecule. Targets can be naturally - occurring or man - made molecules. Also, they can be in their unaltered state or used as aggregates with other species. Targets can be attached to the binding member covalently or non - covalently, either directly or via a specific binding substance. Examples of targets that can be used in any aspect of the present invention are methylated and unmethylated CpG sites. In the art, targets are sometimes referred to as anti - probes. When using the term target herein, no difference in meaning is intended.

[0026] In particular, probe molecules according to any aspect of the present invention comprise nucleic acid sequences complementary to different CpG sites. Thus, an array according to any aspect of the present invention includes several different or distinct positions, where each position contains a specific probe molecule complementary to a different CpG site of an animal species. The array thus includes a plurality of positions, each having a specific probe molecule complementary to a different CpG site of an animal species. In particular, an array according to any aspect of the present invention includes different positions, where each position contains a specific probe molecule complementary to different CpG sites of at least two animal species. An array according to any aspect of the present invention thus includes different positions with specific probe molecules, where each probe molecule is complementary to different CpG sites from at least two animal species.

[0027] As used herein, the term "complementary" refers to hybridization or base - pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double - stranded DNA molecule or between an oligonucleotide primer and a primer - binding site on a single - stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are typically A and T (or A and U), or C and G. Two single - stranded RNA or DNA molecules are generally considered to be complementary when at least about 80% of the nucleotides of one strand pair with the nucleotides of the other strand when optimally aligned and compared with appropriate nucleotide insertions or deletions, typically at least about 90% to 95%, and more preferably about 98 to 100%. Complete complementarity refers to 100% complementarity over the length of the sequence. For example, a 25 - base probe is completely complementary to a target when all 25 bases of the probe are complementary to an adjacent 25 - base sequence of the target with no mismatches over the length of the probe.

[0028] As used herein, a "CpG site" or "methylation site" is a nucleotide within a nucleic acid (DNA or RNA) that is susceptible to methylation by an event occurring naturally in vivo or by an event that chemically methylates the nucleotide in vitro. In cells, some of these sites may be hypermethylated, while some may be hypomethylated.

[0029] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0030] As used herein, a "methylated nucleotide" or "methylated nucleobase" refers to the presence of a methyl moiety on a nucleobase, where the methyl moiety is not typically present in a recognized canonical nucleobase. For example, cytosine in its normal form does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Thus, cytosine in its normal form may not be considered a methylated nucleotide, while 5-methylcytosine may be considered a methylated nucleotide. In another example, thymine may contain a methyl moiety at position 5 of its pyrimidine ring; however, for the purposes of this disclosure, when thymine is present in DNA, it may not be considered a methylated nucleotide. The canonical nucleobases of DNA are thymine, adenine, cytosine, and guanine. The canonical bases of RNA are uracil, adenine, cytosine, and guanine. Accordingly, a "methylation site" is a position within a nucleic acid region of a target gene where methylation has the potential to occur. For example, a position containing CpG is a methylation site where cytosine may or may not be methylated. In particular, the term "methylated nucleotide" refers to a nucleotide carrying a methyl group that is attached to a position of the nucleotide that is susceptible to methylation. These methylated nucleotides typically occur in nature and, to date, have predominantly occurred in the context of the dinucleotide CpG, but methylated cytosines in the context of CpNpG- and CpNpN-sequences may also be considered the most common. In principle, other naturally occurring nucleotides may also be methylated, but they will not be considered for any aspect of the present invention.

[0031] As used herein, a "CpG island" describes a DNA sequence segment that contains a CpG density that is functionally or structurally distinct from the norm. For example, Yamada et al. have described a set of criteria for determining CpG islands: it must be at least 400 nucleotides in length, have a GC content greater than 50% and an OCF / ECF ratio greater than 0.6 (Yamada et al., 2004, Genome Research, 14, 247-266). Others have less stringently defined CpG islands as sequences that are at least 200 nucleotides in length, have a GC content greater than 50% and an OCF / ECF ratio greater than 0.6 (Takai et al., 2002, Proc. Natl. Acad. Sci. USA, 99, 3740-3745). In the context of the present invention, the terms "methylation profile", "methylation pattern", "methylation status" or "methylation state" are used herein to describe the condition, situation or circumstance of genomic sequence methylation, and such terms refer to the characteristics of a DNA segment at a specific genomic locus related to methylation. Such characteristics include, but are not limited to, whether any cytosine (C) residue in the DNA sequence is methylated, the position of one or more methylated C residues, the percentage of methylated C at any particular residue sequence segment, and allelic differences in methylation attributable to, for example, differences in allelic origin.

[0032] The term "methylation state" refers to the state of a particular methylation site (i.e., methylated versus non-methylated), which means that a residue or methylation site is either methylated or not methylated. Then, based on the methylation state of one or more methylation sites, a methylation profile can be determined.

[0033] The term "methylation level" refers to the level of a specific methylation site, which can range from 0 (= unmethylated) to 1 (= fully methylated). Thus, based on the methylation levels of one or more methylation sites, a methylation profile can be determined. Accordingly, the term "methylation profile" or also "methylation pattern" refers to the relative or absolute concentration of methylated C or unmethylated C at any specific residue sequence segment in a biological sample. For example, if one or more cytosine (C) residues that are generally not methylated in a DNA sequence in a sample are more methylated, it can be referred to as "hypermethylated"; while if one or more cytosine (C) residues that are generally methylated in a DNA sequence are less methylated, it can be referred to as "hypomethylated". Similarly, if one or more cytosine (C) residues in a DNA sequence (e.g., sample nucleic acid) are more methylated when compared to another sequence from a different region or different individual (e.g., relative to normal nucleic acid), the said sequence is considered hypermethylated compared to the other sequence. On the other hand, if one or more cytosine (C) residues in a DNA sequence are less methylated when compared to another sequence from a different region or different individual, the said sequence is considered hypomethylated compared to the other sequence. These sequences are referred to as "differentially methylated". For example, when the methylation status is different between inflamed tissue and non-inflamed tissue, the sequence is considered "differentially methylated". The measurement of the differential methylation level can be carried out in a variety of ways known to those skilled in the art. As a non-limiting example, one method is to measure the methylation level of individual queried CpG sites determined by bisulfite sequencing methods.

[0034] "Bisulfite treatment" of genomic DNA, which is used interchangeably with the term 'bisulfite modification', refers to treating genomic DNA with a deaminating agent such as bisulfite, which can be used to treat all DNA, methylated or non-methylated. In particular, as used herein, the term "bisulfite" includes any suitable type of bisulfite, such as sodium bisulfite, or other chemical reagents that are capable of chemically converting cytosine (C) to uracil (U) without chemically modifying methylated cytosine and can thus be used to differentially modify DNA sequences based on the methylation state of the DNA, e.g., U.S. Patent Publication US2010 / 0112595. As used herein, a reagent that "differentially modifies" methylated or non-methylated DNA includes any reagent that modifies methylated and / or unmethylated DNA in a process that produces distinguishable products from methylated and non-methylated DNA, thereby allowing the identification of the DNA methylation state. Such processes can include, but are not limited to, chemical reactions (e.g., C to U conversion by bisulfite) and enzymatic treatments (e.g., cleavage by methylation-dependent endonucleases). Thus, an enzyme that preferentially cleaves or digests methylated DNA is an enzyme that is capable of cleaving or digesting a DNA molecule with much higher efficiency when the DNA is methylated, while an enzyme that preferentially cleaves or digests non-methylated DNA exhibits significantly higher efficiency when the DNA is non-methylated.

[0035] Thus, prior to performing step (a) of any aspect of the present invention, the genomic DNA contained in or obtained from a cell or extracted from a cell is first subjected to bisulfite treatment.

[0036] Alternative methods available in the art can be used in place of bisulfite treatment. Skilled artisans will understand which other methods to use. In one example, TET-assisted pyridine borane sequencing (TAPS) can be used to detect 5mC and 5hmC (Yibin Liu et al., Nature Biotechnology, 37:424-429 (2019)).

[0037] As used herein, the term "genomic material" refers to a nucleic acid molecule or fragment of the genome of an animal according to any aspect of the present invention. In particular, such nucleic acid molecule or fragment is DNA or RNA or a hybrid thereof, and most preferably a molecule of the DNA genome of the subject or subject group.

[0038] As used herein, the term 'biological sample' can be selected from muscle, organ tissue, milk, blood, brain, sperm, and any other tissue or sample that provides genomic DNA for use in the methods according to any aspect of the present invention. In particular, a biological sample can include any biological material containing DNA obtained from a subject, and can be liquid, solid, or both, can be tissue or bone, or a body fluid, such as blood, lymph, etc. In particular, the biological sample for use in the present invention can include biological cells or fragments thereof.

[0039] As used herein, a "DNA sample" refers to DNA extracted from animal cells according to any aspect of the present invention using methods known in the art.

[0040] As used herein, the term 'animal-derived product' refers to a product derived from an animal. In particular, the term 'test animal-derived product' refers to a sample or subject that is to be considered for introduction into an array according to any aspect of the present invention. These products derived from animals can include meat and meat products, and also include fat, meat, blood, processed meat, and lesser-known products such as fish gelatin and crude rennet, poultry products (meat and eggs), dairy products (milk and cheese), and non-food products such as fibers (wool, mohair, cashmere, leather, etc.). Animal-derived products can also include products that can be prepared using animal products (e.g., fat), such as soap, cream, etc. In one example, the animal-derived product is meat, eggs, blood, brain, sperm, milk, and any other tissue or sample that provides genomic DNA. In particular, the animal-derived product is meat. In one example, an animal-derived product sample can be a single type of meat, different types of meat, a single part of one type of meat, different parts of a single type of meat, or different parts of different types of meat. If the animal is an aquatic animal, then these products derived from the animal can include meat and meat products, and also include eggs, fat, meat, blood, processed meat, and lesser-known products, as well as non-food products such as fibers (shells, scales, etc.). Animal-derived products can also include products prepared using animal products (e.g., fish oil), such as tablets, powders, etc. In one example, the animal-derived product is meat, eggs, blood, brain, shells, scales, skin, tissue, abdominal muscle tissue, or any other tissue or sample that provides genomic DNA. In particular, the animal-derived product is meat, skin, blood, trimmings, or any organ from an aquatic animal. In particular, the trimmings can be used as a by-product of fish meal / fish oil, which ultimately enters the animal feed industry or pets. The sample can be from any biological entity having a DNA genome and DNA genome methylation. In particular, the methylation site is a CpG site.

[0041] As used herein, the term "test" when used in conjunction with the term "animal" refers to an animal introduced into an array according to any aspect of the present invention and which is the basis for the analytical applications of the present invention. Thus, a "(single) test subject", "(single) group of test subjects" or "test profile" or "product derived from a test animal" is a (single) subject or group of subjects tested according to the present invention or a profile obtained or generated in the context thereof. Similarly, the term'sample' and / or 'product sample derived from a test animal' as used according to any aspect of the present invention refers to an entity that can be subjected to the methods of the present invention. In contrast, the term "reference" or 'control' shall denote a generally pre-determined entity used for comparison with a test entity. In particular, a sample can be any (test) animal-derived product that can be subjected to the methods of the present invention to determine any characteristic of an animal (i.e., biological age, geographical origin, method of rearing, etc.) by first determining a DNA methylation profile and then comparing the test methylation profile with a control, and 'control' refers to an animal in which the characteristic as mentioned above is known and in which the methylation status is known and used as a reference.

[0042] As used herein, the term 'contact' means bringing a sample of genomic material into direct contact with a DNA methylation-based array. For example, the sample of genomic material can be DNA extracted from a biological sample from a test animal and which is brought into direct contact with the probes in a DNA methylation-based array.

[0043] Monogastric livestock according to any aspect of the present invention includes terrestrial and aquatic livestock having only a single-chambered stomach. In particular, the livestock can be a farm animal selected from terrestrial and aquatic livestock. Even more particularly, monogastric livestock does not include animals having a compartmentalized stomach called ruminants, which include goats, sheep, cattle, bison, etc. In particular, monogastric terrestrial livestock can include pigs, horses, donkeys, mules, rabbits, chickens, turkeys and other gallinaceous birds, ducks, geese, quails, etc. The term monogastric terrestrial livestock refers to the same animals as monogastric farm animals.

[0044] As used herein, the term "aquatic monogastric livestock" refers to any organism that is raised entirely in water or lives primarily in water, especially as compared to terrestrial animals with a single-chambered stomach. These aquatic monogastric livestock may live in different water forms, such as seas, oceans, rivers, lakes, ponds, etc. More particularly, the aquatic monogastric livestock according to any aspect of the present invention can be any fish, cephalopod, aquatic mollusk or aquatic crustacean at all life stages, including eggs, sperm and gametes. Even more particularly, 'aquatic monogastric livestock' means animals of the following species: (i) fish belonging to the superclass Agnatha and the classes Chondrichthyes, Sarcopterygii and Actinopterygii; and (ii) aquatic crustaceans belonging to the subphylum Crustacea. Even more particularly, the aquatic monogastric livestock according to any aspect of the present invention can be aquatic livestock for aquaculture. Some non-limiting examples of the aquatic monogastric livestock according to any aspect of the present invention include vertebrate fish such as lungfish, carp, catfish, halibut, marbled crayfish, marine and brackish fishes, pangasius, rainbow trout, salmonids, black bass, sea bream, tilapia and turbot. Another monogastric livestock can also include sea shrimp, mitten crabs, shellfish, oysters, scallops, soft-shell crabs, turtles, tiger prawns, white-leg prawns, shrimp, octopuses, squids and other decapod crustaceans, bivalves and gastropods. In another example, the test animals used in the method according to any aspect of the present invention can be monogastric livestock (terrestrial and aquatic) as well as crustaceans, bivalves and gastropods.

[0045] According to another aspect of the present invention, there is provided a method for identifying the geographical origin of an animal-derived product, the method comprising the following steps: (a) Performing a method according to any aspect of the present invention to determine the test methylation profile of the animal-derived product being tested; and (b) Comparing the test methylation profile determined in (a) with one or more predetermined reference methylation profiles, wherein each of the one or more predetermined reference methylation profiles is specific for a particular geographical origin of a subject having the same taxonomic unit as the test animal; Wherein if the tested methylation profile is significantly similar to one of the predetermined reference methylation profiles, the product derived from the tested animal has a geographical origin similar to that of a subject having the predetermined reference methylation profile; and / or wherein if the tested methylation profile is different from one of the predetermined reference methylation profiles, the product derived from the tested animal has a geographical origin different from that of a subject having the predetermined reference methylation profile, and wherein the tested animal is a monogastric livestock.

[0046] As used herein, the term "geographical origin" refers to a geographical location that is distinguishable from other geographical locations by one or more environmental parameters of the tested animal. Such environmental parameters depend on the habitat of the animal and may be different if the animal lives or is cultivated in water, on soil, or in soil, or may be selected from food or air parameters, etc. In one example, for freshwater crabs (such as marbled crayfish), the relevant environmental parameters may be selected from pH, water hardness, manganese content, iron content, and aluminum content. However, the relevant environmental parameters may vary significantly depending on the taxonomic unit or species of the animal. Similarly, the habitats of animals living in water may also vary. For example, these habitats may be selected from stagnant or flowing water, such as lakes, rivers, aqua farms, other pools or water bodies, or ponds. The geographical origin should be understood as the geographical location regarded as the habitat where the tested animal was born, hatched, and / or raised, or at least raised for a significant period of its life.

[0047] As used herein, the term "preselected methylation site" refers to a methylation site selected from a gene or region that showed the highest degree of methylation variation during method training and meets certain quality criteria, such as considering a minimum sequencing coverage of ≥5x and ≥5 qualified CpG sites. In addition, genes with an average methylation level of <0.1 or >0.9 can be excluded due to their limited dynamic range. Multivariate statistical methods, such as principal component analysis or Multi-Dimensional Scaling, can be used to define a "reference methylation profile" based on multiple training samples.

[0048] As used herein, and particularly in the context of a comparison of methylation profiles (e.g., a test profile from a comparison between one or more test subjects and a reference profile), the term "significantly similar" shall mean a similarity observed by statistical methods (i.e., by using bioinformatics) and / or also by using eye observation. For example, significant similarity is observed if the test profile overlaps with a reference profile defined by multivariate statistical methods such as principal component analysis or multidimensional scaling analysis through multiple training samples. In particular, if more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the methylation patterns / profiles overlap with the reference profile, the test profile is significantly similar to the predetermined reference profile. The similarity of the test profile to more than one, such as two, three or even all reference profiles reduces the significance of the similarity.

[0049] The term "predetermined reference profile" as used herein refers to a typical or standard methylation profile of genomic material of a living organism having specific characteristics depending on the context in which the term is used. In one example, for a method for identifying the geographical origin of a product derived from a test animal according to any aspect of the present invention, the term "predetermined reference profile" refers to a typical or standard methylation profile of genomic material of a living organism having a specific geographical origin. A predetermined reference profile can be obtained from a control subject. For example, the control subject can be a living organism of the same species as the test subject having a known geographical origin. On the other hand, a predetermined reference profile can be obtained from a plurality of organisms living in a specific geographical origin. The methylation profiles of different organisms having a specific geographical origin may be the same. There may be compilations of several predetermined reference profiles, and comparing the methylation profile of the test subject with the predetermined reference profiles in the compilation can enable identification of a specific predetermined reference profile similar to the methylation profile of the test subject, and then the geographical origin of the test subject can be inferred to be the geographical origin of the predetermined reference profile.

[0050] The term "similar" as used in relation to geographical origin means that the habitat or geographical origin of one or more test subjects is based on the habitat or geographical origin of the organism from which the predetermined reference profile is obtained. The term'similar' can refer to the type of habitat, environmental parameters of the habitat, the country in which the habitat is located, etc. Based on at least one or more environmental parameters as defined above under 'geographical origin', the geographical origin of the test subject may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% similar to the geographical origin of the predetermined reference profile.

[0051] Further disclosures of DNA methylation changes and technical details of using these to determine the link between geographical origins are disclosed in WO2022 / 023208.

[0052] According to another aspect of the present invention, there is provided an in vitro method for predicting the biological age of a test animal from which a product is derived, the method comprising the following steps: (a) Performing a method according to any aspect of the present invention to determine the test methylation profile of the test animal; and (b) Comparing the test methylation profile from (a) with the methylation profile of an age-related reference sample, thereby establishing an epigenetic age and predicting the biological age of the test animal from which the product is derived; and wherein the test animal is a monogastric livestock.

[0053] The age-related reference sample serves as a control and represents the average methylation level at a predetermined and specific chronological age.

[0054] The term 'chronological age' refers to the calendar time that has elapsed since birth / hatching.

[0055] The epigenetic age depends on the biological condition or situation of an individual or a population and takes into account the living environment (such as stress, nutrition, etc.). The terms "epigenetic age", "methylation age" and "biological age" have the same meaning and can be used interchangeably.

[0056] The epigenetic age may or may not match the chronological age. The deviation of the epigenetic age compared to the chronological age is age acceleration or age deceleration. Therefore, the epigenetic age can also be determined by comparing the methylation level of methylation markers (i.e., CpG sites) in the genomic DNA of the sample to be tested with the methylation status of the same markers (i.e., CpG sites) of an age-related reference sample.

[0057] In one example, for an in vitro method for predicting the biological age of a test animal from which a product is derived according to any aspect of the present invention, the term "predetermined reference profile" refers to a typical or standard methylation profile of genomic material of a living organism with a known chronological age. The predetermined reference profile can be obtained from a control subject or a population of control subjects with known animal ages. There may be several compilations of predetermined reference profiles, and comparing the methylation profile of the test subject with the predetermined reference profiles in the compilation can enable the identification of a specific predetermined reference profile similar to the methylation profile of the test subject, and then the biological age of the test subject can be inferred as the biological age of the predetermined reference profile.

[0058] Further disclosures of the technical details of the link between DNA methylation changes and the use of these changes to determine the biological age of an animal under consideration are provided at least in WO 2021 / 148593 and WO 2021 / 148601.

[0059] The method according to the aspect of the invention can also be used to determine (test) the health status of an animal. In particular, the health status can refer to whether the animal has a disease, the animal welfare of the animal based on the environment, and / or the food safety of the animal. Methylation profiles for each of these components can be developed to create a reference methylation profile set, which can then be used to determine the health status of the test animal.

[0060] According to a further aspect of the invention, there is provided a method for determining whether a test animal from which a product is derived has been treated with at least one antibiotic and / or veterinary chemical and / or is currently undergoing said treatment, the method comprising: (a) performing a method according to any aspect of the invention to determine a test methylation profile of the test animal; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal or a product derived from a control animal, wherein the control animal has not been treated with at least one antibiotic and / or veterinary chemical and / or is not currently undergoing said treatment, wherein a difference in the test methylation profile of (a) compared to the reference methylation profile from the control animal indicates that the test animal has been treated with at least one antibiotic and / or veterinary chemical and / or is currently undergoing said treatment; and / or wherein a significant similarity of the test methylation profile of (a) compared to the reference methylation profile indicates that the test animal has not been treated with at least one antibiotic and / or veterinary chemical and / or is not currently undergoing said treatment; and wherein the test animal is a monogastric livestock.

[0061] As used herein, the term 'antibiotic' refers to any pharmaceutical that can be fed to a terrestrial animal for therapeutic and / or prophylactic purposes. Antibiotics can be administered by any method known in the art. Antibiotics can be orally fed to a terrestrial animal according to any aspect of the present invention in animal feed or water such that they are ingested. In another example, the antibiotic can be injected into the animal. In one example, the antibiotic can be introduced into a terrestrial animal via intramammary injection. A person skilled in the art will know the best way to provide an antibiotic to an animal based on the particular taxonomic unit of the animal, the type of antibiotic, and the disease to be treated or prevented. In particular, the antibiotic according to any aspect of the present invention can be selected from the classes amphenicols, aminocyclitols, aminoglycosides, ansamycins, β-lactams, carbaephem, carbapenems, cephalosporins, chloramphenicol, fluoroquinolones, glycopeptides, glycylcyclines, ketolides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, nitroimidazoles, oxazolidinones, penicillins, phosphonic acid derivatives, pleuromutilins, polymyxins, polypeptides, quinolones, rifamycins, rimino fenazines, steroid antibacterial agents, streptogramins, sulfonamides, tetracyclines, and trimethoprim. More particularly, the antibiotic can be selected from tetracycline hydrochloride, amoxicillin, and colistin (Colist).

[0062] At least one or more of the above antibiotics can be fed to a test animal according to any aspect of the present invention either simultaneously or sequentially. Contact of the antibiotic with the terrestrial animal can cause epigenetic changes, at least DNA methylation changes, which can then be determined using the method according to any aspect of the present invention. The concentration of the antibiotic in each dose and / or the time period for which the antibiotic has been administered to the test animal can affect the degree of differential methylation of the test animal relative to a control animal. Determining the concentration per dose and the antibiotic exposure time period required for the test animal depending on whether the antibiotic is administered for prophylactic or therapeutic measures is within the knowledge of a person skilled in the art.

[0063] As used herein, the term 'veterinary chemical' refers to a drug or medicine for treating or preventing diseases, injuries and pests in animals. In particular, 'veterinary chemical' can refer to anthelmintics, antiviral drugs, feed additives, water additives, disinfectants, glutaraldehyde, formalin, mixtures thereof, etc. In one example, the feed additive can be a coccidiostat or an ionophore. Water additives refer to chemicals that can be added to water pipes rather than terrestrial animal feed. Veterinary chemicals can be administered to terrestrial animals by any method known in the art.

[0064] In the method according to any aspect of the invention, the test animals used can be in contact with both antibiotics and veterinary chemicals simultaneously and / or thereby. Changes in the internal environment of the test animals result in epigenetic changes, and this can be determined using the method according to any aspect of the invention.

[0065] In particular, in the method according to any aspect of the invention, in step (a), the methylation status of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 CpG sites is determined. A person skilled in the art will be able to determine the number of CpG sites to be used in step (a) according to any aspect of the invention. Even more particularly, in step (a) of the method according to any aspect of the invention, the methylation status of at least two CpG sites is determined.

[0066] According to yet another aspect of the invention, there is provided a method for determining whether a test animal from which a product is derived has been treated with at least one antibiotic and / or is currently undergoing said treatment, and if so, determining the particular class of antibiotic with which the test animal is being treated and / or is currently undergoing said treatment, the method comprising: (a) performing the method according to any aspect of the invention to determine the test methylation profile of the test animal; and (b) comparing the test methylation profile obtained from (a) with one or more predetermined reference methylation profiles, wherein each predetermined reference methylation profile is from a different animal having the same taxonomic unit as the test animal, and each different animal is treated with a different class of antibiotic, wherein if the test methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, it is confirmed that the test animal from which the product is derived is being treated with the same class of antibiotic as the animal having the predetermined reference profile was treated with, and it has been confirmed that the test animal has been treated with the particular class of antibiotic and / or is currently undergoing said treatment; and / or Wherein if the tested methylation profile of (a) is significantly different from one of the predetermined reference methylation profiles, it is confirmed that the test animal from which the product is derived is not being treated with the same class of antibiotics as the animals having the predetermined reference profile are being treated with, and it has been confirmed that the test animal has not been treated with the special class of antibiotics and / or is currently undergoing said treatment; and Wherein the test animal is a monogastric livestock.

[0067] In particular, the special class of antibiotics are amide alcohols, aminocyclitols, aminoglycosides, ansamycins, β-lactams, carbacephems, carbapenems, cephalosporins, chloramphenicol, fluoroquinolones, glycopeptides, glycylcyclines, ketolides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, nitroimidazoles, oxazolidinones, penicillins, phosphorus-containing antibiotics; and / or the veterinary chemicals are antiparasitics, antivirals, feed additives, disinfectants, glutaraldehyde and / or formalin.

[0068] In one example, a set of predetermined reference profiles can be prepared for different animals to be used as controls, wherein each animal has been exposed to different classes of antibiotics and / or each part of the animal (i.e., tissue, muscle, blood, skin) has its own unique predetermined methylation reference profile, which also forms part of the set of predetermined reference profiles. Different animals from the same taxonomic unit as the test animal, each treated with a different class of antibiotics, may have their own set of predetermined reference profiles for the parts of the animal or animal-derived product used as genomic material. For example, each set may be specific for a single animal exposed to a first antibiotic and / or veterinary chemical, wherein each reference profile may be specific for a part of the animal from which the genomic material is extracted. Thus there will be a compilation of sets of predetermined reference profiles, each set being specific for a control animal having the same taxonomic unit as the test animal, the control animal being exposed to or having been exposed to the first, second, third, etc. antibiotics and / or veterinary chemicals. When a tested methylation profile is obtained from a sample of an animal-derived product of unknown origin, it is then compared with the different sets of predetermined reference profiles of the same taxonomic unit as the test animal to determine the special class of antibiotics and / or veterinary chemicals to which the test animal has been exposed or was previously exposed.

[0069] According to yet another aspect of the present invention, there is provided a method for determining whether a test animal and / or a test animal from which a product is derived has been treated with at least one antibiotic and / or is currently undergoing said treatment, and if so, determining whether the antibiotic is used as a growth promotant or a therapeutant, the method comprising: (a) Performing a method according to any aspect of the present invention to determine a test methylation profile of the test animal; and (b) Comparing the test methylation profile obtained from (a) with one or more predetermined reference methylation profiles, wherein each predetermined reference methylation profile is from a different control animal having the same taxonomic unit as the test animal, and each different control animal is treated with an antibiotic used as a growth promoter or therapeutic agent, wherein if the test methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, it is confirmed that the test animal is being treated with an antibiotic in the same manner as the control animal having a similar predetermined reference profile, and it has been confirmed that the test animal has been treated with an antibiotic used as a growth promoter or therapeutic agent and / or is currently undergoing such treatment.

[0070] As used herein, the term 'growth promoter' refers to an antibiotic used to help improve animal production efficiency by increasing weight gain and product yield. In contrast to being used as a therapeutic agent (i.e., for treating diseases), an antibiotic can be used as a growth promoter.

[0071] According to a further aspect of the present invention, there is provided a method for determining whether a test animal from which a product is derived has undergone a withdrawal period without treatment with at least one antibiotic and / or veterinary chemical prior to obtaining the product, the method comprising: (a) Performing a method according to any aspect of the present invention to determine a test methylation profile of the test animal; and (b) Comparing the test methylation profile obtained from (a) with at least two predetermined reference methylation profiles, wherein at least one of the predetermined reference methylation profiles is from a control animal that has undergone a withdrawal period, and the other predetermined reference methylation profile is from a control animal having the same taxonomic unit as the test animal that has not undergone a withdrawal period prior to obtaining the product, wherein if the test methylation profile of (a) is significantly similar to the predetermined reference methylation profiles, it is confirmed whether the test animal has undergone a withdrawal period or not.

[0072] As used herein, the term 'withdrawal period' refers to the time period from the point in time when an animal is no longer fed antibiotics and / or veterinary chemicals until the moment when the remaining antibiotics are broken down in the body until they become non-functional agents and are finally excreted from the animal's body. The withdrawal periods for different antibiotics can vary from 1 or 2 days to several weeks. A 'withdrawal' period is required from the time of administration of the antibiotic until it is legal to slaughter the animal or derive products from the animal. Thus, the time taken for the body to break down the antibiotic until it is no longer functional or present is called the withdrawal time (or withdrawal period). Once the withdrawal period has passed, the antibiotic has been eliminated from the animal's system.

[0073] According to a further aspect of the invention, there is provided a method of assaying a sample of an animal-derived product, the method comprising the steps of: (a) performing a method according to any aspect of the invention to determine the test methylation profile of the test animal; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal having the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal was not slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea and / or the oesophagus, and / or the control animal did not die bleeding, and wherein a difference in the test methylation profile from (a) compared to the reference methylation profile from the control animal indicates that the test animal has been slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea and / or the oesophagus, and / or the test animal has died bleeding, and the animal-derived product is assayed as such; and / or wherein a significant similarity in the test methylation profile from (a) compared to the reference methylation profile from the control animal indicates that the test animal has not been slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea and / or the oesophagus, and / or the test animal has died bleeding, and the animal-derived product is assayed as such; and wherein the test animal is a monogastric livestock.

[0074] The term 'quality inspection' refers to a certificate or attestation given by a designated inspection body, which guarantees the quality of products derived from specific animals, including food for human use and / or consumption. The term 'quality inspection' can be used interchangeably with the term 'inspection'. These inspections usually appear on the packaging of products derived from animals (including food for consumption) and are printed by the product manufacturer. 'Rearing method (Haltungsform)', 'animal welfare (Tierwohl)', 'non-genetically engineered (OhneGentechnik)', 'halal', 'kosher', 'organic', 'free-range', 'pasture-raised', 'grass-fed', 'grain-fed','vegetarian', 'raised without hormones', etc. There are also different inspections based on the country. For example, like the rearing method in Germany, other inspections include Red Tractor (UK), Label Rouge (France), USDA grades (USA), etc., as well as other safety labels that confirm that the products for sale have been prepared in accordance with specific religious or safety regulations. Specifically, the term 'food quality inspection' refers to a certificate or attestation given by a designated inspection body, which guarantees the quality, origin or slaughter method of specific food for human consumption. According to any aspect of the present invention, the quality of the inspection can be a special inspected food quality, or a special inspection, and this can be kosher, non-kosher, halal or non-halal.

[0075] In one example, the special inspection or inspection of sample X according to any aspect of the present invention can be kosher, non-kosher, halal or non-halal. More particularly, kosher or halal refers to sample X from an animal slaughtered by a single cut across the throat severing the two carotid arteries, two jugular veins, two vagus nerves, the trachea and / or the esophagus. Even more particularly, the animal is exsanguinated.

[0076] The term 'kosher food' used in connection with food according to any aspect of the present invention refers to food that complies with the dietary laws (kashrut) of Judaism or food that may be consumed according to the halakha (law). Kosher food used in connection with meat particularly relates to the way in which an animal is prepared for consumption. According to Jewish tradition, when meat comes from an animal slaughtered according to Jewish law, the meat is considered kosher food, where the animal is killed by a single cut across the throat to an exact depth, severing the two carotid arteries, two jugular veins, two vagus nerves, the trachea and the esophagus, not higher than the epiglottis and not lower than the starting point of the cilia in the trachea, thus causing the animal to bleed to death. This slaughter is to be carried out using a sharp large knife, which is inspected before each slaughter to ensure that it has no irregularities (such as nicks and dents). The slaughter is usually also carried out by a butcher (shochet) or a rabbi. Kosher food meat generally refers to most meat excluding pigs. In particular, kosher food meat may be selected from beef, chicken, lamb, mutton, goat meat and mixtures thereof. Kosher food meat does not include shelled aquatic animals, which are not permitted for consumption according to Jewish tradition. Although Jewish tradition permits the consumption of vertebrate fish, since there is no special method for slaughtering vertebrate fish, all vertebrate fish may be considered kosher food. Any food or meat that does not fall within the definition of 'kosher food' is then considered 'non-kosher food'.

[0077] The term 'halal' used in connection with food according to any aspect of the present invention refers to food that complies with the Islamic dietary laws, and in particular meat prepared according to those requirements. Similar to the way in which kosher food meat is prepared, in Islamic tradition, the animal is according to For slaughtering, an animal is slaughtered by a clean cut with a sharp blade without a serrated edge across the neck, resulting in an incision that cuts the front of the larynx, the esophagus, and the jugular vein but not the spinal cord. Except as per the guidelines, the animal to be slaughtered should be done so while reciting the Islamic prayer Bismillah. The animal must also be bled out after slaughter. The slaughter must be performed by an adult Muslim. Halal meat generally refers to most meats excluding pork. In particular, halal meat can be selected from beef, chicken, lamb, mutton, goat meat, and mixtures thereof. Although Islamic tradition permits the consumption of shelled aquatic animals and vertebrate fish, since there is no special method for preparing shelled aquatic animals, all shelled aquatic animals and vertebrate fish can be considered halal. Any food or meat that does not fall within the definition of 'halal' is thus considered "non-halal". The definition of halal is further provided at https: / / www.smiic.org / en / project / 24 (Organisation of Islamic Cooperation (OIC) / Standards and Metrology Institute for the Islamic Countries (SMIIC), OIC / SMIIC 1:2019 General Requirements for Halal Food. Accessed on June 8, 2022).

[0078] As used herein, the term "predetermined reference profile" refers to the typical or standard methylation profile of genomic material of a class of reference animal-derived products that have been verified to be correctly labeled or assayed. In one example, the predetermined reference profile can be used in the context of a control animal that has been correctly assayed (i.e., where the control animal has neither been slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea, and / or the esophagus, nor died by bleeding). The control animal can thus have been slaughtered using a non-halal and non-kosher food slaughtering method, which can be referred to as a normal slaughtering method. The set of predetermined reference profiles for the control animal can include profiles from different samples obtained from different parts of the control animal (that has not been slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea, and / or the esophagus, nor died by bleeding). For example, the set of predetermined reference profiles can include the profile of at least one egg, the profile of at least one meat (muscle, tissue, organ, etc.), the profile of at least one milk, etc. Each of these samples may have its own unique predetermined methylation reference profile, which also forms part of the set of predetermined reference profiles.

[0079] In another instance, a predetermined reference spectrum can be used in the context of a reference animal that may have been slaughtered by a single cut across the larynx severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea, and / or esophagus and / or bled to death. A set of predetermined reference spectra can be prepared for different samples from animals that have been confirmed to have been slaughtered by a single cut across the larynx severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea, and / or esophagus and / or bled to death. Here again, there can be a set of predetermined reference spectra for each product, where each product is derived from an animal that has been slaughtered by a single cut across the larynx severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea, and / or esophagus and / or bled to death. For example, the set of predetermined reference spectra can include spectra of at least one egg, at least one meat (muscle, tissue, organ, etc.), at least one milk, etc. from an animal that has been slaughtered by a single cut across the larynx severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea, and / or esophagus and / or bled to death. Each of these samples may have its own unique predetermined methylation reference spectrum, which also forms part of the set of predetermined reference spectra.

[0080] For example, a control can be a piece of meat of the same species or the same animal taxonomic unit as the animal-derived product sample or unknown sample with a known assay. On the other hand, a predetermined reference spectrum can be obtained from different types of meat with a known assay. In one instance, the control can be chicken meat certified as 'halal'. Halal chicken meat can be a mixture of different parts of the chicken (i.e., breast, thigh, kidney, liver, etc.). In another instance, the halal chicken meat may come from one part of the chicken. A methylation spectrum can be obtained from the halal chicken meat as a predetermined reference spectrum (control), which can be used to determine whether the test sample (meat) is halal or non-halal by determining whether the methylation spectrum of the test sample is significantly similar to the predetermined reference spectrum. The methylation spectra of different types of halal meat from one animal species may be the same. The methylation spectra of different types of halal meat from different animal species may also be the same. In one instance, the control can be chicken meat certified as 'kosher'. Kosher chicken meat can be a mixture of different parts of the chicken (i.e., breast, thigh, kidney, liver, etc.). In another instance, the kosher chicken meat may come from one part of the chicken. A methylation spectrum can be obtained from the kosher chicken meat as a predetermined reference spectrum (control), which can be used to determine whether the test sample (meat) is kosher or non-kosher by determining whether the methylation spectrum of the test sample is significantly similar to the predetermined reference spectrum. The methylation spectra of different types of kosher meat from one animal species may be the same. The methylation spectra of different types of kosher meat from different animal species may also be the same.

[0081] There may be compilations of several predetermined reference spectra, and comparing the methylation spectrum of a test sample with the predetermined reference spectra in the compilation enables identification of a specific predetermined reference spectrum that is (significantly) similar to the methylation spectrum of the test sample, and then it can be confirmed whether the test sample was slaughtered by a single cut across the larynx severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea, and / or esophagus and / or bled to death. In one example, the predetermined reference spectra may include methylation spectra of different parts (i.e., chest, thigh, kidney, liver, shoulder, rib, intestine, etc.) of meat from animals (chickens, goats, cows, lambs, sheep, etc.) that have been slaughtered by a single cut across the larynx severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea, and / or esophagus and / or bled to death.

[0082] In particular, the set of predetermined reference methylation spectra according to any aspect of the present invention is specific for products derived from different test animals. That is, each predetermined reference methylation spectrum is specific for a product derived from a single animal. Thus, the set of predetermined reference methylation spectra may include many different predetermined reference methylation spectra from different parts of animals or several animals that have the same taxonomic unit as the test animal. For different taxonomic units of animals, there will also be different sets of predetermined reference methylation spectra, and the relevant set of predetermined reference methylation spectra unique to a taxonomic unit of an animal will depend on the taxonomic unit of the test animal.

[0083] According to a further aspect of the present invention, there is provided a method for assaying a sample of a product derived from a test animal, the method comprising the steps of: (a) Performing a method according to any aspect of the present invention to determine the test methylation spectrum of the test animal; and (b) Comparing the test methylation spectrum obtained from (a) with a reference methylation spectrum obtained from a control animal having the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal is reared under a particular type of animal husbandry; wherein a significant similarity of the test methylation spectrum from (a) compared to the reference methylation spectrum from the control animal indicates that the test animal has been reared under the same particular type of animal husbandry as the control animal; and / or wherein a difference of the test methylation spectrum from (a) compared to the reference methylation spectrum of the control animal indicates that the test animal has been reared under a different particular type of animal husbandry compared to the control animal; and wherein the test animal is a monogastric livestock.

[0084] In one instance, the special or specific determination of sample X can be based on the type of animal husbandry in which the test animals are kept. In Germany, this is referred to as the 'keeping method'. There are at least four types / conditions in which animals can be kept. These four levels of animal husbandry include stable housing (Stallhaltung), stable housing plus (StallhaltungPlus), outside climate (Auβenklima), and premium, which are also referred to as keeping methods 1, 2, 3, and 4, respectively. Animal products derived from animals reared under different animal husbandry conditions can result in different DNA methylation profiles. The specific type of animal husbandry can vary depending on the country in which the method according to any aspect of the present invention is implemented. Regardless of the different terms used in different countries to describe different specific animal husbandry practices, the general concept of the method according to any aspect of the present invention is the same and applicable in any of these countries.

[0085] For example, in Germany, different special types of livestock husbandry techniques applied to livestock and poultry can be referred to as ‘keeping methods’, and as mentioned above, are officially and industrially accepted as being divided into at least four types / conditions of animals that can be kept. These four levels of livestock husbandry include cage / barn keeping (Stallhaltung), cage / barn keeping upgraded (StallhaltungPlus), outdoor climate (Auβenklima) and premium. Similarly, in France, livestock and poultry may be labelled with ‘French Red Label’, ‘organic’ or other pictograms that show the method of keeping the animals before obtaining products derived from the animals. Among livestock and poultry in the UK, there is the Red Tractor Food Assurance accreditation scheme, which includes at least three levels of livestock husbandry, including Certified Standards, Enhanced Welfare and Free Range. Other existing labels in the UK include RSPCA Assured, which accredits specific animal welfare standards, and several organic meat accreditation schemes, such as the Organic Farmers and Growers Certification and the Soil Association Organic Standard. Examples of meat accreditation in the United States of America (USA) include those provided by the United States Department of Agriculture (USDA), which as examples include Carcass Quality and organic accreditation. The USDA has also approved some third-party accreditation schemes, such as those provided by the non-profit A Greener World, which includes Certified Animal Welfare Approved, which defines the accreditation of livestock husbandry related to animal welfare, and Certified Grassfed, which defines the accreditation of specific types of feed in livestock husbandry.

[0086] The term "predetermined reference spectrum" in the examples described can be used in the context of control animals, where the control animals have been properly characterized (i.e., based on the animal husbandry techniques for breeding or raising the control animals. The control animals may thus have been raised under stall keeping (Stallhaltung), enhanced stall keeping (StallhaltungPlus), outdoor climate (Auβenklima), and premium conditions. The set of predetermined reference spectra for the control animals can also include spectra of different samples obtained from different parts of the control animals (animals raised under at least one, two, three, or four animal husbandry conditions). For example, the set of predetermined reference spectra can include the spectrum of at least one egg, the spectrum of at least one type of meat (muscle, tissue, organ, etc.), the spectrum of at least one type of milk, etc. Each of these samples may have its own unique predetermined methylation reference spectrum, which also forms part of the set of predetermined reference spectra. The set can also include the predetermined reference spectrum for each of these animal-derived products specific to each of the four animal husbandry techniques).

[0087] A set of predetermined reference spectra can be prepared for different samples from animals proven to have been raised under at least one of these 4 animal husbandry techniques. Here again, there may be a set of predetermined reference spectra for each product derived from animals raised under at least one of these four animal husbandry techniques. For example, the set of predetermined reference spectra can include the spectrum of at least one egg, the spectrum of at least one type of meat (muscle, tissue, organ, etc.), the spectrum of at least one type of milk, etc., from animals raised under animal husbandry belonging to stall keeping (Stallhaltung). Each of these samples may have its own unique predetermined methylation reference spectrum, which also forms part of the set of predetermined reference spectra. A second set of predetermined reference spectra can include the spectrum of at least one egg, the spectrum of at least one type of meat (muscle, tissue, organ, etc.), the spectrum of at least one type of milk, etc., from animals raised under animal husbandry belonging to enhanced stall keeping (StallhaltungPlus). A third set of predetermined reference spectra can include the spectrum of at least one egg, the spectrum of at least one type of meat (muscle, tissue, organ, etc.), the spectrum of at least one type of milk, etc., from animals raised under animal husbandry belonging to outdoor climate (Auβenklima). A fourth set of predetermined reference spectra can include the spectrum of at least one egg, the spectrum of at least one type of meat (muscle, tissue, organ, etc.), the spectrum of at least one type of milk, etc., from animals raised under animal husbandry belonging to premium conditions. In one example, the set of predetermined reference spectra can include all four different sets. In yet another example, the sets can be based on different animal husbandry techniques found in a particular region, country, or geographical location. The number of sets of predetermined reference spectra can vary depending on the location where the method is implemented and what the animal rearing and / or animal husbandry techniques are implemented in the country or region).

[0088] The methylation profiles of different types of meat from animals grown under specific livestock husbandry techniques can be the same. The methylation profiles of different types of meat from different species of animals raised under specific livestock husbandry techniques can also be the same.

[0089] There may be compilations of several predefined reference profiles, and comparing the methylation profile of a test sample with the predefined reference profiles in the compilation can enable the identification of a specific predefined reference profile that is (significantly) similar to the methylation profile of the test sample, and then it can be confirmed whether the test sample has been raised under a particular livestock husbandry technique. In one example, the predefined reference profiles can include the methylation profiles of different parts (i.e., chest, thigh, kidney, liver, shoulder, rib, intestine, etc.) of the meat from animals (such as chickens, goats, cows, lambs, sheep, etc.) that have been raised under a particular livestock husbandry technique.

[0090] In particular, the set of predefined reference methylation profiles according to any aspect of the present invention is specific for products derived from different test animals. That is, each predefined reference methylation profile is specific for a product derived from a single animal. Thus, the set of predefined reference methylation profiles can include many different predefined reference methylation profiles from different parts of animals or several animals that belong to the same taxonomic unit as the test animal. For different animal taxonomic units, there will also be different sets of predefined reference methylation profiles, and the relevant set of predefined reference methylation profiles unique to the animal taxonomic unit will depend on the animal taxonomic unit of the test animal.

[0091] According to another aspect of the present invention, there is provided a method for assaying a sample of a product derived from a test animal, the method comprising the following steps: (a) Performing a method according to any aspect of the present invention to determine the test methylation profile of the test animal; and (b) Comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal that belongs to the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal is raised using conventional husbandry techniques; wherein the difference between the test methylation profile of (a) compared with the reference methylation profile from the control animal indicates that the test animal has been raised according to organic standards, and the product derived from the test animal is assayed as such.

[0092] According to one aspect of the present invention, there is provided a method for determining whether a product derived from a test animal has been raised or wild-caught, the method comprising the following steps: (a) Performing a method according to any aspect of the present invention to determine the test methylation profile of the product derived from the test animal; and (b) comparing the test methylation profile determined in (a) to one or more predetermined reference methylation profiles, wherein each of the one or more predetermined reference methylation profiles is specific for a wild-caught or farmed subject of the same taxonomic unit as the test animal; Wherein if the test methylation profile is significantly similar to one of the predetermined reference methylation profiles, then the product derived from the test animal is wild caught or farm raised.

[0093] According to a further aspect of the present invention, there is provided a method for determining a supplier from which a sample of a product derived from a test animal comes, the method comprising the steps of: (a) performing a method according to any aspect of the invention to determine a test methylation profile of a product derived from said test animal; and (b) comparing the test methylation profile determined in (a) to a set of predetermined reference methylation profiles of the same taxonomic unit of the test animal from which the product sample was derived, wherein each predetermined reference methylation profile is from a different reference animal and / or a different supplier, wherein if the test methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, then it is confirmed that the product sample derived from the test animal is from a first supplier and that a first reference animal having a predetermined reference profile was obtained from the first supplier; and / or wherein if the test methylation profile of (a) is different from one of the predetermined reference methylation profiles, it is confirmed that the test animal-derived product sample is not from a first supplier from which a first reference animal having a predetermined reference profile was obtained; and wherein the test animal is a monogastric livestock.

[0094] Methods according to aspects of the invention may therefore enable tracing of the original slaughterhouse, farm, product, supplier etc. from which the test animal material came, as well as products derived from the test animal.

[0095] The method according to said aspect of the invention can be used to identify unknown samples (i.e., animal-derived product samples) based on DNA methylation patterns. These DNA methylation patterns can then be compared with reference DNA methylation patterns to trace the animal-derived product samples back to the slaughterhouse or farmhouse from which they were produced, and then determine whether the unknown sample corresponds to an animal that has been slaughtered according to Kosher, Halal, non-kosher, or non-halal. In this way, a purchaser or consumer of meat can verify that the meat sold or promoted as Kosher or Halal is indeed what it claims to be.

[0096] As used herein, "supplier" refers to an entity that supplies animal-derived products. This can be considered a slaughterhouse in accordance with any aspect of the present invention, as a slaughterhouse is where animals are killed according to specific practices and from which different animal-derived products, including animal products and animal by-products, originate. A slaughterhouse or slaughter facility generally slaughters animals and then freezes, ages, and cuts the carcasses into various meat cuts, and packages those meat cuts for shipment to wholesalers and retailers. A slaughterhouse is also where non-food products originate. A supplier can be certified. "Certified supplier" means a supplier that has been evaluated for quality, commercial, technical, environmental, health, and safety considerations and has then been approved by the government or one or more third-party agencies. In one example, a supplier may be certified as "halal". This means that all animal-derived products supplied by the certified supplier will be certified as "halal". In another example, a supplier may be certified as "kosher". This means that all animal-derived products supplied by or originating from the certified supplier will be certified as "kosher". Where an animal is slaughtered by a single cut across the throat severing both carotid arteries, both jugular veins, both vagus nerves, the trachea, and / or the esophagus, the supplier is certified as kosher or halal. Additionally, or alternatively, where an animal is exsanguinated, the supplier is certified as kosher or halal. The animal can be selected from cattle, sheep, goats, camels, chickens, geese, ducks, and turkeys.

[0097] The term "predetermined reference profile" as used in accordance with this aspect of the present invention can be obtained from a control subject. For example, the control can be a piece of meat from an animal that has been slaughtered by a certified supplier. A group of predetermined reference profiles can be prepared for each certified supplier, where each predetermined profile is unique for each animal that has been slaughtered by a certified supplier and / or each part of the animal (i.e., tissue, muscle, blood, skin) has its own unique predetermined methylation reference profile, which also forms part of the group of predetermined reference profiles. Different certified suppliers can then have different groups of predetermined reference profiles. For example, each group may be specific to a single animal, where each reference profile may be specific to a part of the animal from which genomic material was extracted. When a test methylation profile is obtained from a sample of an animal-derived product of unknown origin, it is then compared to the different groups of predetermined reference profiles to trace the actual animal from which the animal-derived product sample originated. If the test methylation profile is significantly similar to one of the predetermined reference profiles, the origin of the animal-derived product sample can be determined, and the sample can be given an appropriate certification, or the sample can be certified for its quality. If the test methylation profile is found not to be significantly similar to any of the predetermined reference profiles, it is confirmed that the animal-derived product sample does not originate from the list of certified suppliers and cannot be given a certification or cannot be certified for its quality.

[0098] For example, the control can be a kind of meat that belongs to the same species or the same animal taxonomic unit as the animal-derived product sample or unknown sample with a known assay or food quality assay. On the other hand, a predetermined reference spectrum can be obtained from different types of meat with known food quality assays. In one example, the control can be chicken that is 'halal' as an assay from a supplier of a first assay. The halal chicken can be a mixture of different parts of the chicken from a single chicken (i.e., breast, thigh, kidney, liver, etc.). In another example, the halal chicken may be a mixture of different parts of the chicken from several chickens from a supplier of a first assay (i.e., breast, thigh, kidney, liver, etc.). In yet another example, the halal chicken may come from one part of a single chicken from a supplier of a first assay. A methylation spectrum can be obtained from the halal chicken as a predetermined reference spectrum (control), which can be used to determine whether the test sample (meat) is halal or non-halal by determining whether the test sample comes from a supplier of a first assay. The methylation spectra of different types of halal meat from one animal species may be the same. The methylation spectra of different types of halal meat from different animal species may also be the same. In another example, the control can be chicken that is 'kosher' as an assay. The kosher chicken can be a mixture of different parts of the chicken from a single chicken from a supplier of a second assay (i.e., breast, thigh, kidney, liver, etc.). In another example, the kosher chicken may be a mixture of different parts of the chicken from several chickens from a supplier of a second assay (i.e., breast, thigh, kidney, liver, etc.). In yet another example, the kosher chicken may come from one part of a single chicken from a supplier of a second assay. A methylation spectrum can be obtained from the kosher chicken as a predetermined reference spectrum (control), which can be used to determine whether the test sample (meat) is kosher or non-kosher by first determining whether the test sample comes from a supplier of a second assay. The methylation spectra of different types of kosher meat from one animal species may be the same. The methylation spectra of different types of kosher meat from different animal species may also be the same.

[0099] There may be compilations of several predetermined reference spectra, and comparing the methylation spectrum of a test sample with the predetermined reference spectra in the compilation can enable identification of a specific predetermined reference spectrum that is (significantly) similar to the methylation spectrum of the test sample, and then the test sample can be traced back to its origin or supplier and thereby it can be inferred that the quality verification of the test sample is the quality verification of the predetermined reference spectrum. In one example, the predetermined reference spectra can include methylation spectra of different parts (i.e., chest, thigh, kidney, liver, shoulder, rib, intestine, etc.) of meat from a single animal (chicken, goat, cow, lamb, sheep, etc.) or at least two animals of the same species or at least two animals of different species. The different unique methylation spectra are reference epigenetic characteristics of meat from a specific source

[0100] In particular, the set of predetermined reference methylation spectra according to any aspect of the present invention is specific for products derived from different test animals. That is, each predetermined reference methylation spectrum is specific for a product derived from a single animal from a unique supplier. Thus, the set of predetermined reference methylation spectra can include many different predetermined reference methylation spectra of different parts of a single animal from a unique supplier, many different predetermined reference methylation spectra of different animals from a unique supplier, and many different predetermined reference methylation spectra of different parts of different animals from different suppliers. For different animal taxa, there will also be different sets of predetermined reference methylation spectra, and the relevant set of predetermined reference methylation spectra unique to an animal taxon will depend on the animal taxon of the test animal.

[0101] In particular, a first supplier according to any aspect of the present invention is verified, and a product sample derived from a test animal has the same verification as a first reference animal from which a significantly similar predetermined reference spectrum is obtained.

[0102] More particularly, in the case where an animal is slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea and / or the esophagus, a first supplier according to any aspect of the present invention is verified as kosher or halal. Additionally, or alternatively, in the case where an animal is exsanguinated, the first supplier is verified as kosher or halal.

[0103] A method for determining whether a product sample derived from a test animal is derived from an animal suitable for selective breeding, the method comprising the steps of: (a) Performing a method according to any aspect of the present invention to determine the test methylation spectrum of the test animal; and (b) Compare the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal of the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal comprises at least one phenotypic trait of interest for selective breeding, and wherein a significant similarity of the test methylation profile from (a) compared to the reference methylation profile from the control animal indicates that the test animal is suitable for selective breeding; and / or wherein a difference of the test methylation profile from (a) compared to the reference methylation profile from the control animal indicates that the test animal is not suitable for selective breeding; and wherein the test animal is a monogastric livestock.

[0104] In particular, methylation profiling can be used to determine whether a test animal contains a phenotypic trait of interest or will develop a phenotypic trait of interest at a later stage during the early stage of growth of the test animal. Thus, the method according to the aspect of the present invention can be used to determine whether a test animal will develop a phenotypic trait of interest at an early stage, rather than raising all animals to adulthood to search for a phenotypic trait of interest. This can save the time and resources typically required to raise animals to adulthood. The method according to the aspect of the present invention provides a method for tracking the heritability of epigenetic patterns over time.

[0105] The term "selective breeding" as used herein, also referred to as 'animal selection', refers to the process of animal breeding performed by humans to selectively develop specific phenotypic traits (characteristics) by selecting generally which animal males and females will sexually reproduce and jointly have offspring. With respect to domesticated animals, this is referred to as a breed. In animal breeding, techniques such as inbreeding, line breeding, and outcrossing are used. These phenotypic traits, also referred to herein as 'one or more phenotypic traits of interest', refer to different variants of the phenotypic characteristics of an organism that are desired to be present in the selectively bred animal. In particular, the phenotypic traits of interest in the selectively bred animal are valuable traits that the breeder desires to be present in the selectively bred animal. Some examples of phenotypic traits of interest in cattle may be high milk production, high-quality meat, etc., and for chickens, the phenotypic traits of interest may include eggs, meat, and high production of new young poultry for further reproduction. Other phenotypic traits may include feed efficiency (the amount of feed required to produce a limited amount of meat / eggs), meat quality characteristics (texture, taste, intramuscular fat), disease resistance, lack of physiological problems (bone, joint, or breast meat problems), etc.

[0106] According to this aspect of the invention, the term reference methylation profile may be a methylation profile set, wherein each methylation profile in the set is associated with at least one specific phenotype of interest. For example, in a methylation profile set of chickens, there may be a reference methylation profile for chickens that lay many eggs, there may be a reference methylation profile for chickens with high-quality meat, and there may be a reference methylation profile for chickens that can reproduce and produce many offspring. Thus, the reference methylation profile set of chickens may include a reference methylation profile for each of these phenotypes of interest.

[0107] One or more of the preselected methylation sites in (a) are methylation sites related to tissue-specific gene expression, preferably wherein the preselected methylation sites are related to the gene expression of a particular tissue.

[0108] The tissue may be selected from (i) metabolic tissues, such as intestinal tissue, which is preferably ileum or jejunum, (ii) muscle tissue, (iii) skin tissue, and (iv) organ tissues, which are preferably liver and / or pancreatic tissues.

[0109] According to yet another aspect of the invention, there is provided a DNA methylation-based array for performing the method according to any aspect of the invention. Brief Description of the Drawings Figure 1 Is a PCA using the positions of differentially methylated determined by the sequencing in Example 3, wherein clustering based on the feeding method is shown.

[0111] Figure 2 Is the result of a PCA plot using the Beadchip data of Example 3 to distinguish three feeding conditions.

[0112] Figure 3 Is a graph showing the first two principal components of the differentially methylated analysis CpGs, revealing meaningful sample clustering of halal and non-halal samples.

[0113] Figure 4 Is a PCA plot using the Beadchip data to distinguish halal and non-halal groups.

[0114] Figure 5 Is a graph showing the first two principal components of the differentially methylated analysis CpGs, revealing meaningful clustering of samples from different geographical origins.

[0115] Figure 6 Is a PCA plot using the Beadchip data to distinguish different locations. Examples

[0116] The foregoing describes preferred embodiments which, as will be understood by those skilled in the art, may undergo changes or modifications in design, construction or operation without departing from the scope of the claims. Such changes are intended to be included within the scope of the claims, for example.

[0117] Example 1 Geographical origin traceability of salmon meat Fillet muscle tissue was excised from two-year-old Atlantic salmon slaughtered at the sale weight. Six replicate samples each were obtained from salmon reared in Canada, Chile or Norway.

[0118] DNA extraction DNA was extracted using the PureLink Genomic DNA Isolation Minikit (Invitrogen) according to the manufacturer's instructions, including RNase treatment. The DNA quantity was measured by PicoGreen assay and the DNA quality was evaluated via NanoDrop (Thermo Scientific) to ensure an A260 / 280 ratio ≤ 1.8. Then, a small amount of the sample was also analyzed on an agarose gel to ensure that each sample contained high molecular weight DNA.

[0119] Bisulfite conversion and BeadChip analysis The genomic DNA samples were then subjected to bisulfite conversion using the EZ DNA Methylation-Gold TM Kit (ZymoResearch). The methylation levels were then quantified using our custom methylation BeadChip kit (Illumina), which can quantitatively analyze over 50,000 methylation sites throughout the genome at single nucleotide resolution.

[0120] Data processing: Custom chip array data processing was performed in R version 4.1.2 using sesame version 1.14.2. The DNA methylation level at each locus was calculated as a methylation β-value. The β-value is defined as the methylated signal / (methylated signal + unmethylated signal). It can be calculated using the getBetas function. The SeSAMe pipeline (Zhou et al., 2018) was used to generate normalized β-values and for quality control. pOOBAH was used for low-intensity detection calling and generation (based on p-values). Background subtraction based on normal-exponential deconvolution using out-of-band probes noob (Triche et al., 2013) was also implemented, and optionally with additional bleed-through subtraction.

[0121] Differential methylation analysis on sample groups was also performed using Sesame.

[0122] The results showed that the methylation profiles of salmon meat samples from Canada, Chile, or Norway had position-dependent methylation profiles, i.e., samples from the same country were similar to each other and could be distinguished from samples from different countries.

[0123] Example 2 Confirmation of Halal Verification of Broiler Chicken Meat Breast muscle samples were collected from 6 conventionally slaughtered broiler chickens and 6 broiler chickens slaughtered by the method of Halal verification. Halal slaughter of chickens is defined as a single cut across the larynx that severs both carotid arteries, both jugular veins, both vagus nerves, the trachea, and the esophagus and results in the death of the chicken by bleeding.

[0124] DNA extraction, bisulfite conversion, BeadChip analysis, quality control, data processing, and differential methylation analysis were as outlined in Example 1.

[0125] The results showed that the breast meat from broiler chickens slaughtered by the conventional method had different CpG methylation profiles compared to broiler chickens slaughtered by the method of Halal verification.

[0126] Example 3 Assessment of Pork to Confirm the Antibiotic Treatment Status in Pigs Loin samples were collected from the carcasses of 6 pigs at market weight treated orally with tylosin and 6 pigs of similar age and weight raised without antibiotics.

[0127] DNA extraction, bisulfite conversion, BeadChip analysis, quality control, data processing, and differential methylation analysis were as outlined in Example 1.

[0128] The results showed that pigs treated orally with tylosin had different methylation profiles in loin DNA when compared to pigs raised in antibiotic-free production.

[0129] Example 4 Rearing Conditions Wet-Lab Methods Breast meat from broiler chickens was obtained from three different German supermarkets to obtain as many replicate samples as possible of the German verification standard called rearing method. There are four special rearing method categories for broiler chicken meat in Germany, which include Rearing Method 1 (cage / barn rearing), 2 (upgraded cage / barn rearing), 3 (outdoor climate), and 4 (premium). From Rearing Methods 1 - 4, the rearing conditions increasingly improve in line with the animal welfare requirements being evaluated. For example, the space requirement for the largest 39 kg chicken in cage / barn rearing is 2Increased to 21 kg / m in the premium category 2 Additional requirements defining each rearing method category include the genetic lineage of the broiler chickens, the duration of rearing (the last 81 days in rearing methods 3 and 4), the amount of enrichment and outdoor activity access available, and the type of ingredients fed to the animals.

[0130] Based on the sample collection, two replicate samples of breast meat from rearing method 2 from each of three grocery stores for a total of six replicate samples were obtained, three replicate samples of breast meat from rearing method 2 from one grocery store were obtained, and three replicate samples of breast meat from rearing method 4 from one grocery store were obtained. Unfortunately, it was not possible to obtain clearly labeled rearing method 1 samples, so that category was excluded. In total, there were 12 samples, including three of the four available rearing method categories (Table 1).

[0131] Table 1. Sample identification of 12 breast meat samples obtained from three of four special rearing method conditions. DNA sample id Sequencing sample name Type Supermarket Rearing conditions Replicate sample CHKN-0015 SR_15 Frozen Aldi Rearing method 2 Sample 1 CHKN-0016 SR_16 Frozen Aldi Rearing method 2 Sample 2 CHKN-0018 SR_18 Frozen Lidl Rearing method 2 Sample 1 CHKN-0019 SR_19 Frozen Lidl Rearing method 2 Sample 2 CHKN-0021 SR_21 Frozen Rewe Rearing method 2 Sample 1 CHKN-0022 SR_22 Frozen Rewe Rearing method 2 Sample 2 CHKN-0024 SR_24 Frozen Aldi Rearing method 3 Sample 1 CHKN-0025 SR_25 Frozen Aldi Rearing method 3 Sample 2 CHKN-0026 SR_26 Frozen Aldi Rearing method 3 Sample 3 CHKN-0027 SR_27 Frozen Lidl Rearing method 4 Sample 1 CHKN-0028 SR_28 Frozen Lidl Rearing method 4 Sample 2 CHKN-0029 SR_29 Frozen Lidl Rearing method 4 Sample 3

[0132] DNA extraction DNA was extracted using the PureLink Genomic DNA Isolation Minikit kit (Invitrogen) according to the manufacturer's instructions, including RNase treatment. The DNA quantity was measured by PicoGreen assay and the DNA quality was evaluated via NanoDrop (Thermo Scientific) to ensure an A260 / 280 ratio ≤ 1.8. Then a small amount of the samples was also analyzed by automated electrophoresis on a TapeStation (Agilent) to ensure that each sample contained high molecular weight DNA.

[0133] Sequencing analysis Genomic DNA was purified from breast tissue samples using the DNeasy Blood and Tissue Kit (Qiagen) and quantified using PicroGreen or NanoDrop TM 2000.

[0134] Genomic DNA (500 ng) from breast tissue samples was used to prepare libraries for whole-genome bisulfite sequencing (WGBS). Sequencing of the libraries was performed by a third party on the NovaSeq platform, which generated 125 GB of data per sample with 20X coverage.

[0135] Bisulfite conversion and BeadChip analysis Run sample subsets using beadchip. Two samples per rearing condition were analyzed in triplicate runs.

[0136] Table 2. Sample identification of six chicken breast samples obtained from three of four special rearing methods. DNA sample ID Type Supermarket Rearing conditions CHKN-0009 Fresh Rewe 2 CHKN-0022 Frozen Rewe 2 CHKN-0011 Fresh Aldi 3 CHKN-0025 Frozen Aldi 3 CHKN-0013 Fresh Lidl 4 CHKN-0029 Frozen Lidl 4

[0137] Genomic DNA samples were then subjected to bisulfite conversion using the EZ DNA Methylation-Gold TM Kit (ZymoResearch). Methylation levels were then quantified using our custom methylation BeadChip kit (Illumina), which can quantitatively analyze over 50,000 methylation sites throughout the genome at single nucleotide resolution. After bisulfite conversion, samples were processed through a three-day workflow, including sample amplification, fragmentation, precipitation, hybridization to the BeadChip, and X-stain according to the Infinium HD Methylation Assay (Illumina, Document #15019519v07), and then imaged on the iScan (Illumina), where intensity files for calculating β-values were generated.

[0138] Data processing: Processing of sequencing data: Using the assembled version 5.0 of the chicken (Gallus gallus) genome as the reference sequence, use BSMAP 1 Version 2.5 to trim and map sequencing reads. After using picard 2 to remove duplicates (deduplication), use the Python script (methratio.py) distributed with the BSMAP package to determine the methylation ratio. For all further analyses, only CpGs covered by at least ten reads were considered, which resulted in 6,458,063 CpG sites (not provided).

[0139] Differential methylation analysis Perform differential methylation analysis between different rearing method groups using MethylKit 3 (version 1.12.0). MethylKit uses logistic regression to calculate p-values and the sliding linear model method 4 to adjust the p-values to q-values.

[0140] CpG sites with an FDR below 0.05 and a methylation change greater than 25% between groups were considered significantly differentially methylated positions (DMPs), resulting in 201,246 CpG sites (not provided). methylKit generates a 'prcomp' object, which can be used to extract and plot the principal components. Principal component analysis (PCA) is a dimensionality reduction method that can transform a large dataset into a few principal components. The first few principal components usually retain most of the variation present in the dataset and are useful for highlighting the grouping structure in the data.

[0141] Processing of Beadchip data: Custom chip array data processing was performed in R version 4.1.2 using sesame version 1.14.2. The DNA methylation level at each locus was calculated as the methylation beta-value. The beta-value is defined as the methylated signal / (methylated signal + unmethylated signal). It can be calculated using the getBetas function. The SeSAMe pipeline (Zhou et al., 2018) was used to generate normalized beta-values and for quality control. pOOBAH was used for low-intensity detection calling and generation (based on p-values). Background subtraction based on normal-exponential deconvolution using out-of-band probes noob was also implemented (Triche et al., 2013), and optionally with additional bleed-through subtraction. After obtaining the beta-value table, probes related to the 'feeding condition' subclass were selected, and probes with NA were removed. PCA plots were drawn for 1517 loci (Tables 3a - 3g).

[0142] Table 3a. 1517 CpG sites on the methylation array for determining the feeding conditions of chickens Table 3b. 1517 CpG sites on the methylation array for determining the feeding conditions of chickens Table 3c. 1517 CpG sites on the methylation array for determining the feeding conditions of chickens Table 3d. 1517 CpG sites on the methylation array for determining the feeding conditions of chickens Table 3e. 1517 CpG sites on the methylation array for determining the feeding conditions of chickens Table 3f. 1517 CpG sites on the methylation array for determining the feeding conditions of chickens Table 3g. 1517 CpG sites on the methylation array for determining the rearing conditions of chickens Results When running the second PCA using the differentially methylated positions, the clustering based on the rearing method, also known as the rearing conditions, became even clearer ( Figure 1 ).

[0143] Run PCA plots using Beadchip data to distinguish three rearing conditions ( Figure 2 ).

[0144] In summary, the plot of the first two principal components of the CpGs revealed a meaningful clustering of the samples processed by sequencing and beadchip data. The CpG sites were able to effectively cluster different groups in a meaningful way. We could effectively reproduce the results of the rearing method application using the beadchip.

[0145] Example 5 Halal 25 wet laboratory method halal and non - halal chicken breasts were purchased from five different suppliers. The suppliers of halal meat included Pasar, ZAC Butchery, Hego premium, RedMart, and Keesong, while non - halal meat was purchased from Pasar, Tegel, Farm fresh, Ryan, and AW’s in Singapore. Halal Non-Halal Brand Brand Pasar Pasar ZAC Butchery Tegel Hego premium Farm fresh RedMart yan Keesong AW's

[0146] DNA extraction, sequencing analysis, bisulfite conversion, and BeadChip analysis and data processing were performed according to Example 4.

[0147] After obtaining the β - value table of the samples, the probes related to the 'halal' subclass were selected, and the probes with NA were removed. PCA plots were drawn for 506 sites (Table 4).

[0148] Table 4a. 506 CpG sites on the DNA methylation array for determining halal or non - halal Table 4b. 506 CpG sites on the DNA methylation array for determining halal or non - halal Table 4c. 506 CpG sites on the DNA methylation array for determining halal or non - halal Results Plots of the first two principal components of CpGs before and after differential methylation analysis revealed meaningful clustering of the samples. Positions of differentially methylated (DMPs) were able to effectively cluster the halal and non - halal groups in a meaningful way.

[0149] Plots of the first two principal components of CpGs in differential methylation analysis revealed meaningful clustering of the samples ( Figure 3 ).

[0150] PCA plots were run using Beadchip data to distinguish halal and non - halal groups ( Figure 4 ).

[0151] In summary, plots of the first two principal components of CpGs revealed meaningful clustering of samples processed by sequencing and Beadchip data. CpG sites were able to effectively cluster different groups in a meaningful way. We could effectively replicate the results of halal applications using Beadchip.

[0152] Example 6 Traceability of origin - Identification of differentially methylated CpG sites in chickens To identify differentially methylated CpG sites in chickens, the function "calculateDiffMeth" from the R package MethylKit was used on degenerate representative bisulfite sequencing (RRBS) data.

[0153] Materials and methods Different service laboratories in the respective sample - source countries provided isolated and purified genomic DNA from pectoral muscle tissue. Quality was checked using the 2200 TapeStation (Agilent).

[0154] RRBS library preparation was performed as described in the Zymo - Seq RRBS TM Library Kit User Manual Ver.1.0.0. Quality control was performed and sample concentrations were measured on the 2200 TapeStation (Agilent). Multiplexed samples were sequenced on the HiSeq 4000 system (Illumina).

[0155] The read lengths were quality trimmed using trimmomatic version 0.38 and mapped to the Red Jungle Fowl genome assembly version 5.0 using BSMAP 2.90. The methylation ratios were calculated using a python script (methratio.py) distributed with the BSMAP package. All CpG sites related to sex chromosomes and CpG sites overlapping with SNPs in the Red Jungle Fowl genome 25 were filtered out from further analysis. Prior to the said analysis, the data was filtered for SNPs and a coverage cut-off of at least 10 was applied for each CpG site. Differential methylation analysis was performed using the R package MethylKit (Akalin et al. (2012), Genome Biology, 13(10), R87).

[0156] MethylKit uses logistic regression to calculate p-values and a sliding linear model method to adjust the p-values to q-values. CpG sites with an FDR below 0.05 and a methylation change greater than 25% between groups were considered significantly differentially methylated positions (DMPs), resulting in 8500 CpG sites.

[0157] MethylKit generates a 'prcomp' object which can be used to extract and plot the principal components. The use of MethylKit is disclosed in Akalin A et al. Genome Biol. 2012; 13(10):R87. Principal component analysis (PCA) is a dimensionality reduction method which can transform a large dataset into a few principal components. The first few principal components usually retain most of the variation present in the dataset and are useful for highlighting the grouping structure in the data.

[0158] Bisulfite conversion and BeadChip analysis and the processing of Beadchip data were performed according to Example 3.

[0159] After obtaining the β-value table of the samples, the probes related to the 'origin traceability' subclass were selected and the probes with NA were removed. Based on the remaining probes, the variance of each probe was calculated. A PCA plot was also created using the top 1000 probes (out of 12466 probes). (Table 5) Table 5a The top 1000 probes (out of 12466 probes) used in the chip for determining geographical origin Table 5b The top 1000 probes (out of 12466 probes) used in the chip for determining geographical origin Table 5c The top 1000 probes (out of 12466 probes) used in the chip for determining geographical origin Table 5d Top 1000 probes (out of 12,466 probes) used in the chip for determining geographical origin Table 5e Top 1000 probes (out of 12,466 probes) used in the chip for determining geographical origin Results Plots of the first two principal components of CpGs before and after differential methylation analysis revealed meaningful clustering of the samples. Positions of differential methylation (DMPs) were able to effectively cluster different locations in a meaningful way.

[0160] Plots of the first two principal components of differential methylation analysis of CpGs revealed meaningful clustering of the samples ( Figure 5 ).

[0161] PCA plots were run using Beadchip data to distinguish different locations ( Figure 6 ).

[0162] In summary, plots of the first two principal components of CpGs revealed meaningful clustering of samples processed by sequencing and Beadchip data. CpG sites were able to effectively cluster different locations in a meaningful way. Results of the origin traceability application using Beadchip were shown to be reproducible.

Claims

1. A method for detecting DNA methylation of genomic material contained in a biological sample obtained from a product derived from a test animal and / or determining a test methylation profile of genomic material contained in a biological sample obtained from a product derived from a test animal, the method comprising the steps of: - contacting a genomic material sample from a product derived from the test animal with a DNA methylation array specific to the test animal species, wherein the test animal is a monogastric livestock.

2. A method for identifying the geographical origin of a product derived from a test animal, the method comprising the steps of: (a) performing the method according to claim 1 to determine the test methylation profile of the product derived from the test animal; and (b) comparing the test methylation profile determined in (a) with one or more predetermined reference methylation profiles, wherein each of the one or more predetermined reference methylation profiles is specific to a particular geographical origin of a subject having the same taxonomic unit as the test animal; wherein if the test methylation profile is significantly similar to one of the predetermined reference methylation profiles, the product derived from the test animal has a geographical origin similar to that of the subject having the predetermined reference methylation profile; and / or wherein if the test methylation profile is different from one of the predetermined reference methylation profiles, the product derived from the test animal has a geographical origin different from that of the subject having the predetermined reference methylation profile, wherein the test animal is a monogastric livestock.

3. The method according to claim 2, wherein the particular geographical origin is a geographical location regarded as a habitat where the test animal was born, hatched and / or raised, or at least raised for a significant period of its life.

4. An in vitro method for predicting the biological age of a test animal from which a product is derived, the method comprising the steps of: (a) performing the method according to claim 1 to determine the test methylation profile of the test animal; and (b) comparing the test methylation profile from (a) with the methylation profile from an age-related reference sample, thereby establishing an epigenetic age and predicting the biological age of the test animal from which the product is derived; and wherein the test animal is a monogastric livestock.

5. A method for determining whether a test animal from which a product is derived has been treated with at least one antibiotic and / or veterinary chemical and / or is currently undergoing such treatment, the method comprising: (a) performing the method according to claim 1 to determine the test methylation profile of the test animal; and (b) comparing the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal or a product derived from a control animal, wherein the control animal has not been treated with at least one antibiotic and / or veterinary chemical and / or is not currently undergoing such treatment, wherein a difference in the tested methylation profile of (a) as compared to the reference methylation profile from the control animal indicates that the test animal has been treated with at least one antibiotic and / or veterinary chemical and / or is currently undergoing such treatment; and / or wherein a significant similarity of the tested methylation profile of (a) as compared to the reference methylation profile indicates that the test animal has not been treated with at least one antibiotic and / or veterinary chemical and / or is not currently undergoing such treatment; and wherein the test animal is a monogastric livestock.

6. A method for determining whether a test animal from which a product is derived has been treated with at least one antibiotic and / or is currently undergoing such treatment, and if so, determining the particular class of antibiotic with which the test animal has been treated and / or is currently undergoing such treatment, the method comprising: (a) performing the method according to claim 1 to determine the tested methylation profile of the test animal; and (b) comparing the tested methylation profile obtained from (a) with one or more predetermined reference methylation profiles, wherein each predetermined reference methylation profile is from a different animal having the same taxonomic unit as the test animal, and each different animal has been treated with a different class of antibiotic, wherein if the tested methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, it is confirmed that the test animal from which the product is derived is being treated with the same class of antibiotic as the animal having the predetermined reference profile and has been treated with the particular class of antibiotic and / or is currently undergoing such treatment; and / or wherein if the tested methylation profile of (a) is significantly different from one of the predetermined reference methylation profiles, it is confirmed that the test animal from which the product is derived is not being treated with the same class of antibiotic as the animal having the predetermined reference profile and has not been treated with the particular class of antibiotic and / or is not currently undergoing such treatment; and wherein the test animal is a monogastric livestock.

7. The method according to claim 5 or 6, wherein - The special category of antibiotics is aminoglycosides, aminocyclitols, aminoglycosides, ansamycins, β-lactams, carbacephems, carbapenems, cephalosporins, chloramphenicol, fluoroquinolones, glycopeptides, glycylcyclines, ketolides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, nitroimidazoles, oxazolidinones, penicillins, phosphorus-containing antibiotics; and / or - the veterinary chemical is anthelmintic, antiviral, feed additive, disinfectant, glutaraldehyde and / or formalin.

8. A method for assaying a product sample derived from a test animal, the method comprising the steps of: (a) contacting genomic material contained in a biological sample from a product derived from the test animal with a DNA methylation-based array to determine the tested methylation profile of the test animal; and (b) comparing the tested methylation profile obtained from (a) with a reference methylation profile obtained from a control animal having the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal is not slaughtered by a single cut across the larynx severing the bilateral carotid arteries, bilateral jugular veins, bilateral vagus nerves, trachea and / or esophagus, and / or the control animal does not die from bleeding wherein a difference between the tested methylation profile of (a) and a reference methylation profile from the control animal indicates that the tested animal has been slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea, and / or the esophagus, and / or that the tested animal has bled to death, and the product derived from the tested animal is so certified; and / or wherein a significant similarity between the tested methylation profile of (a) and a reference methylation profile from the control animal indicates that the tested animal has not been slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea, and / or the esophagus, and / or that the tested animal has bled to death, and the product derived from the tested animal is so certified; and wherein the tested animal is a monogastric livestock.

9. A method for determining the supplier from which a sample of a product derived from a tested animal is obtained, the method comprising the steps of: (a) performing the method according to claim 1 to determine a tested methylation profile of the product derived from the tested animal; and (b) comparing the tested methylation profile determined in (a) with a set of predetermined reference methylation profiles of the same taxonomic unit of the tested animal from which the product sample is derived, wherein each predetermined reference methylation profile is from a different supplier, wherein if the tested methylation profile of (a) is significantly similar to one of the predetermined reference methylation profiles, it is confirmed that the product sample derived from the tested animal is from a first supplier, and a first reference animal with the predetermined reference profile is obtained from the first supplier; and / or wherein if the tested methylation profile of (a) is different from one of the predetermined reference methylation profiles, it is confirmed that the product sample derived from the tested animal is not from the first supplier, and a first reference animal with the predetermined reference profile is obtained from the first supplier; and wherein the tested animal is a monogastric livestock.

10. A method for certifying a product sample derived from a tested animal, the method comprising the steps of: (a) performing the method according to claim 1 to determine a tested methylation profile of the tested animal; and (b) comparing the tested methylation profile obtained from (a) with a reference methylation profile obtained from a control animal of the same taxonomic unit as the tested animal from which the product sample is derived, wherein the control animal is reared under a known specific type of animal husbandry, wherein a significant similarity between the tested methylation profile of (a) and the reference methylation profile from the control animal indicates that the tested animal has been reared under the same specific type of animal husbandry as the control animal and the product derived from the tested animal is so certified; and / or wherein a difference between the tested methylation profile of (a) and the reference methylation profile of the control animal indicates that the tested animal has been reared under a different specific type of animal husbandry compared to the control animal; and wherein the tested animal is a monogastric livestock.

11. A method for determining whether a product sample derived from a tested animal is derived from an animal suitable for selective breeding, the method comprising the steps of: (a) Perform the method according to claim 1 to determine the test methylation profile of the test animal; and (b) Compare the test methylation profile obtained from (a) with a reference methylation profile obtained from a control animal of the same taxonomic unit as the test animal from which the product sample is derived, wherein the control animal comprises at least one phenotype of interest for selective breeding, wherein a significant similarity of the test methylation profile of (a) compared to the reference methylation profile from the control animal indicates that the test animal is suitable for selective breeding; and / or wherein a difference of the test methylation profile of (a) compared to the reference methylation profile from the control animal indicates that the test animal is not suitable for selective breeding; and wherein the test animal is a monogastric livestock.

12. The method according to any one of the preceding claims, wherein the monogastric livestock comprises terrestrial livestock and aquatic livestock.

13. The method according to any one of the preceding claims, wherein the monogastric livestock is selected from pigs, horses, donkeys, rabbits, and mules and / or poultry and vertebrate fish selected from chickens, turkeys, ducks, geese, and quails.

14. The method according to any one of the preceding claims, wherein the animal-derived product is meat, muscle, at least one organ, milk, collagen, feathers, blood, and / or bone.

15. A DNA methylation-based array for performing the method according to any one of claims 1-14.

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