Detection of epigenetic cytosine modifications

Abstract

The present invention includes improved methods and compositions for reducing the C5-C6 double bonds of cytosine. In particular, the improved methods and compositions for reducing the C5-C6 double bonds of cytosine are via an enzymatic means, rather than via a chemical means. In particular, the present disclosure relates to a process for the conversion of 5, 6-dihydro-fC (fC) and / or 5, 6-dihydro-caC to 5, 6-dihydro-U (DHU). In particular, the present disclosure relates to methods of converting 5fC and / or 5caC to DHU. In addition, the present disclosure relates to methods for reducing the C5-C6 double bonds of cytosine using alkene reductases to detect epigenetic cytosine modifications, particularly cytosine methylation.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid-based diagnostics. More specifically, the present invention relates to a method for detecting epigenetic cytosine modifications in nucleic acids, wherein the epigenetic modifications may have biological and / or clinical significance. Background Art

[0002] Mapping epigenetic modifications of DNA and RNA is becoming increasingly important as these modifications play a role in a variety of biological processes and diseases, including development, aging, cancer, etc. Most relevant for identifying epigenetic modifications is their detection by sequencing-based methods.

[0003] The distinction between cytosine (C) and 5-methylcytosine (5mC) is described by direct sequencing of DNA through nanopores (without pre-amplification) (e.g., by Pacific Bioscience single-molecule real-time (SMRT) sequencing technology), thereby reading out the different kinetics of nucleotide incorporation relative to C and mC by the polymerase. Other techniques include the use of methods that convert C or mC to T (U) equivalents and subsequently amplify them to distinguish when comparing untreated and converted sample DNA (see, Zhao et al., "Mapping the epigenetic modifications of DNA and RNA", Protein & Cell 11(11):792-808 (2020). The most important methods are: (a) bisulfite sequencing (converting C to uracil (U) by bisulfite treatment); (b) New England Biolab (NEB) EM-seq method (mC is oxidized by TET2 enzyme and glycosylated by β-glucosyltransferase (to block the enzymatic deaminase reaction) and C is converted to U by APOBEC deaminase); (c) TAPS method (TET-assisted pyridine borane sequencing), which uses TET enzyme to oxidize mC and then reduce the oxidized mC species by pyridine borane to obtain dihydrocytidine nucleosides that are easily deaminated to give dihydrouracil (T equivalent); and (d) CLEVER method, which is based on the oxidation of mC by TET enzyme and the subsequent reaction of 5-formyl-C with malononitrile to obtain an adduct that is mainly used as a T equivalent in subsequent PCR amplification (Zhu et al., "Single-Cell 5-Formylcytosine Landscapes of Mammalian Early Embryos and ESCsat Single-Base Resolution", Cell Stem Cell 2008. Cell 20: 720-731 (2017). Instead of enzymatic oxidation of mC by TET enzymes, oxidation can also be performed by chemical means using, for example, potassium perruthenate (KRuO4). There are also methods for distinguishing between modification of 5-hydroxymethyl-dC (5hmC), 5-formyl-dC (5fC) or 5-carboxyl-dC (5caC) by partially modifying the above method.

[0004] The TAPS method uses TET enzyme-based oxidation of 5-methylcytosine to 5-formylcytosine and / or 5-carboxylcytosine, and subsequent reduction of the C5-C6 double bond of cytosine under deformylation and decarboxylation, respectively. The formed 5,6-dihydrocytosine is easily deaminated at the N-4 position to give 5,6-dihydrouracil (DHU). The polymerase reads DHU as T, and therefore incorporates A relative to DHU, which is able to distinguish epigenetic C modifications (e.g., mC, hmC, fC or caC) from unmodified C (see, Liu et al., "Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution", Nat. Biotechnol. 37(4): 424-429 (2019)).

[0005] The above TAPS method uses chemical reduction of the C5-C6 double bond of cytosine. Borane (particularly pyridine borane or 2-methylpyridine borane) is used as a reducing agent. However, these chemical reagents and chemical methods are disadvantageous because they are toxic. Therefore, there is a need in the art for a safe and environmentally friendly method to reduce the C5-C6 double bond of cytosine.

[0006] This patent application relates to the use of enzymatic reduction of the C5-C6 double bond of cytosine, rather than reduction by chemical means (as in the TAPS process). Thus, chemical reduction by toxic reagents (in the TAPS process) is replaced by an environmentally friendly enzymatic reduction process. Summary of the invention

[0007] Therefore, there is a need in the art for a safe and environmentally friendly method for reducing the C5-C6 double bond of cytosine. The present patent application relates to the use of enzymatic reduction of the C5-C6 double bond of cytosine, rather than reduction by chemical means (as in the TAPS method). Thus, chemical reduction by toxic reagents (in the TAPS method) is replaced by an environmentally friendly enzymatic reduction method.

[0008] The enzyme used is an enzyme from the group of ene reductases, which is able to reduce electron-deficient C-C double bonds, especially double bonds with electron-withdrawing substituents. In the case of fC or caC, both the formyl group and the carboxyl group are electron-withdrawing groups, and thus can reduce fC and / or caC, but not unsubstituted C and U or mC and T. Figure 1 The reduction of 5,6-dihydro-fC (fC) to 5,6-dihydro-U (DHU) is shown. Figure 2The reduction of 5,6-dihydro-caC (caC) to 5,6-dihydro-U (DHU) is shown. Ene reductase (ERED) is commercially available (eg, Codexis, Redwood City, CA, US). Figure 3 The reaction of interest is shown, in which an ene reductase (ERED) reduces a double bond (excerpted from CODEXIS, Codex ERED Screening Kit, Screening Protocol, Document No. PRO-004-003, page 1 (accessed on June 10, 2022, at: https: / / www.codexis-estore.com / _files / ugd / 5a7b2a_5fbf41f3ae 2741a081894f64424552d1.pdf ).

[0009] One embodiment relates to a method for reducing the C5-C6 double bond of cytosine in a nucleic acid, wherein the method comprises contacting the cytosine in the nucleic acid with an ene reductase. In one embodiment, the cytosine is 5-formylcytosine (5fC). In one embodiment, the cytosine is 5-carboxylcytosine (5caC). In one embodiment, the cytosine is 5,6-dihydro-fC. In one embodiment, the cytosine is 5,6-dihydro-caC. In one embodiment, the cytosine is 2'-deoxy-5-formylcytidine. In one embodiment, the cytosine is 2'-deoxy-5-carboxycytidine. In one embodiment, the cytosine is 5-formyl-2'-deoxycytidine. In one embodiment, the cytosine is 5-formyl-dC. In one embodiment, the cytosine is 5-carboxyl-2'-deoxycytidine. In one embodiment, the cytosine is 5-carboxyl-dC. In one embodiment, the cytosine in the nucleic acid is contacted with an ene reductase to reduce the cytosine to 5,6-dihydro-U (DHU). In one embodiment, DHU is subsequently converted to thymine.In one embodiment, reduction of the C5-C6 double bond of cytosine in a nucleic acid by contacting the cytosine in the nucleic acid with an ene reductase is part of a method for detecting epigenetic cytosine modifications.

[0010] Another embodiment relates to a method for detecting epigenetic cytosine modifications, wherein the method includes at least a step of reducing the C5-C6 double bond of a cytosine in a nucleic acid by contacting the cytosine in the nucleic acid with an ene reductase. In one embodiment, the cytosine is 5-formylcytosine (5fC). In one embodiment, the cytosine is 5-carboxylcytosine (5caC). In one embodiment, the cytosine is 5,6-dihydro-fC. In one embodiment, the cytosine is 5,6-dihydro-caC. In one embodiment, the cytosine is 2'-deoxy-5-formylcytidine. In one embodiment, the cytosine is 2'-deoxy-5-carboxycytidine. In one embodiment, the cytosine is 5-formyl-2'-deoxycytidine. In one embodiment, the cytosine is 5-formyl-dC. In one embodiment, the cytosine is 5-carboxyl-2'-deoxycytidine. In one embodiment, the cytosine is 5-carboxyl-dC. In one embodiment, cytosine in the nucleic acid is contacted with an ene reductase to reduce the cytosine to 5,6-dihydro-U (DHU). In one embodiment, the DHU is subsequently converted to thymine.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although in the practice or testing of this theme, methods and materials similar or equivalent to those described herein can be used, suitable methods and materials are described below. In addition, materials, methods and examples are only illustrative and are not intended to be limiting. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail.

[0012] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] Figure 1 The reduction of 5,6-dihydro-fC (fC) to 5,6-dihydro-U (DHU) is shown.

[0015] Figure 2 The reduction of 5,6-dihydro-caC (caC) to 5,6-dihydro-U (DHU) is shown.

[0016] Figure 3The reaction of interest is shown, in which an ene reductase (ERED) reduces a double bond (excerpted from CODEXIS, Codex ERED Screening Kit, Screening Protocol, Document No. PRO-004-003, page 1 (accessed on June 10, 2022, at: https: / / www.codexis-estore.com / _files / ugd / 5a7b2a_5fbf41f3ae2741a081894f64424552d1.p df ). DETAILED DESCRIPTION

[0017] abbreviation

[0018] Listed below are some abbreviations used throughout this disclosure.

[0019] C – Cytosine

[0020] T – Thymine

[0021] U – uracil

[0022] DHU-Dihydrouracil

[0023] 5mC – 5-methylcytosine

[0024] 5hmC – 5-hydroxymethylcytosine

[0025] 5ghmC-5-glucosyl-hydroxymethylcytosine

[0026] 5fC – 5-Formylcytosine

[0027] 5caC – 5-carboxycytosine

[0028] TET – 10-11 translocation dioxygenase

[0029] TAPS–TET-assisted methylpyridine-borane sequencing

[0030] CAPS – Chemically Assisted Picoline-Borane Sequencing

[0031] oxBS or oxBS-Seq – Oxidative bisulfite sequencing

[0032] 5-Methylcytosine and 5-hydroxymethylcytosine (5mC and 5hmC) are important epigenetic biomarkers with many clinical applications in oncology, prenatal testing and other fields. Until recently, methylated base level detection was achieved by reacting unmethylated cytosine with bisulfite, followed by PCR, array hybridization or sequencing. Unmethylated cytosine (C) will be read as thymine (T) after reaction with bisulfite, while methylated cytosine (5mC and 5hmC) will be read as C. Unfortunately, bisulfite treatment causes most sample nucleic acids to degrade, making it unsuitable for applications requiring high sensitivity. For example, this method is not suitable for the latest applications of analyzing cell-free nucleic acids such as cell-free DNA.

[0033] Recently, less harsh methods for detecting methylated cytosine have been disclosed. The latest methods involve modifying methylated cytosine instead of non-methylated cytosine, such as in the case of bisulfite treatment (Liu et al., Nature Biotechnology 37: 424-429 (2019) (hereinafter referred to as "Liu et al. (2019)")). In the presence of Fe (II) ions and α-ketoglutarate, methylcytosine (5mC) is gradually oxidized to formyl cytosine (5fC) and carboxyl cytosine (5caC) via 5-hydroxymethyl cytosine (5hmC) using 10-11 translocation dioxygenase (TET).

[0034] Liu et al. (2019) further described the use of borane derivatives (such as pyridine borane, methyl pyridine borane, etc.) to reduce 5fC (and 5caC) to dihydrouracil (DHU). Then, in subsequent amplification and sequencing, DHU is read as T by uracil-tolerant nucleic acid polymerase. As a result, methylated C is read as T, while unmethylated C remains unchanged. This TET and methyl pyridine-borane-based method is called TAPS (TET-assisted methyl pyridine-borane sequencing), which does not cause DNA degradation like bisulfite treatment, and allows direct detection of signals instead of subtracting background to obtain signals. Both advantages will allow higher alignment rates, potentially lower sequencing depths, and recovery of higher molecular diversity from samples.

[0035] Another technique called CAPS (chemically assisted methylpyridine borane sequencing) involves the selective conversion of 5hmC to 5fC using potassium perruthenate (KRuO4). The use of KRuO4 as a chemical alternative to TET is known from a technique called oxidative bisulfite sequencing or oxBS-seq (see, Booth et al., Science 336(6083):934-937(2012) (hereinafter referred to as "Booth et al. (2012)")). The 5fC obtained by potassium perruthenate conversion becomes a favorable target for further processing by, for example, borane treatment or any other downstream method.

[0036] Another sequencing technique is an alternative method for reducing 5fC with borane. The method involves forming a 5fC adduct that is identified as T. The adduct is formed using malononitrile (see, Zhu et al. Cell Stem Cell 20:720-731 (2017) (hereinafter referred to as "Zhu et al. (2017)")). The TAPS, CAPS and malononitrile methods of Zhu et al. (2017) are superior to the bisulfite method because they avoid harsh chemical treatments and the resulting loss of sample nucleic acids.

[0037] However, the disadvantage of these methods is that chemical reduction of the C5-C6 double bond of cytosine is usually used. Borane (particularly pyridine borane or 2-picoline borane) is used as a reducing agent. However, these chemical reagents and chemical methods are disadvantageous because they are toxic. Therefore, there is a need in the art for a safe and environmentally friendly method to reduce the C5-C6 double bond of cytosine.

[0038] This patent application relates to the use of enzymatic reduction of the C5-C6 double bond of cytosine, rather than reduction by chemical means (as in the TAPS process). Thus, chemical reduction by toxic reagents (in the TAPS process) is replaced by an environmentally friendly enzymatic reduction process.

[0039] In some embodiments, the present invention is a method for detecting epigenetic modifications (specifically, epigenetic cytosine modifications in nucleic acids (including but not limited to cytosine methylation)). Prior art methods for detecting epigenetic cytosine modifications may include the use of harsh chemical methods to reduce C5-C6 double bonds. However, the present patent application provides improvements in the art. The present patent application relates to enzymatic reduction (i.e., not chemical reduction) of the C5-C6 double bond using cytosine.

[0040] The present invention relates to a method for manipulating nucleic acid of a sample. In certain embodiments, the sample is obtained from a subject or a patient. In certain embodiments, the sample may include, for example, a fragment of a solid tissue or solid tumor derived from a subject or a patient by biopsy. The sample may also include a body fluid (for example, urine, sputum, serum, blood or blood components (i.e., plasma), lymph, saliva, sputum, sweat, tears, cerebrospinal fluid, amniotic fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, cystic fluid, bile, gastric juice, intestinal fluid or fecal sample) that may contain nucleic acid. In other embodiments, the sample is a culture sample, for example, a tissue culture containing cells and liquids from which nucleic acid can be isolated. In certain embodiments, the target nucleic acid in the sample is from an infectious agent, such as a virus, bacterium, protozoa or fungus.

[0041] The present invention relates to the operation of separating or extracting the isolated nucleic acid from a sample. The method of nucleic acid extraction is well known in the art (see, Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2nd edition, Cold Spring Harbor Laboratory Press: New York, NY. Various kits are commercially available for extracting nucleic acids (DNA or RNA) from biological samples (e.g., KAPA expression extracts (Roche Sequencing Solutions, Pleasanton, Cal.) and other similar products from BD Biosciences Clontech (Palo Alto, Cal.), Epicentre Technologies (Madison, Wisc.), Gentra Systems (Minneapolis, Minn.) and Qiagen (Valencia, Cal.), Ambion (Austin, Tex.), BioRad Laboratories (Hercules, Cal.), etc.

[0042] In some embodiments, nucleic acids are extracted, separated by size and optionally concentrated by accelerated electrophoresis as described, for example, in International Patent Publication Nos. WO2019 / 092269 and WO2020 / 074742.

[0043] The present invention relates to detecting epigenetic modifications (specifically, epigenetic cytosine modifications in nucleic acids (including but not limited to cytosine methylation)). Nucleic acid sequences that undergo conditional epigenetic modifications are target sequences analyzed by the methods disclosed herein. The same nucleic acid sequence may or may not have an epigenetic modification characterized by methylation of cytosine at position 5 (5mC or 5hmC). In some embodiments, a set or group of target nucleic acids is detected for the presence or absence of methylation. For example, methylation of a biomarker in a set of methylation biomarkers indicates that the patient has colorectal cancer, as shown in the following documents: Patai et al., "Comprehensive DNA Methylation Analysis Reveals a Common Ten-Gene Methylation Signature in Colorectal Adenomas and Carcinomas" PLOS ONE 10(8):e0133836(2015); and Onwuka et al., "A panel of DNA methylation signature from peripheral blood may predict colorectal cancer susceptibility", BMC Cancer 20, 692(2020). Thus, it is contemplated that any known or future panel of methylation biomarkers may be tested for prognostic or diagnostic purposes using the methods disclosed herein.

[0044] In some embodiments, the entire genome of an organism is probed for the presence of methylation. The methods of the present invention include using sequence analysis and artificial intelligence tools such as those described in the following literature to detect methylation in all sites of the entire genome of an organism to diagnose a disease or disorder or a tendency to a disease or disorder: Shull et al., "Sequencing the cancer methylome", Methods Mol Biol. 1238: 627-635 (2015).

[0045] In some embodiments, the present invention includes an improved step for detecting methylated cytosine in nucleic acids by forming and detecting 5-carboxycytosine (5caC) and 5-formylcytosine (5fC), wherein 5fC, 5caC, or a mixture of 5fC and 5caC is formed by one of the methods described above. The method involves contacting a sample containing nucleic acids comprising 5fC and / or 5caC with an enzyme, particularly an ene reductase, to form dihydrouracil (DHU).

[0046] After forming the DHU, the nucleic acid with the DHU is sequenced. In some embodiments, the sequencing is performed by a next-generation massively parallel sequencing process. The sequencing results in the test sequence, wherein the DHU is read as thymine (T), that is, the sequencing polymerase is able to accommodate the adduct or DHU in the replicated chain and incorporates an adenine (A) opposite to the DHU. The method further includes the step of comparing the test sequence with a reference sequence, wherein a change from a cytosine (C) in the reference sequence to a thymine (T) in a corresponding position in the test sequence indicates the presence of methylated cytosine in the test nucleic acid.

[0047] In some embodiments, the nucleic acid in the sample is amplified prior to sequencing. In some embodiments, the amplification utilizes a B family polymerase to effectively incorporate an adenine (A) nucleotide opposite to DHU. In this embodiment, sequencing can be performed with any polymerase suitable for the sequencing process, because DHU is already recognized as T by the amplification polymerase.

[0048] In some embodiments, nucleic acids in the sample are ligated to adaptors, wherein the adaptors comprise elements that can be used for amplification and sequencing. The adaptors comprise at least one of: a barcode, a primer binding site, and a ligation site.

[0049] In some embodiments, the present invention is an improved method for detecting methylated cytosine nucleotides in nucleic acids, the method comprising: (i) ligating an adaptor to nucleic acids in a sample, wherein the adaptor comprises an amplification primer binding site; (ii) forming a reaction mixture by contacting the sample containing the adaptor-ligated nucleic acids with a TET capable of converting methylated cytosine in the nucleic acids into a mixture of 5-carboxycytosine (5caC) or 5-formylcytosine (5fC) and 5caC; (iii) contacting the reaction mixture with an enzyme capable of reacting with 5fC and 5caC in the nucleic acids to form DHU; (iv) contacting the reaction mixture with an enzyme capable of reacting with 5fC and 5caC in the nucleic acids to form DHU; The reaction mixture is incubated for no more than about 1 hour, wherein at least 90% of the 5fC and 5caC have formed DHU (including but not limited to ene reductase); (v) amplifying the linked nucleic acid using a DNA polymerase and a primer capable of binding to the primer binding site, wherein the DNA polymerase reads DHU as thymine (T) during amplification; (vi) sequencing the amplified nucleic acid to obtain a test sequence; (vii) comparing the test sequence to a reference sequence, wherein a change from cytosine (C) in the reference sequence to thymine (T) in the corresponding position in the test sequence indicates the presence of 5mC in the nucleic acid. In some embodiments, in steps (iii) and (iv), the borane derivative is present in a non-aqueous solvent such as ethanol or methanol. In some embodiments, in steps (iii) and (iv), the borane derivative is present in a solution comprising an organic acid such as acetic acid.

[0050] In some embodiments, the present invention utilizes adapters added to one or both ends of a nucleic acid or nucleic acid chain. Adapters with various shapes and functions are known in the art (see, e.g., International Patent Application No. PCT / EP2019 / 05515, U.S. Patent Nos. 8,822,150 and 8,455,193, filed on February 28, 2019). In some embodiments, the function of the adapter is to introduce the desired element into the nucleic acid. The adapter carries an element including at least one of a nucleic acid barcode, a primer binding site, or a connection enabling site.

[0051] The adapter can be double-stranded, partially single-stranded, or single-stranded. In some embodiments, a Y-shaped, hairpin adapter, or stem-loop adapter is used, wherein the double-stranded portion of the adapter is ligated to a double-stranded nucleic acid formed as described herein.

[0052] In some embodiments, the adapter molecule is an artificial sequence synthesized in vitro. In other embodiments, the adapter molecule is a naturally occurring sequence synthesized in vitro. In other embodiments, the adapter molecule is an isolated naturally occurring molecule or an isolated non-naturally occurring molecule.

[0053] Double-stranded or partially double-stranded adapter oligonucleotides may have overhangs or flat ends. In certain embodiments, double-stranded DNA may include flat ends, and flat end connections may be applied to the flat ends to connect flat end adapters. In other embodiments, flat end DNA undergoes A tailing, wherein a single A nucleotide is added to the flat end to match the adapter, and the adapter is designed to have a single T nucleotide extending from the flat end to facilitate the connection between DNA and the adapter. Commercially available kits for performing adapter connections include AVENIO ctDNA library preparation kits or KAPA HyperPrep and HyperPlus kits (Roche Sequencing Solutions, Pleasanton, California). In certain embodiments, the (connected) DNA of the adapter connection can be separated from the adapter and unconnected DNA in excess.

[0054] In some embodiments, the present invention includes the use of barcodes. In some embodiments, the method for detecting epigenetic modification includes sequencing. The nucleic acid treated as described herein is sequenced; preferably, large-scale parallel single molecule sequencing. Analysis of single molecules by large-scale parallel sequencing usually requires a separate level of barcoding for sample identification and error correction. The use of molecular barcodes, such as described in U.S. Patents Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678 and 8,722,368. A unique molecular barcode is added to each molecule to be sequenced to mark the molecule and its offspring (e.g., the original molecule and its amplicon generated by PCR). Unique molecular barcodes (UIDs) have multiple uses, including calculating the number and error correction of the original target molecule in the sample (Newman, A. et al., "An ultrasensitive method for quantitating circulating tumor DNA with broadpatient coverage", Nature Medicine doi: 10.1038 / nm.3519 (2014)).

[0055] In some embodiments, a unique molecular barcode (UID) is used for sequencing error correction. The entire progeny of a single target molecule is labeled with the same barcode and forms a barcode family. The variation in the sequence not shared by all members of the barcoded family is discarded as an artifact. Barcodes can also be used for positional deduplication and target quantification because the entire family represents a single molecule in the original sample (Newman, A. et al., "Integrated digital error suppression for improved detection of circulating tumor DNA", Nature Biotechnology 34: 547 (2016)).

[0056] In some embodiments of the present invention, the adapter connected to one or both ends of the barcoded target nucleic acid comprises one or more barcodes for sequencing. The barcode can be a UID or a multiple sample ID (MID or SID) for identifying the source of the sample in the mixed (multiple) sample. The barcode can also be a combination of a UID and a MID. In some embodiments, a single barcode is used as a UID and a MID. In some embodiments, each barcode comprises a predefined sequence. In other embodiments, the barcode comprises a random sequence. In some embodiments of the present invention, the length of the barcode is between about 4-20 bases, so that 96 to 384 different adapters are added to the human genome sample, each adapter having a different identical barcode pair. In some embodiments, the number of UIDs in the reaction can exceed the number of molecules to be labeled. Those of ordinary skill will recognize that the number of barcodes depends on the complexity of the sample (i.e., the expected number of unique target molecules), and will be able to create a suitable number of barcodes for each experiment.

[0057] In some embodiments, the method involves forming a library comprising nucleic acids from a sample. The library consists of a plurality of nucleic acids ready for sequencing or another type of detection method (e.g., PCR). The library can be stored and used multiple times for further processing, such as amplification or sequencing of the nucleic acids in the library. In some embodiments, the library is an input nucleic acid, wherein methylation is detected by the methods described herein. In other embodiments, the library is formed by nucleic acids that have undergone methylation detection reactions described herein.

[0058] In some embodiments, nucleic acids processed according to the methods described herein for detecting epigenetic modifications are sequenced. Any of a variety of sequencing techniques or sequencing analyses can be used. As used herein, the term "Next Generation Sequencing (NGS)" refers to a sequencing method that allows for large-scale parallel sequencing of molecules and single nucleic acid molecules that are cloned and amplified.

[0059] Non-limiting examples of sequence determination suitable for use with the methods disclosed herein include nanopore sequencing (U.S. Patent Publication Nos. US2013 / 0244340, US2013 / 0264207, US2014 / 0134616, US2015 / 0119259, and US2015 / 0337366), Sanger sequencing, capillary array sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid phase sequencing (Zimmerman et al., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing using mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., Nature 500). Biotech., 16:381-384 (1998)), sequencing by hybridization (Drmanac et al., Nature Biotech., 16:54-58 (1998)), and NGS methods, including but not limited to sequencing by synthesis (e.g., HiSeq TM 、MiSeq TM or Genome Analyzer, both available from Illumina), by ligation sequencing (e.g., SOLiD TM , Life Technologies), ion semiconductor sequencing (e.g., Ion Torrent TM , Life Technologies) and Sequencing (eg, Pacific Biosciences).

[0060] Commercially available sequencing technologies include sequencing by hybridization platform from Affymetrix Inc. (Sunnyvale, Calif.), sequencing by synthesis platform from Illumina / Solexa (San Diego, Calif.) and Helicos Biosciences (Cambridge, Mass.), sequencing by ligation platform from Applied Biosystems (Foster City, Calif.) Other sequencing technologies include, but are not limited to, Ion Torrent technology (Thermo Fisher Scientific) and nanopore sequencing (Genia Technology from Roche Sequencing Solutions, Santa Clara, Calif.) and Oxford Nanopore Technologies (Oxford, UK).

[0061] In some embodiments, the sequencing step involves sequence analysis. In some embodiments, a consensus sequence is determined from multiple sequences (e.g., multiple sequences with the same unique molecule ID (UID)) using alignment. The molecule ID is a barcode that can be added to each molecule before sequencing, or if an amplification step is included, before the amplification step. In some embodiments, the UID is present in the 5' portion of the RT primer. Similarly, the UID can appear at the 5' end of the last barcode subunit to be added to the composite barcode. In other embodiments, the UID is present in an adapter and is added to one or both ends of the target nucleic acid by connection.

[0062] In some embodiments, the consensus sequence is determined from multiple sequences that all have the same UID. It is inferred that the sequences with the same UID are derived from the same original molecule by amplification. In other embodiments, the UID is used to eliminate artifacts, that is, variations (characterized by specific UIDs) present in single molecule offspring. Such artifacts derived from PCR errors or sequencing errors can be eliminated using UIDs.

[0063] In some embodiments, the number of each sequence in the sample can be quantified by quantifying the relative number of sequences of each UID in a population with the same multiple sample ID (MID). Each UID represents a single molecule in the original sample, and counting the different UIDs associated with each sequence variant can determine the ratio of each sequence variant in the original sample, wherein all molecules share the same MID. Those skilled in the art will be able to determine the number of sequence reads necessary for determining the consensus sequence. In some embodiments, for accurate quantitative results, each UID ("sequence depth") needs to read the relevant number. In some embodiments, the desired depth is 5-50 reads per UID.

[0064] In some embodiments, the present invention is a kit comprising components and tools for performing the improved method for detecting DNA methylation as described herein. In some embodiments, the kit includes components for detecting cytosine methylation in nucleic acids by detecting the product of 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) oxidized in vitro. In some embodiments, the product is 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC). In other embodiments, the kit further includes components for performing in vitro oxidation of 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) to 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC).

[0065] In some embodiments, the kit includes an enzyme. In some embodiments, the enzyme is an ene reductase. In some embodiments, the kit further includes an organic acid. In some embodiments, the kit includes instructions for use of an organic acid (such as acetic acid) in a method for detecting DNA methylation including a borane derivative in a non-aqueous solvent as described herein. In some embodiments, the kit further includes a buffer such as MES or TRIS.

[0066] In some embodiments, the kit includes malononitrile and a non-aqueous solvent. The non-aqueous solvent is selected from ethanol and methanol. In other embodiments, the kit does not include a non-aqueous solvent, but includes instructions for use of a non-aqueous solvent (such as ethanol or methanol) in a method for detecting DNA methylation with malononitrile as described herein. In some embodiments, the kit further includes an organic acid and a primary amine, a secondary amine or a tertiary amine. The organic acid can be acetic acid, and the amine can be triethanolamine. In other embodiments, the kit includes instructions for use of an organic acid and an amine (such as acetic acid and triethanolamine) in a method for detecting DNA methylation with malononitrile as described herein. In some embodiments, the kit further includes a buffer such as MES or TRIS.

[0067] In some embodiments, the kit further comprises a TET enzyme for in vitro oxidation of 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) to 5-carboxycytosine (5caC). In some embodiments, TET is selected from mouse TET1, TET2 or TET3 (mTET1, mTET2 or mTET3); human TET1, TET2 or TET3 (hTET1, hTET2 or hTET3); Naegleria TET (NgTET); Coprinus cinereus (CcTET) or any other analog or equivalent thereof having similar or equivalent enzyme activity. In some embodiments, TET is a Naegleria TET-like oxygenase (NgTET1). In some embodiments, TET is a wild-type protein. In other embodiments, TET is a mutant protein. In some embodiments, the kit further comprises one or more cofactors selected from α-ketoglutarate and a source of Fe(II) ions.

[0068] In some embodiments, as an alternative to TET, the kit includes a chemical oxidizing agent such as potassium perruthenate (KRuO4) or potassium ruthenate (K2RuO4).

[0069] In some embodiments, the kit further includes a reagent for chemically blocking 5hmC to prevent it from reacting with 5mC. In some embodiments, the kit includes a glucose compound and a glucosyltransferase capable of transferring the glucose moiety to the 5-hydroxyl moiety of 5hmC. In some embodiments, the kit includes β-glucosyltransferase (BGT) and UDP-glucose. In some embodiments, BGT is T4 BGT.

[0070] In some embodiments, the method further includes assessing the patient's state based on the methylation state of one or more genetic loci in the subject's (e.g., patient) genome. In some embodiments, the method includes determining the genomic location in the patient sample and optionally determining the amount of methylated cytosine (5mC and / or 5hmC and / or 5fC and / or 5caC) in the genome. In some embodiments, the methylation of the genetic loci known as disease biomarkers is assessed. The method further includes diagnosing the patient's disease or illness or selecting or changing treatment based on the presence or amount of methylation in the nucleic acid isolated from the patient.

[0071] There are a variety of methods for identifying disease or condition specific methylation loci, and the methylation of the loci can be assessed using the methods disclosed herein, see, for example, U.S. Patent Publication Nos. US2020 / 0385813 "Systems and methods for estimating cell source fractions using methylation information"; US2020 / 0239965 "Source of origin deconvolution based on methylation fragments in cell-free DNA samples"; US2019 / 0287652 "Anomalous fragment detection and classification" (methylation markers indicate disease state); US2019 / 0316209 "Multi-assay prediction model for cancer detection"; US2019 / 0390257A1 "Tissue-specific methylation marker"; WO2011 / 070441 "Categorization of DNA samples"; WO2011 / 101728 "Identification of source of DNA samples"; WO2020 / 188561"Methods and systems for detecting methylation changes in DNA samples".

[0072] In some embodiments, the present invention includes methods for detecting tissue-specific DNA methylation patterns using the methylation detection methods disclosed herein. In one aspect of this embodiment, the method may further include identifying the tissue of origin of the methylated DNA present in the sample. In some embodiments, the method further includes identifying the tissue of origin of the cell-free DNA separated from the blood. In another aspect of this embodiment, the present invention includes using the methylation pattern of cell-free DNA to detect organ failure or organ damage, including organ transplant rejection in transplant recipients. The present invention includes detecting circulating cell-free DNA with organ-specific methylation patterns, wherein the presence of such cell-free DNA indicates organ transplant rejection. In some embodiments, the present invention includes monitoring transplant rejection by regularly sampling circulating cell-free DNA and measuring changes in cell-free DNA levels with organ-specific methylation patterns, wherein the increase in such cell-free DNA levels indicates organ transplant rejection.

[0073] In some embodiments, the present invention includes a method for diagnosing or screening for the presence of cancerous tumors in patients or subjects. In some embodiments, the present invention includes using the methylation detection method disclosed herein to detect tumors using the methylation pattern of cell-free DNA. In some embodiments, the present invention includes detecting tumors derived from specific tissues or organs by detecting circulating cell-free DNA with tissue- or organ-specific methylation patterns using the methylation detection method disclosed herein, wherein the presence of such cell-free DNA indicates the presence of tumors derived from the tissue or organ. In some embodiments, the present invention includes monitoring the growth or reduction of tumors by regularly sampling circulating cell-free DNA and measuring changes in cell-free DNA levels with tumor-specific methylation patterns, wherein an increase in such cell-free DNA levels indicates tumor growth, and a decrease in such cell-free DNA levels indicates tumor reduction.

[0074] In some embodiments, the present invention includes a method for monitoring the effectiveness of cancer treatment in a patient or subject. In some embodiments, the present invention includes using the methylation pattern of cell-free DNA to detect tumor dynamics associated with treatment, and the methylation pattern is detected using the methylation detection method disclosed herein. In some embodiments, the present invention includes detecting the therapeutic effect on tumors derived from specific tissues or organs by regularly sampling circulating cell-free DNA and measuring changes in the level of free DNA with tissue- or organ-specific methylation patterns, wherein an increase in the level of such cell-free DNA indicates tumor growth and ineffective treatment, and a decrease in the level of such cell-free DNA indicates tumor shrinkage and treatment effectiveness, and a stable level of such cell-free DNA indicates disease stability and treatment effectiveness.

[0075] In some embodiments, the present invention includes a method for diagnosing minimal residual disease (MRD) in a cancer patient after treatment. The National Cancer Institute of the United States defines MRD as a very small number of cancer cells remaining in the body during or after treatment when the patient has no signs or symptoms of the disease. In some embodiments, the present invention includes a method for detecting MRD using a methylation pattern of cell-free DNA, which is detected using a methylation detection method disclosed herein. In some embodiments, the present invention includes detecting MRD from a tumor originating from a specific tissue or organ by detecting circulating cell-free DNA with a tissue- or organ-specific methylation pattern, wherein the presence of such cell-free DNA indicates the presence of MRD from the tumor.

[0076] In some embodiments, the present invention includes a method for diagnosing or screening the presence or state of an autoimmune disease in a patient or subject. In some embodiments, the present invention includes detecting a tumor using a methylation pattern of cell-free DNA, which is detected using a methylation detection method disclosed herein. In some embodiments, the present invention includes detecting an autoimmune disease characterized by damage to a specific tissue or organ by detecting circulating cell-free DNA with a tissue- or organ-specific methylation pattern, wherein the presence of such cell-free DNA indicates organ damage and the presence of an autoimmune disease caused by an autoimmune disease. In some embodiments, the present invention includes monitoring the onset or remission of an autoimmune disease by regularly sampling circulating cell-free DNA and measuring changes in the level of free DNA with a tissue- or organ-specific methylation pattern, wherein an increase in the level of such cell-free DNA indicates an increase in the onset of organ damage and autoimmune disease, and a decrease in the level of such cell-free DNA indicates a decrease in organ damage and the remission of an autoimmune disease.

Claims

1. A method for reducing the C5-C6 double bond of cytosine in a nucleic acid, wherein the method comprises contacting the cytosine in the nucleic acid with an ene reductase.

2. The method of claim 1, wherein the cytosine is selected from the group consisting of 5-formylcytosine (5fC), 5-carboxycytosine (5caC), 5,6-dihydro-fC, 5,6-dihydro-caC, 2'-deoxy-5-formylcytidine, 2'-deoxy-5-carboxycytidine, 5-formyl-2'-deoxycytidine, 5-formyl-dC, 5-carboxy-2'-deoxycytidine and 5-carboxy-dC, 3. The method of claim 1, wherein the cytosine in the nucleic acid is contacted with an ene reductase to reduce the cytosine to 5,6-dihydro-U (DHU).

4. The method according to claim 3, wherein the DHU is subsequently converted to thymine.

5. The method of claim 1, wherein reduction of the C5-C6 double bond of cytosine in a nucleic acid by contacting the cytosine in the nucleic acid with an ene reductase is part of a method for detecting epigenetic cytosine modifications.

6. A method for detecting epigenetic cytosine modification, wherein the method comprises at least the step of reducing the C5-C6 double bond of cytosine in the nucleic acid by contacting the cytosine in the nucleic acid with an ene reductase.

7. The method of claim 6, wherein the cytosine is selected from the group consisting of 5-formylcytosine (5fC), 5-carboxycytosine (5caC), 5,6-dihydro-fC, 5,6-dihydro-caC, 2'-deoxy-5-formylcytidine, 2'-deoxy-5-carboxycytidine, 5-formyl-2'-deoxycytidine, 5-formyl-dC, 5-carboxy-2'-deoxycytidine and 5-carboxy-dC.

8. The method of claim 6, wherein the cytosine in the nucleic acid is contacted with an ene reductase to reduce the cytosine to 5,6-dihydro-U (DHU).

9. The method of claim 8, wherein the DHU is subsequently converted to thymine.

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