Multi-species chip for detecting DNA-methylation

Abstract

The present invention relates to a DNA methylation-based array comprising at least: a first plurality of different positions, each position having at least one probe molecule comprising a nucleic acid sequence complementary to a CpG site of a first plurality of CpG sites from a first animal species; and a second plurality of different positions, each position having at least one probe molecule comprising a nucleic acid sequence complementary to a CpG site from a second plurality of CpG sites of a second animal species wherein the first and second animal species are each independently selected from the group consisting of viruses, mammals, birds, and aquatic animals, and the mammal is at least one livestock or animal cell line; -said birds are at least one poultry; and-said aquatic animal is at least one crustacean, cephalopoda or fish, and wherein said first plurality of CpG sites comprises at least 1000 CpG sites of said first animal species; and the second plurality of CpG sites comprises at least 1000 CpG sites of the second animal species.

Description

Field of the Invention

[0001] The present invention relates to a multi-species chip. In particular, the multi-species chip is a methylation-based array that includes probes complementary to nucleic acids having CpG sites specific for more than one animal species. The animal species can be from any animal class selected from invertebrates and vertebrates, where the vertebrates can be mammals, birds, fish, etc. Background of the Invention Epigenetics is the study of heritable traits caused by mechanisms other than changes in the underlying DNA sequence. In other words, epigenetic marks "orchestrate" our genes. Epigenetic marks can be chemical marks (e.g., methylation), protein-based marks (e.g., histones), or a combination of both. During development and cell differentiation, DNA methylation is dynamic, but some DNA methylation patterns can be maintained, accumulated, and / or inherited as a form of epigenetic memory to the next generation. Those changes may be responsible for heritable changes in gene activity, as DNA methylation events have been shown to be regulatory mechanisms associated with gene silencing, expression, chromatin remodeling, or imprinting. Epigenetics is attractive for animal breeding because it can identify the causal relationships and heritability of complex traits and diseases. DNA methylation patterns are modified during an individual's life by environmental forces such as diet, stress, drugs, or pollution, among others. Some environments are more likely to increase certain methylation patterns, and these patterns can contribute to epigenetic and / or phenotypic variation between individuals.

[0003] Epigenetic techniques can thus be used, for example, to study the correlation between the epigenome and a specific phenotype, or to develop composite biomarkers for differentiating between different environmentally treated groups, as is currently done in Epigenome-Wide Association Studies (EWAS).

[0004] 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 to uracil, leaving methylated and hemimethylated cytosine nucleotides unchanged (Stevens et al., 2013). Next-generation sequencing is performed, and the resulting 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.

[0005] DNA-methylation-based arrays allow for high-throughput and robust methods to determine semi-quantitative / quantitative DNA-methylation information from small samples of extracted DNA of interest. These custom designed arrays use Illumina iScan and Infinium platform technologies, which allow for hundreds of thousands of different bead types covalently bound to DNA-methylation probes, for example, 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 an Illumina Infinium array chip. Once on the chip, the DNA hybridizes to the beads at each CpG site, enabling the methylation status of each site to be specifically detected by single nucleotide extension.

[0006] So far, DNA methylation-based arrays have been available only for a limited number of species of high research interest (e.g., mouse, human), and most arrays are only available for the single specific species they are designed for. While a few arrays have been designed for use with multiple species, those that exist use CpG sites conserved for a subgroup of animals (e.g., class Mammalia) (Arneson, A. et al., Nat Commun 13, 783 (2022)). In this design scenario, CpG sites cannot be added unless they are conserved across the entire class, regardless of whether they are highly relevant and meaningful for assessment in one or several of the species represented. Moreover, the method restricts the design to similar taxonomic groups.

[0007] Accordingly, there remains a lack of such methylation-based arrays that contain probes specific for multiple species and that make the process of detecting methylation changes in DNA cost-effective, robust, reliable, and efficient. Brief Description of the Drawings Figure 1 Relates to the design of an array with three different animal species, Chinese hamster ovary cell line (CHO), chicken, and freshwater crayfish. % shows the % of CpG sites found in the array that belong to a particular species. For example, 16% of the total CpG sites are from freshwater crayfish, 36% are from CHO cells, and 48% are from chicken.

[0009] Figure 2 Is a distribution plot of the average β values of the titrated chicken samples of Example 3.

[0010] Figure 3 Is a distribution plot of the average β values of the titrated CHO samples of Example 4.

[0011] Description of the Invention The present invention solves the above problems by providing a methylation-based array containing probes for multiple species, wherein the probes are specific for CpG targets found on each individual species on a multi-species chip. This is particularly advantageous because the results of methylation-based arrays are accurate and reproducible. In particular, the probes on a multi-species chip according to any aspect of the present invention are specific for CpG targets from different species that are not conserved among all species represented on the chip, and / or the probes are not only designed for different purposes in different species represented on the multi-species chip, but they are also new and specific. Further, a multi-species chip according to any aspect of the present invention includes species from different classes of animals (mammals, vertebrates, invertebrates) together on a single array on a chip comprising one or more (e.g., 12, 24, 48 or 96) arrays. This provides the flexibility to generate data simultaneously from multiple samples from a single species or several species in a faster and more cost-effective manner.

[0012] A multi-species chip according to any aspect of the present invention will also improve the cost savings, flexibility and efficiency of a research laboratory by allowing the laboratory to save time and effort, particularly for multiple species, which time and effort are spent on developing and stocking multiple chips and waiting for sufficient samples of each individual species to be available to run a full chip for the most cost-effective analysis or using traditional sequencing techniques.

[0013] Further, 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 computation 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.

[0014] 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 passed from one generation to the next.

[0015] According to one aspect of the present invention, there is provided a DNA methylation-based array comprising at least: - A first plurality of distinct positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a CpG site of a first plurality of CpG sites from a first animal species; and - A second plurality of distinct positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a CpG site of a second plurality of CpG sites from a second animal species wherein the first and second animal species are each independently selected from viruses, mammals, birds, and aquatic animals, and - the mammal is at least one domestic animal or an animal cell line; - The bird is at least one poultry; and - The aquatic animal is at least one crustacean, cephalopod, or fish, and wherein the first plurality of CpG sites includes at least 1000 CpG sites of the first animal species; and the second plurality of CpG sites includes at least 1000 CpG sites of the second animal species.

[0016] An array according to any aspect of the present invention is particularly advantageous because the CpG sites to which the nucleic acid sequences of the array bind are specific and particular to the first or second animal species and are not common or universal to different species. This makes an array according to any aspect of the present invention accurate and effective in identifying not only the species of the animal being tested but also other characteristics of the test animal, such as epigenetic age, geographical origin, whether the animal has been exposed to antibiotics and / or veterinary chemicals, whether the test animal has been bred under specific conditions, etc.

[0017] As used herein, the term "array" refers to an intentionally created collection of probe molecules, which can be prepared synthetically or biosynthetically. The probe molecules in the array can be identical or different from each other. The array can take various forms, for example, a library of soluble molecules; a library of compounds bound to resin beads, silicon chips, or other solid supports.

[0018] In particular, DNA methylation-based arrays 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 includes a plurality of different probe molecules, which may be attached to a substrate or otherwise spatially distinguishable in the array. Examples of arrays that may be used in any aspect of the present invention include glass slide arrays, silicon wafer arrays, liquid arrays, bead-based arrays, and the like. In one example, the array technology used in any aspect of the present invention combines a miniaturized array platform, a high level of assay multiplexing, and scalable automation for sample handling and data processing.

[0019] In particular, an array according to any aspect of the present invention may be an array of arrays, also referred to as a composite array, which has a plurality of individual arrays configured to allow simultaneous processing of multiple samples. Examples of composite arrays and the technology behind them are disclosed at least in US 6,429,027 and US2002 / 0102578. The substrate of the composite array may include a plurality of individual array positions, each having a plurality of probes and being 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 may 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).

[0020] In one example, the array substrate may 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 may allow probes to be 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 and methods for attaching beads to substrates that may be used in an array according to any aspect of the present invention.

[0021] In one example, the surface of the substrate may be physically altered to enable attachment of probes or generation of array positions. For example, the surface of the substrate may be modified to contain chemically modified sites for covalently or non-covalently attaching probe molecules or particles having attached probe molecules. The probes may 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 using a mask. WO2004110246 discloses these techniques in more detail.

[0022] 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.). A bead array useful according to any aspect of the present invention can also be in fluid form, such as the fluid flow 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.

[0023] 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.

[0024] A DNA methylation array according to any aspect of the present invention can be a very high-density array, for example, 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 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.

[0025] 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 differentiate the methylation status of the locus.

[0026] An array according to any aspect of the present invention includes: - A first plurality of different positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a CpG site of a first plurality of CpG sites from a first animal species, and the first plurality of CpG sites includes at least 1000 CpG sites of the first animal species; and - A second plurality of different positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a CpG site of a second plurality of CpG sites from a second animal species, and the second plurality of CpG sites comprising at least 1000 CpG sites of the second animal species.

[0027] 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 non - methylated allele of a CpG site, or both.

[0028] 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 artificial molecules. Also, they can be in their unaltered condition 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 non - methylated 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.

[0029] In particular, the probe molecule according to any aspect of the present invention comprises a nucleic acid sequence complementary to a different CpG site of a first animal species. Thus, the 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 position having a specific probe molecule complementary to a different CpG site of an animal species. In particular, the 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. The array according to any aspect of the present invention thus includes different positions having specific probe molecules, where each probe molecule is complementary to different CpG sites from at least two animal species.

[0030] In particular, the plurality of CpG sites for each animal species includes at least about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800 or 10,000 CpG sites. More particularly, the first plurality of CpG sites includes at least 1000 CpG sites of the first animal species, and the second plurality of CpG sites includes at least 1000 CpG sites of the second animal species.

[0031] 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 generally 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 without any mismatches over the length of the probe.

[0032] 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 a cell, some of these sites may be hypermethylated while some may be hypomethylated.

[0033] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0034] 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 a CpG island as a sequence that is at least 200 nucleotides in length, has 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 circumstances of genomic sequence methylation, and such terms refer to the characteristics of a DNA segment at a particular 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.

[0035] The term "methylation state" refers to the state (i.e., methylated versus non-methylated) of a particular methylation site, which means that the residue or methylation site is methylated or not methylated. Then, based on the methylation state of one or more methylation sites, a methylation profile can be determined.

[0036] The term "methylation level" refers to the level at 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 unmethylated in a DNA sequence in a sample are more methylated, it can be termed "hypermethylated"; while if one or more cytosine (C) residues that are generally methylated in a DNA sequence are less methylated, it can be termed "hypomethylated". Similarly, if one or more cytosine (C) residues in a DNA sequence (e.g., a 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 called "differentially methylated". For example, when the methylation status is different between an inflamed tissue and a 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 method.

[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 a group of subjects.

[0038] The term 'biological sample' as used herein can be selected from muscle, organ tissue, milk, blood, brain, sperm, and any other tissue or sample that provides genomic DNA to be used in a method 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, a 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] The "bisulfite treatment" of genomic DNA, which is used interchangeably with the term 'bisulfite modification', refers to the treatment of 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 DNA methylation status, for example, 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 status. Such processes can include, but are not limited to, chemical reactions (e.g., the 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 one that can cleave or digest a DNA molecule with much higher efficiency when the DNA is methylated, while an enzyme that preferentially cleaves or digests unmethylated DNA exhibits significantly higher efficiency when the DNA is unmethylated.

[0041] Alternative methods available in the art can be used in place of bisulfite treatment. Those skilled in the art 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).

[0042] As used herein, "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 herein, 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 in a target gene nucleic acid region where methylation has the potential to occur. For example, a position containing CpG is a methylation site, where the 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 amenable to methylation. These methylated nucleotides typically occur in nature and, to date, have mainly 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.

[0043] 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 particular 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.

[0044] The term "hypermethylation" refers to the average methylation status corresponding to the presence of an increase in 5-mCyt at one or more CpG dinucleotides in the DNA sequence of a test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample.

[0045] The term "hypomethylation" refers to the average methylation state corresponding to the presence of a decrease in 5-mCyt at one or more CpG dinucleotides in the DNA sequence of a test DNA sample relative to the amount of 5-mCyt found at the corresponding CpG dinucleotides in a normal control DNA sample.

[0046] In particular, the first and second animal species are selected from viruses, mammals, birds, and / or aquatic animals. More particularly, - the mammal is at least one domestic animal and / or an animal cell line; - the bird is at least one domestic fowl; and / or - the aquatic animal is at least one crustacean, cephalopod, and / or fish.

[0047] The domestic animal can be a farm animal selected from domestic animals or domestic fowls. In particular, the domestic animal can include cows, sheep, pigs, goats, horses, camels, donkeys, mules, rabbits, etc., and the domestic fowl can include chickens, turkeys, and other gallinaceous birds, ducks, geese, quails, etc. As used herein, the term 'domestic animal' can also include domestic fowls and refers to any farm animal or animal that can be used in agriculture.

[0048] As used herein, the term "aquatic animal" refers to any organism that lives entirely or mainly in water, especially as compared to terrestrial animals. In particular, an aquatic animal according to any aspect of the present invention can be any animal in the animal kingdom that mainly lives in water. These aquatic animals may live in different water forms, such as seas, oceans, rivers, lakes, ponds, etc. More particularly, an aquatic animal 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 animal' means an animal of the following species: (i) fish belonging to the superclass Agnatha and the classes Chondrichthyes, Sarcopterygii and Actinopterygii; (ii) aquatic mollusks belonging to the phylum Mollusca; and (iii) aquatic crustaceans belonging to the subphylum Crustacea. Even more particularly, an aquatic animal according to any aspect of the present invention can be an aquatic animal for aquaculture. Some non-limiting examples of aquatic animals according to any aspect of the present invention include the Australian lungfish, carp, catfish, halibut, marbled crayfish, marine and brackish fishes, sea shrimp, mitten crabs, shellfish, oysters, pangasius, rainbow trout, salmonids, scallops, black bass, sea bream, soft-shell crabs, turtles, tiger prawns, tilapia, turbot, white-leg prawn, shrimp, octopus, squid and other decapod crustaceans, bivalves and gastropods.

[0049] Animal cell lines can be the immortalized Chinese hamster ovary cell line (CHO) derived from the Chinese hamster (Cricetulus griseus), the Vero cell line isolated from kidney epithelial cells extracted from African green monkeys (i.e., Chlorocebus sp.), the immortalized HeLa cell line from humans, etc.

[0050] In one example, the mammal can be a human, and the array can include CpG sites for different parts of a human, such as CpG sites associated with human skin.

[0051] In particular, the array according to any aspect of the present invention can include first and second animal species selected from salmon, shrimp, pig, chicken, freshwater crayfish, CHO and at least one virus.

[0052] The CpG sites specific to each species are specifically selected. In particular, the CpG sites of each species are specifically selected based on their methylation values observed from early experimental data. This includes environmentally specific CpG sites, also known as dynamic sites; CpG sites from dynamic regions such as hypomethylated regions (LMRs) and differentially methylated regions (DMRs), and CpG sites from candidate gene regulatory regions of pathways important in certain biological contexts. Examples of biological contexts that may affect CpG site methylation include exposure of animals to antibiotic treatment compared to animals without antibiotics, animals experiencing inflammation compared to healthy controls, and animals raised in different geographical regions. Almost all CpG targets that are highly valuable for both mammals and invertebrates are represented on arrays according to any aspect of the present invention using the described design strategy. In particular, the CpG targets on arrays according to any aspect of the present invention are not conserved across all species represented and / or have different uses across the different species presented.

[0053] "Environmentally specific CpG sites" refer to CpG sites that are differentially methylated in response to environmental conditions or exposures. For example, CpG site methylation may be affected by environmental forces such as diet, stress, drugs, or pollution, among others. As disclosed in WO2022023208, environmentally specific "epigenetic fingerprints" have been found on the genomes of various animals, and these environmentally specific CpG sites can be used to identify the geographical origin of these animals. Thus, the term "environmentally specific CpG sites" as used herein refers to CpG sites of an animal genome, and these may vary greatly depending on the taxonomic unit or species of the animal, which can be used to distinguish one geographical location from another based on one or more environmental parameters. These environmentally specific CpG sites may also be referred to as dynamic CpG sites. The methylation status of these environmentally specific CpG sites may thus vary depending on changes in the environment or specific environmental parameters. Such environmental parameters depend on the habitat of the animal and may be different if the animal is cultured in water or grown in soil, or may be selected from food or air parameters, etc. For example, for freshwater crabs (e.g., marbled crayfish), the environmental parameters may be selected from pH, water hardness, manganese content, iron content, and aluminum content. In another example, the habitat of an animal living in water may be selected from stagnant or flowing water, such as a lake, river, aqua farm, other water pools or bodies, or a pond. Geographical origin should be understood as the geographical location considered to be the habitat where the animal spawns and / or is cultured, or at least cultured for a significant period of its life. Thus, 'environmentally specific CpG sites' refer to CpG sites, i.e., dynamic CpG sites whose methylation changes based on different environmental parameters.

[0054] Low-methylated regions (LMRs) are regions in the genome where fewer than 60% of the CpGs are methylated. More particularly, fewer than 50%, 40%, 30%, 20% or 10% of the CpGs in LMRs are methylated. Any method known in the art can be used to identify or detect LMRs in genomic DNA. Well-known methods include using a program such as MethylSeekR. In particular, LMRs in genomic DNA have at least three consecutive CpGs and no single nucleotide polymorphisms (SNPs) at any of the CpG positions. Even more particularly, LMRs in genomic DNA are identified based on methods disclosed at least in Burger, L., (2013) Nucleic Acids Research, 41(16):e155 and / or Stadler, M., (2011) Nature 480, 490-495. LMRs are known to have an average methylation ranging from 10% to 50%; be low CG density regions; tend to be enriched in H3K4me1, DHSs and p300 / CBP; and / or be mainly located distally to promoters within intergenic or intronic regions. In particular, LMRs: - have an average methylation ranging from 10% to 50%, - are low CG density regions; - are enriched in histone H3 monomethylated at lysine 4 (H3K4me1), DNase I hypersensitive sites (DHSs) and the transcriptional coactivator CREB-binding protein (CPB) and p300; - are mainly located distally to promoters within intergenic or intronic regions; and / or - have no single nucleotide polymorphisms (SNPs) at any of the CpG positions.

[0055] Low-methylated regions (LMRs) represent a key feature of the dynamic methylome. LMRs are local decreases in the DNA methylation landscape and represent CpG-poor distal regulatory regions, which often reflect the binding of transcription factors and other DNA-binding proteins. LMRs were initially described in mice (Stadler et al. (2011) Nature: 480, 490-95). The evolutionary conservation of LMRs outside mammals remains unexplored.

[0056] Differentially methylated regions (DMRs) are genomic regions with different methylation states in multiple biological samples such as tissues, cells, individuals, etc. These are genomic regions where there are differences between phenotypes. When considering adjacent DMPs as a whole together, the statistical power may be greater [Gu H et al. (2010) Nat Methods 2010; 7:133 - 6]. The length range of DMRs can be from several hundred to several thousand base pairs [Rakyan et al. (2011) Nat Rev Genet 12:529 - 41, 2011, Bock C (2012) Nat Rev Genet 2012; 13:705 - 19].

[0057] DMRs can occur throughout the genome but have been specifically identified around the promoter regions of genes, within gene bodies, and at intergenic regulatory regions. There are two types of regions: predefined or user - defined. Regions with special biological significance such as CpG islands, CpG shores, UTRs, etc., are predefined. Many traditional statistical tests, including t - tests and Wilcoxon rank - sum tests, can be performed at the region level. For user - defined regions, criteria such as fixed region length, fixed number of significant and adjacent CpG sites, significant and smooth estimated effect size, etc.

[0058] The array according to any aspect of the present invention further comprises - 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.

[0059] These probes specific for SNPs can be used for SNP genotyping, which is a measure of the genetic variation of SNPs between members of a species. In particular, an SNP is a single - base - pair mutation at a specific locus that typically consists of two alleles that are conserved during evolution (where the rare - allele frequency > 1%). These probes enable the identification of species, particularly breeds of species. In particular, when a DNA sample is introduced into the 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.

[0060] An array according to any aspect of the invention includes at least a third plurality of distinct positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a CpG site of a third plurality of CpG sites from a third animal species. In the examples, the third animal species can be chicken, freshwater crayfish, and CHO cells.

[0061] The first animal species can be chicken, and the plurality of CpG sites includes at least: - CpG sites in dynamic CpG sites, CpG sites of promoters, and / or low methylation regions (LMRs).

[0062] The term 'CpG site of a promoter' herein refers to any CpG site that can be found on gene promoter regions such as immune system genes (IRF5, STAT3, TBK1, SOCS1), feed-related genes (RAC2, VCAM1, CTSS, and TLR4), antibiotic-related genes (RRAD, PDK4, SGK1, PTPRT), pectoralis muscle development genes (myopathy) (ARNT2, EYA2, PTGS1, CADM1), etc. As used herein, the term "promoter" or "gene promoter", which may be used interchangeably with the term'regulatory region' or'regulatory sequence', refers to the corresponding contiguous gene DNA sequence extending from 1.5 kb upstream to 1.5 kb downstream relative to the transcription start site (TSS), or an adjacent portion thereof. In particular, the'regulatory region' refers to the corresponding contiguous gene DNA sequence extending from 1.5 kb upstream to 0.5 kb downstream relative to the TSS. In some examples, the'regulatory region' refers to the corresponding contiguous gene DNA sequence extending from 1.5 kb upstream to the downstream edge of the CpG island, which overlaps with the region from 1.5 kb upstream to 1.5 kb downstream of the TSS (and in this case, may extend even further downstream beyond 1.5 kb), and an adjacent portion thereof.

[0063] The second animal species can be freshwater crayfish, and the plurality of CpG sites includes at least: - Dynamic CpG sites, CpG sites in methylated repetitive sequences in the freshwater crayfish genome, and / or CpG sites in immune system-related genes, meiosis genes, and DNMT1; and / or The term 'CpG sites in dynamic repetitive sequences in the freshwater crayfish genome' as used herein refers to CpG sites in transposable elements that show differential methylation between Procambarus virginalis and Procambarus fallax.

[0064] Other CpG sites of interest from freshwater crayfish include CpG sites in immune system-related genes, meiosis genes, and DNA methyltransferase 1 (DNMT1).

[0065] 'Immune system-related genes' in freshwater crayfish include genes such as HSPB1, CL17A, ARSH, SPB9, AGO2.

[0066] 'Meiosis genes' related to freshwater crayfish include genes such as POLO-G, CDK10, RECQ4, and RAD54.

[0067] The 'CpG sites in DNA methyltransferase 1 (DNMT1)' used in this article refer to CpG sites that are differentially methylated between control and DNMT1 knockout animals.

[0068] The third animal species is CHO cells, and the multiple CpG sites include at least: - Dynamic CpG sites, and CpG sites found in the promoters of metabolism-related genes, protein production-related genes, cell growth and division-related genes, epigenetic-related genes, and viral promoters.

[0069] 'Environment-specific CpG sites' in the context of CHO cell background refer to CpG sites that are differentially methylated in different CHO cell lines. The cell lines used in the analysis include CHO-K1 (ATCC), CHO-DG44 (Thermo Fisher Scientific), CHO-DXB11 (ATCC), ExpiCHO-S TM cells (Thermo Fisher Scientific), FreeStyle TM CHO-S TM cells (Thermo Fisher Scientific), CHO 1-15[subscript 500] (ATCC), and Agarabi CHO (ATCC).

[0070] 'Metabolism-related genes' in the context of CHO cells in this article refer to genes related to several metabolic pathways, such as glycolysis, TCA cycle, pentose phosphate pathway, malate-aspartate shuttle, amino acid metabolism, lactate metabolism, cholesterol biosynthesis, nucleotide biosynthesis, nucleotide sugar biosynthesis, etc. Several examples of such genes include Hk2, Pgk1, Idh3a, Pgm1, and Pdha1. A person skilled in the art will easily identify the genes found in CHO cells belonging to the said category.

[0071] As used herein in the context of CHO cells, the 'protein production-related genes' refer to genes associated with cellular processes such as DNA replication and repair, mRNA transcription, mRNA translation, post-translational modification, and protein folding and export. Several examples of such genes include Gatb, Sec61a2, Ube2e3, Exosc1, Dna2, Pold1, etc. A person skilled in the art will readily be able to identify other genes found in CHO cells that belong to the said category.

[0072] As used herein in the context of CHO cells, the 'cell growth and division-related genes' refer to genes associated with cellular processes such as cell cycle regulation, cytoskeleton-related elements, cell signaling, nucleotide metabolism, and cell death. Several examples of such genes include Camk1, Cd82, Cdk4, Col1a1, and Ctsb. Again, a person skilled in the art will readily be able to identify other genes found in CHO cells that belong to the said category.

[0073] As used herein in the context of CHO cells, the 'epigenetic-related genes' refer to genes associated with epigenetic modifications such as DNA methylation pathways, DNA demethylation pathways, folate and methionine cycles, and histone modifications. Several examples of such genes include Hat1, Shmt1, Bhmt, Dnmt1, and Ehmt1. A person skilled in the art will readily be able to identify other genes found in CHO cells that belong to the said category.

[0074] As used herein in the context of CHO cells, the term 'viral promoter' refers to the promoters and enhancers of at least cytomegalovirus (CMV) and simian vacuolating virus 40 (SV40).

[0075] An array according to any aspect of the present invention can include at least 4, 5, 6, 7, 8, 9, 10, and even more different positions limited by the size of the array.

[0076] In one example, an array according to any aspect of the present invention can include at least three different positions, each of which is specific for a CpG site of an animal species, and wherein the three animal species can be chicken, freshwater crayfish, and CHO cells.

[0077] In the said example, when the first animal species is chicken, the multiple CpG sites at least include: - Approximately 60, 55, 50, 45, 40, 35, 30, 25, 20% of dynamic CpG sites or 15 - 60, 15 - 55, 15 - 50, 15 - 45, 15 - 40, 15 - 35, 15 - 30, 15 - 25, 15 - 20, 20 - 60, 20 - 55, 20 - 50, 20 - 45, 20 - 40, 20 - 35, 20 - 30, 20 - 25, 25 - 60, 25 - 55, 25 - 50, 25 - 45, 25 - 40, 25 - 35, 25 - 30, 30 - 60, 30 - 55, 30 - 50, 35 - 60, 35 - 55, 35 - 50, 35 - 45, 35 - 40, 40 - 60, 40 - 55, 40 - 50, 40 - 45, 45 - 60, 45 - 55, 45 - 50% of dynamic CpG sites; - Approximately 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20% of CpG sites of promoters and / or genes or 30 - 80, 30 - 75, 30 - 70, 30 - 65, 30 - 60, 30 - 55, 30 - 50, 30 - 45, 30 - 40, 35 - 80, 35 - 75, 35 - 70, 35 - 65, 35 - 60, 35 - 55, 35 - 50, 35 - 45, 35 - 40, 40 - 80, 40 - 75, 40 - 70, 40 - 65, 40 - 60, 40 - 55, 40 - 50, 40 - 45, 45 - 80, 45 - 75, 45 - 70, 45 - 65, 45 - 60, 45 - 55, 45 - 50, 50 - 80, 50 - 75, 50 - 70, 50 - 65, 50 - 60, 50 - 55, 55 - 80, 55 - 75, 55 - 70, 55 - 65, 55 - 60, 60 - 80, 60 - 75, 60 - 70, 60 - 65, 65 - 80, 65 - 75, 65 - 70, 70 - 80, 70 - 75% of CpG sites of promoters and / or genes; and / or - Approximately 30, 25, 20, 15, 10, 5% of CpG sites in LMRs, or 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 30, 10 - 25, 10 - 20, 10 - 15% of CpG sites in LMRs.

[0078] In particular, the first animal species in the array according to any aspect of the present invention is a chicken, and the plurality of CpG sites includes at least: - Approximately 52% of dynamic CpG sites, - Approximately 36% of CpG sites of promoters, and / or - Approximately 12% of CpG sites in hypomethylated regions (LMRs) In the example, when the second animal species is a freshwater crayfish, the plurality of CpG sites includes at least: - Approximately 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30% of dynamic CpG sites or 30 - 90, 30 - 85, 30 - 80, 30 - 75, 30 - 70, 30 - 65, 30 - 60, 30 - 55, 30 - 50, 30 - 45, 30 - 40, 35 - 90, 35 - 85, 35 - 80, 35 - 75, 35 - 70, 35 - 65, 35 - 60, 35 - 55, 35 - 50, 35 - 45, 35 - 40, 40 - 90, 40 - 85, 40 - 80, 40 - 75, 40 - 70, 40 - 65, 40 - 60, 40 - 55, 40 - 50, 40 - 45, 45 - 90, 45 - 85, 45 - 80, 45 - 75, 45 - 70, 45 - 65, 45 - 60, 45 - 55, 45 - 50, 50 - 90, 50 - 85, 50 - 80, 50 - 75, 50 - 70, 50 - 65, 50 - 60, 50 - 55, 55 - 80, 55 - 75, 55 - 70, 55 - 65, 55 - 60, 60 - 90, 60 - 85, 60 - 80, 60 - 75, 60 - 70, 60 - 65, 65 - 80, 65 - 75, 65 - 70, 70 - 80, 70 - 75% of dynamic CpG sites; - Approximately 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1% of CpG sites found in methylated repetitive sequences in the freshwater crayfish genome or 5 - 50, 5 - 45, 5 - 40, 5 - 35, 5 - 30, 5 - 25, 5 - 20, 5 - 15, 5 - 10, 10 - 50, 10 - 45, 10 - 40, 10 - 35, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 15 - 50, 15 - 45, 15 - 40, 15 - 35, 15 - 30, 15 - 25, 15 - 20, 20 - 50, 20 - 45, 20 - 40, 20 - 35, 20 - 30, 20 - 25, 25 - 50, 25 - 45, 25 - 40, 25 - 35, 25 - 30, 30 - 50, 35 - 45, 35 - 40, 40 - 50, 40 - 45, 45 - 50% of CpG sites found in methylated repetitive sequences in the freshwater crayfish genome; - Approximately 30, 25, 20, 15, 10, 5% of the CpG sites in immune system-related genes, meiosis genes, and DNMT1, or 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15% of the CpG sites in immune system-related genes, meiosis genes, and DNMT1. In particular, approximately 1, 2, 3, 4, 5% of the CpG sites are related to the DNMT1 gene, and approximately 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5% of the CpG sites are related to immune system-related genes and / or meiosis genes.

[0079] In particular, the second animal species in the array according to any aspect of the present invention is a freshwater crayfish, and the plurality of CpG sites at least includes: - Approximately 83% of the dynamic CpG sites, - Approximately 5% of the CpG sites found in the methylated repetitive sequences in the freshwater crayfish genome, and / or - approximately 10% of the CpG sites in immune system-related genes, meiosis genes, and / or - Approximately 3% of the CpG sites in DNMT1.

[0080] In the said example, when the third animal species is a CHO cell, the plurality of CpG sites at least includes: - Approximately 50, 45, 40, 35, 30, 25, 20, 15, 10, 5% of the dynamic CpG sites or 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45, 25-40, 25-35, 25-30, 30-50, 35-45, 35-40, 40-50, 40-45, 45-50% of the dynamic CpG sites; and - Approximately 95, 90, 85, 80, 75, 70, 65, 60, 55, 50% of the CpG sites found in promoters, metabolism-related genes, protein production-related genes, cell growth / division-related genes, methylation-related genes, and viral promoters, or 50 - 95, 50 - 90, 50 - 85, 50 - 80, 50 - 75, 50 - 70, 50 - 65, 50 - 60, 50 - 55, 55 - 95, 55 - 90, 55 - 85, 55 - 80, 55 - 75, 55 - 70, 55 - 65, 55 - 60, 60 - 95, 60 - 90, 60 - 85, 60 - 80, 60 - 75, 60 - 70, 60 - 65, 65 - 95, 65 - 90, 65 - 85, 60 - 80, 65 - 75, 65 - 70, 70 - 95, 70 - 90, 70 - 85, 70 - 80, 70 - 75, 65 - 95, 65 - 90, 65 - 85, 65 - 80, 65 - 75, 65 - 70, 65 - 65, 65 - 60, 60 - 95, 60 - 90, 60 - 85, 60 - 80, 60 - 75, 60 - 70, 60 - 65, 65 - 95, 65 - 90, 65 - 85, 65 - 80, 65 - 75, 65 - 70, 70 - 95, 70 - 90, 70 - 85, 70 - 80, 70 - 75, 75 - 95, 75 - 90, 75 - 85, 75 - 80, 80 - 95, 80 - 90, 80 - 85, 85 - 95, 85 - 90, 90 - 95 of the CpG sites found in promoters, metabolism-related genes, protein production-related genes, cell growth / division-related genes, methylation-related genes, and viral promoters.

[0081] In particular, the third animal species in the array according to any aspect of the present invention is a CHO cell, and the plurality of CpG sites includes at least: - Approximately 28% of the dynamic CpG sites, and / or - Approximately 72% of the CpG sites found in promoters, metabolism-related genes, protein production-related genes, cell growth / division-related genes, methylation-related genes, and viral promoters. In particular, approximately 63% of the CpG sites are found in promoters, metabolism-related genes, protein production-related genes, cell growth / division-related genes, and methylation-related genes, and approximately 9% of the CpG sites are found in viral promoters.

[0082] In one example, an array according to any aspect of the present invention may include at least 3 animal species, where the first animal species may be a chicken, and about 45 - 50% of the total number of CpG sites on the array are from chickens, the second animal species may be a freshwater crayfish, and about 10 - 20% of the total number of CpG sites on the array are from freshwater crayfish, and the third animal species may be a CHO cell line, and about 30 - 40% of the total number of CpG sites on the array are from the CHO cell line.

[0083] In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand still ensures the technical effect of the feature under consideration. The terms generally denote a deviation of ±20%, ±15%, ±10% and for example ±5% from the indicated value. As will be understood by a person of ordinary skill in the art, the specific deviation of the value for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects generally have a greater such deviation than artificial or engineering technical effects.

[0084] According to another aspect of the present invention, there is provided the use of an array according to any aspect of the present invention for predicting the biological age of a test animal.

[0085] The term 'chronological age' refers to the calendar time that has elapsed since birth / hatching.

[0086] Biological 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 are interchangeable in the context of the present application.

[0087] As used herein, the term "test" when used in conjunction with the term animal refers to an animal that is introduced into an array according to any aspect of the present invention and is the basis for the analytical application of the present invention. Thus, a "test animal" is an animal that is tested according to any aspect of the present invention or a profile obtained or generated in that context. In contrast, the term "reference" or 'control' shall denote a generally pre - determined entity used for comparison with the test entity. In particular, a 'test animal' refers to an animal that is tested to determine any characteristic of the animal (i.e., biological age, geographical origin, method of rearing, etc.) for which the methylation status must be determined, while a 'control' refers to an animal for which the characteristics as mentioned above are known and for which the methylation status is known and used as a reference.

[0088] According to a further aspect of the present invention, there is provided the use of an array according to any aspect of the present invention 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 veterinary chemical and / or is currently undergoing such treatment.

[0089] As used herein, the term 'antibiotic' refers to any pharmaceutical that can be fed to terrestrial animals for therapeutic and / or prophylactic purposes. Antibiotics can be administered by any method known in the art. Antibiotics can be orally fed to aquatic animals according to any aspect of the present invention in animal feed or in the water for raising aquatic animals so that they are ingested or used as a bath for external body injection. In another example, antibiotics can be injected into aquatic animals. A person skilled in the art will know the best way to provide antibiotics to an animal based on the specific taxonomic unit of the animal, the type of antibiotic, and the disease to be treated or prevented. In particular, antibiotics 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, monocyclic β-lactams, 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 antibiotics can be selected from tetracyclines and fluoroquinolones, especially norfloxacin.

[0090] 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 antibiotics with the test animal may cause epigenetic changes, at least DNA methylation changes, which can then be determined using the methods 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 may 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 measures or for therapeutic measures is within the knowledge of a person skilled in the art.

[0091] As used herein, the term 'veterinary chemical' refers to a drug or pharmaceutical used for treating or preventing diseases, injuries, and pests in animals. In particular, 'veterinary chemical' can refer to antiparasitic drugs, antiviral drugs, feed additives, water additives, disinfectants, glutaraldehyde, formalin, mixtures thereof, etc. The veterinary chemicals can be administered to the test animal by any method known in the art.

[0092] The test animals used in the methods according to any aspect of the present invention can be simultaneously and / or consequently exposed to both antibiotics and veterinary chemicals. Alterations in the internal environment of the test animals result in epigenetic alterations, and these can be determined using an array according to any aspect of the present invention.

[0093] An array according to any aspect of the present invention can also be used to determine 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 such treatment, and if so, to determine the particular class of antibiotic with which the test animal is being treated and / or is currently undergoing such treatment.

[0094] According to yet another aspect of the present invention, there is provided the use of an array to determine 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 such treatment, and if so, to determine whether the antibiotic is used as a growth promotant or as a therapeutant.

[0095] As used herein, the term 'growth promotant' 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 therapeutant (i.e., for treating diseases), an antibiotic can be used as a growth promotant.

[0096] 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 taken into consideration for introduction into an array according to any aspect of the present invention. These products derived from animals can include meat and meat products, as well as fats, 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., fats), such as soaps, creams, 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, as well as eggs, fats, meat, blood, processed meat, and lesser-known products, and non-food products such as fibers (shells, scales, etc.). Animal-derived products can also include products that are 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 for fish meal / fish oil, which ultimately goes into the animal feed industry or pets. The sample can be from any biological entity that has a genomic DNA and DNA genomic methylation. In particular, the methylation site is a CpG site.

[0097] According to a further aspect of the present invention, there is provided the use of an array according to any aspect of the present invention to determine whether a test animal from which a product is derived has undergone a withdrawal period during which it was not treated with at least one antibiotic and / or veterinary chemical prior to obtaining the product.

[0098] As used herein, the term "withdrawal period" refers to the period of time from the point at which an animal is no longer fed antibiotics and / or veterinary chemicals until the remaining antibiotics have decomposed in the body until they become non-functional agents and are finally eliminated 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 the animal is slaughtered or killed or until it is legal to derive products from the animal. Thus, the time it takes for the body to break down the antibiotic until it is no longer functional or present is referred to as the withdrawal time (or withdrawal period). Once the withdrawal period has passed, the antibiotic has been eliminated from the animal's system.

[0099] According to yet a further aspect of the invention, there is provided the use of an array according to any aspect of the invention for the specific verification of a sample of a product derived from a test animal. In particular, the specific verification of a sample of a product derived from an animal is whether the animal has been slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea and the oesophagus and / or whether the test animal has bled to death.

[0100] The term "quality verification" refers to a certificate or attestation given by a designated verification body which guarantees the quality of a specific animal-derived product, including food for human use and / or consumption. The term "quality verification" is used interchangeably with the term "verification". These verifications typically appear on the packaging of animal-derived products (including food for consumption) and are printed by the product manufacturer. Examples of specific food quality verifications can include "rearing method (Haltungsform)", "animal welfare (Tierwohl)", "non-genetically engineered (Ohne Gentechnik)", "halal", "kosher", and other safety labels which attest that the product sold has been prepared in accordance with specific religious or safety regulations. Specifically, the term "food quality verification" refers to a certificate or attestation given by a designated verification body which guarantees the quality, origin or method of slaughter of a specific food for human consumption. According to any aspect of the invention, the quality of the verification can be a specific verified food quality, or a specific verification, and this can be kosher, non-kosher, halal or non-halal.

[0101] In one example, the specific verified food quality or verification of sample X according to any aspect of the 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 larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea and / or the oesophagus. Even more particularly, the animal has been bled out.

[0102] 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 is edible according to the halakha (law). Kosher food used in connection with meat particularly relates to the manner 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, 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 place where the cilia of the trachea begin, 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 meat generally refers to most meat excluding pigs. In particular, kosher meat may be selected from beef, chicken, lamb, mutton, goat meat, and mixtures thereof. Kosher meat does not include shellfish, 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. Any food or meat that does not fall within the definition of 'kosher food' is then considered 'non-kosher food'.

[0103] The term 'halal' used in connection with food according to any aspect of the present invention refers to food that complies with Islamic dietary laws, and in particular meat prepared according to those requirements. Similar to the manner of preparing kosher meat, in Islamic tradition, the animal is according to For slaughtering, an animal is slaughtered by a clean cut with a sharp blade without serrated edges across the neck to produce 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 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 then 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).

[0104] In one instance, the specific verification of an animal-derived product sample according to any aspect of the present invention is based on the type of animal husbandry in which the test animal is raised.

[0105] In one instance, the specific verification or verification of sample X can be based on the type of animal husbandry in which the test animal is raised. In Germany, this is referred to as 'husbandry method'. There are at least four types / conditions in which animals can be raised. These four levels of animal husbandry include stable housing (Stallhaltung), stable housing plus (StallhaltungPlus), outside climate (Außenklima), and premium, which are also known as husbandry methods 1, 2, 3, and 4 respectively. Animal products derived from animals raised 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.

[0106] 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 / shed keeping (Stallhaltung), upgraded cage / shed keeping (StallhaltungPlus), outdoor climate (Auβenklima), and premium. Similarly, in France, livestock and poultry may be labeled with 'French Red Label', 'organic', or other pictograms that show the keeping methods the animals have undergone before obtaining products derived from the animals. Among livestock and poultry in the UK, there is the Red Tractor Food Assurance certification 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 certifies specific animal welfare standards, and several organic meat certification schemes, such as the Organic Farmers and Growers Certification and the Soil Association Organic Standard. Examples of meat certifications in the United States (USA) include those provided by the United States Department of Agriculture (USDA), which include, as examples, Carcass Quality and organic certification. The USDA has also approved some third-party certification schemes, such as those provided by the non-profit A Greener World, which include Certified Animal Welfare Approved, which defines the certification of livestock husbandry related to animal welfare, and Certified Grassfed, which defines the certification of specific feed types in livestock husbandry.

[0107] According to yet another aspect of the present invention, there is provided the use of an array according to any aspect of the present invention for identifying the geographical origin of a test animal-derived product.

[0108] The term "geographical origin" as used herein refers to a geographical location that is distinguished from other geographical locations by one or more environmental parameters of a test animal. Such environmental parameters depend on the habitat of the animal and may be different if the animal lives or is cultured in water, on soil, or in soil, or may be selected from food or air parameters, etc. In one example, for a freshwater crab (e.g., the 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 greatly 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 water or flowing water, such as lakes, rivers, aqua farms, other water pools or bodies of water, or ponds. The geographical origin should be understood as the geographical location regarded as the habitat where the test animal was born, hatched, and / or raised, or at least raised for a considerable period of its life. Example

[0109] The foregoing describes preferred embodiments which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction, or operation without departing from the scope of the claims. Such variations are, for example, intended to be encompassed by the scope of the claims.

[0110] Example 1 A customizable methylation bead BeadChip array (Illumina) was designed for functionality regarding three different animal species: Chinese hamster ovary cell line (CHO), chicken, and freshwater crayfish ( Figure 1 ).

[0111] The final design contained nearly 70,000 CpG sites. To design the array, the following method was used to identify candidate CpG sites for each species for each category (Table 1): Table 1. Breakdown of CpG site categories for each species in the multi-species methylation bead array design.

[0112] Environment-specific CpG sites or dynamic CpG sites Differentially methylated positions (DMPs) were identified from sequencing data (WGBS / RRBS). Samples from various environmental conditions were collected and then the sequencing data was processed and analyzed to identify CpG sites contributing to various environmental factors including location, various treatment conditions, etc. (Tables 2 and 5).

[0113] LMR / clock-based CpG sites These sites were selected using a chicken methylation age clock, which was developed using a penalized regression model to regress chronological age, as described in Raddatz, G., Commun Biol 4, 76 (2021). (Table 4).

[0114] Gene promoter-based CpG sites Three CpG sites in each gene promoter were identified from all promoters (exploratory) and candidate genes based on the following species-specific requirements: · For chicken, probes were designed using unpublished promoters (exploratory) and data on candidate genes focusing on immune system genes, feed-related genes, antibiotic-related genes, and breast muscle development genes such as those associated with white striping (some examples are provided in Table 3).

[0115] · For CHO, probes were designed for all promoters (exploratory) and candidate genes focusing on metabolism-related genes, protein production-related genes, cell growth / division-related genes, methylation-related genes, and viral promoters and enhancers such as CMV and SV40 (some examples are provided in Table 6).

[0116] · For freshwater crayfish, probes were designed for immune system-related genes, meiosis genes, and DNMT1-responsive CpG sites (some examples are provided in Table 7).

[0117] The CpG sites were annotated using the provided Illumina design information, and a final list of CpG sites represented on the array was selected based on the designability score, probe type, and strand type for each site.

[0118] Example 2 A customizable methylation bead BeadChip array (Illumina) was designed for functionality regarding three different animal species: Chinese hamster ovary cell line (CHO), chicken, and freshwater crayfish.

[0119] The final design contains nearly 80,000 different bead types in each array. For Infinium I chemistry, two bead types are used to assay a single DNA-methylation site. Infinium II requires only one bead type. Since many DNA-methylation sites need to be covered with Infinium I chemistry, the number of bead types is different from the number of DNA methylation sites that can be assayed. To design the arrays, the following method was used to identify candidate CpG sites for each species in each category (Table 8): Table 8. Breakdown of CpG site categories for each species in the multi-species methylation bead array design. Species Final DNA-methylation sites on the TALOS chip Chicken 28083 CHO 22598 Freshwater crayfish 9023 Total 59704

[0120] Example 3 Evaluation of methylation values of titrated chicken samples To evaluate the accuracy of the methylation levels detected on the BeadChip of Example 2, DNA methylation controls were obtained from an external vendor, where genomic DNA samples were fully methylated and demethylated from a ready-to-use chicken genomic DNA sample to provide 0% and 100% methylated DNA samples. These methylation controls were then further tested according to the bisulfite conversion, BeadChip analysis, and data processing sections below. The samples were run as three technical replicates across the three BeadChips of Example 2.

[0121] 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. A small amount of the samples was then also analyzed by automated electrophoresis on a TapeStation (Agilent) to ensure that each sample contained high molecular weight DNA.

[0122] Bisulfite conversion and BeadChip analysis Genomic DNA samples were then subjected to use of EZ DNA Methylation-Gold TMBisulfite conversion of the kit (ZymoResearch). Then the methylation level was 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, the 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.

[0123] Data processing: Custom 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 β-value. The β-value was defined as the signal of methylation / (signal of methylation + signal of unmethylation). The SeSAMe pipeline (Zhou et al., 2018) was used to generate normalized β-values and for quality control. The pipeline first infers the Infinium 1 channel, followed by dye bias correction, low-intensity-based detection calling and generation (based on p-values) using pOOBAH. 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. After obtaining the β-values, control probes were filtered out from the data frame. CpG sites with NA β-values were also removed from the data.

[0124] Results The average mean of each chip at each titration level was obtained by first calculating the average β-value of each probe. Once the average β-value of each probe was obtained, the mean of all probes at that titration level was calculated.

[0125] Density plots were created using the geom_density_ridges_gradient function from the package - 'ggridges'. The average β-value of each probe was plotted.

[0126] Table 9 shows the methylation values of the titrated samples of chickens. Figure 2Displays the distribution plot of the average β-values of the samples. Samples with specified methylation levels of -0% and 100% were run as technical replicates (x3) across 3 chips. The average and median β-values of each probe were calculated across 3 technical replicates within the array.

[0127] Table 9. Methylation values of titrated samples of chicken.

[0128] Example 4 Evaluation of methylation values of titrated CHO samples To evaluate the accuracy of the methylation levels detected on the BeadChip, genomic DNA samples from CHO-K1, DXB11, and DG44 were provided to an external vendor for complete methylation and demethylation and then mixed at specific ratios to provide DNA samples with 0%, 50%, 75%, and 100% methylation. These methylation controls were then further tested according to the following bisulfite conversion, BeadChip analysis, and data processing sections. Samples were run across three BeadChips as three technical replicates.

[0129] DNA extraction, bisulfite conversion, BeadChip analysis, quality control, data processing, and differential methylation analysis were as outlined in Example 3.

[0130] Figure 3 Displays the distribution plot of the average β-values of the samples. Samples with specified methylation levels of -0% - 100% were run as technical replicates (x3) across 3 chips. The average and median β-values of each probe were calculated across 3 technical replicates within the array.

[0131] Table 10. Methylation values of titrated samples of CHO samples (CHO-K1, transgenic DXB11, transgenic DG44).

Claims

1. An array based on DNA methylation, at least comprising: - A first plurality of different positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a CpG site of a first plurality of CpG sites from a first animal species; and - A second plurality of different positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a CpG site of a second plurality of CpG sites from a second animal species, wherein the first and second animal species are each independently selected from viruses, mammals, birds, and aquatic animals, and - The mammal is at least one domestic animal or animal cell line; - The bird is at least one poultry; and - The aquatic animal is at least one crustacean, cephalopod, or fish, and wherein the first plurality of CpG sites includes at least 1000 CpG sites of the first animal species; and The second plurality of CpG sites includes at least 1000 CpG sites of the second animal species.

2. The array according to claim 1, wherein: - The domestic animal is selected from cattle, goats, sheep, pigs, horses, donkeys, rabbits, and mules; - The poultry is selected from chickens, turkeys, ducks, geese, and quails; - The crustacean is at least one decapod, preferably freshwater crayfish and / or shrimp, and the cephalopod is at least one octopus and / or squid; and / or - The animal cell line is a Chinese hamster ovary cell line (CHO).

3. The array according to claim 1 or 2, wherein the array is a bead-based array.

4. The array according to any one of the preceding claims, which further comprises: - At least one probe molecule specific for at least one single nucleotide polymorphism (SNP) of the first animal species; and - At least one probe molecule specific for at least one SNP of the second animal species.

5. The array according to any one of the preceding claims, wherein the first and second animal species are selected from salmon, shrimp, pigs, chickens, freshwater crayfish, CHO, and at least one virus.

6. The array according to any one of the preceding claims, wherein at least a portion of the plurality of CpG sites of the first and second species are dynamic CpG sites.

7. The array according to any one of the preceding claims, wherein the array includes at least a third plurality of different positions, each position having at least one probe molecule, the probe molecule comprising a nucleic acid sequence complementary to a CpG site of a third plurality of CpG sites from a third animal species.

8. The array according to claim 7, wherein the third animal species is chicken, freshwater crayfish, and CHO cells.

9. The array according to claim 8, wherein: (a) The first animal species is chicken, and the plurality of CpG sites includes at least: - CpG sites in dynamic CpG sites, CpG sites of promoters, and / or low methylation regions (LMRs), and the CpG sites include at least CpG sites selected from Tables 2, 3, and 4 respectively; (b) The second animal species is freshwater crayfish, and the plurality of CpG sites includes at least: - Dynamically methylated CpG sites, CpG sites found in methylated repetitive sequences in the genome of freshwater crayfish, and / or CpG sites in immune system-related genes, meiosis genes, and DNMT1, and said CpG sites and said CpG sites at least include CpG sites selected from Table 7; and / or (c) The third animal species is a CHO cell, and said plurality of CpG sites at least include: - Dynamically methylated CpG sites, and CpG sites found in promoters, metabolism-related genes, protein production-related genes, cell growth and division-related genes, methylation-related genes, and viral promoters, and said CpG sites at least include CpG sites selected from Tables 5 and 6.

10. Use of the array according to any one of claims 1-9 for predicting the biological age of a test animal.

11. Use of the array according to any one of claims 1-9 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 veterinary chemical and / or is currently undergoing said treatment.

12. Use of the array according to any one of claims 1-9 for determining a special assay of a product sample derived from a test animal.

13. The use according to claim 12, wherein the special assay of the product sample derived from the animal is based on (a) Whether the animal has been slaughtered by a single cut across the larynx severing both carotid arteries, both jugular veins, both vagus nerves, the trachea, and the esophagus and / or whether the test animal has died by bleeding; or (b) The type of livestock farming in which the test animal was raised.

14. Use of the array according to any one of claims 1-9 for identifying the geographical origin of a product derived from a test animal.

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