Methylation signal probe and use thereof

Abstract

Provided are a methylation signal probe and a use thereof, particularly a method for designing and preparing a DNA methylation detection probe and a use thereof, belonging to the technical field of gene detection. A probe designed and prepared according to the method can be used for specifically enriching methylation signals in a low-content gene sample, particularly for specifically enriching low-abundance methylated haplotype regions, so that 1) the capture sensitivity for low-abundance methylated haplotype regions can be improved, and 2) a smaller sequencing data volume is used to obtain the same amount of effective information.

Description

A methylation signal probe and its application Technical Field

[0001] The present invention relates to the field of genetic testing technology, particularly high-throughput genomic methylated DNA detection. In particular, the detection of circulating tumor ctDNA in subjects can be used for the diagnosis of diseases, particularly cancer. Specifically, the present invention provides a probe design and preparation method and diagnostic technology that utilizes probes to specifically enrich cancer methylation signals, a diagnostic technology that effectively improves the signal-to-noise ratio. Background Art

[0002] DNA methylation is a common epigenetic modification in the genomes of prokaryotes and eukaryotes. This DNA modification can regulate gene expression without changing the gene sequence, thereby affecting gene function, transposon silencing, chromatin interactions, cell differentiation, and growth and development.

[0003] In particular, abnormal DNA methylation is associated with many disease processes, including cancer. DNA methylation has its own specific change pattern at various stages of the occurrence and development of many tumors. DNA methylation is gradually becoming an epigenetic marker for a variety of diseases such as mammalian development and cancer. As a non-morphological molecular detection method, gene methylation analysis can avoid the limitations of cell examination and ensure the accuracy of diagnosis. It is increasingly considered to be a valuable diagnostic tool for detecting, diagnosing and / or monitoring cancer. However, DNA methylation analysis using methylation sequencing (e.g., whole genome bisulfite sequencing (WGBS)) has the disadvantages of high cost and difficulty in practical application when applied to DNA detection of species with larger genomes.

[0004] Sequence capture technology is a technique for selectively enriching specific regions of the genome. By isolating and processing the region of interest from the genome and then sequencing the target region, it is possible to study the target gene region more efficiently and in a targeted manner. The DNA probes used in traditional hybridization capture are all 120-nt oligonucleotides, that is, long probes. Long probes have a very strong binding ability to DNA templates. When there is a certain number of base mismatches between the long probe and the DNA template, the long probe can still bind to the template, that is, the long probe will capture tumor-type as well as wild-type or healthy DNA without bias. Compared with the 120nt long probes that have a long history, the design of short probes is more difficult. The key lies in the accuracy of thermodynamic predictions of probe binding to DNA templates of different sequences, which requires a relatively complete model for prediction. Since short probes are more sensitive to base mismatches, improper design may cause false positive and false negative results, and therefore they have not been widely used.

[0005] In cancer diagnosis, early detection and diagnosis of cancer are highly desirable. To this end, the use of cell-free DNA (cfDNA) fragments to target methylation patterns specific to cancer or tissues can provide a cost-effective, non-invasive method. However, the blood-free DNA (cfDNA) of early-stage patients is composed of extremely low levels of circulating tumor DNA (ctDNA, target signal) and extremely high levels of healthy cell-free DNA (wild-type cfDNA, background noise). The long probe's ability to capture these signals and noise without offset means that: 1) trace amounts of ctDNA in blood samples may not be effectively detected, resulting in a decrease in clinical sensitivity; 2) the vast majority of data in the final library is wild-type cfDNA, which contains no tumor information, leading to increased sequencing costs.

[0006] Therefore, there is a need to develop new gene methylation probe design methods that can sensitively detect DNA or RNA methylation and improve methods that can reduce sequencing costs.

[0007] Summary of the Invention

[0008] After in-depth research, the inventors have provided an optimized gene methylation probe design method based on the biological characteristics of methylation haplotypes. In particular, the designed short probes are utilized to solve the problems of high cost, long application time and limited sensitivity of the detection technology based on traditional DNA methylation hybridization capture in the prior art.

[0009] Compared to traditional long-probe DNA capture techniques without bias, this invention can: 1) improve clinical sensitivity; 2) reduce sequencing costs. The solution provided by this invention is a diagnostic technology that can effectively improve the signal-to-noise ratio, particularly addressing the technical challenges of extremely low levels of target circulating tumor DNA (ctDNA) and excessive background noise from non-tumor-associated DNA (wild-type cfDNA) in cfDNA samples from the blood of early-stage cancer patients.

[0010] The inventors exploited the thermodynamic properties of short probes (less than 120 nt, such as 20-100 nt oligonucleotides), which are highly sensitive to base mismatches in DNA templates. This means that short probes bind to specific tumor DNA templates much more efficiently than to non-tumor DNA templates. This results in the vast majority of data in the library after hybridization capture being valid tumor DNA templates. Therefore, short probe technology allows us to simultaneously track numerous methylation variant sites in ctDNA, using less sequencing data than long probes to obtain the same amount of effective information.

[0011] In particular, the probes designed according to the present invention can be used to enrich methylation haplotypes, such as tumor-associated methylation haplotypes in plasma / tissue samples (eg, cfDNA samples).

[0012] In a first aspect of the present invention, a method for designing a methylation probe is provided, particularly a method for designing a probe for specifically enriching (cancer) methylation signals, and a method for preparing a probe for enriching methylation haplotypes is provided. The method comprises:

[0013] 1) Constructing a target DNA interval containing a specific methylation haplotype sequence;

[0014] 2) Design one or more short probes for the target DNA interval based on the methylation haplotype sequence characteristics. In embodiments of the present invention, the short probes are less than 120 nt in length. In preferred embodiments, the probes designed and used in the present invention are 20-100 nt in length.

[0015] In a specific embodiment, the design scheme provided by the present invention can be used for diagnostic technology that effectively improves the signal-to-noise ratio, aiming to solve the technical problems of extremely low target signal circulating tumor DNA (ctDNA) content and excessively high background noise of non-tumor-related DNA (Wild-type cfDNA) in cfDNA samples of blood of early-stage cancer patients.

[0016] In some embodiments, during the design and preparation of the probes of the present invention, the probes are configured to hybridize to methylated sequences in the target DNA region.

[0017] In a preferred embodiment, the probes designed and prepared according to the methods of the present invention hybridize to a region including one or more CpG sites.

[0018] In a more preferred embodiment, the probes designed and prepared according to the methods of the present invention hybridize as a whole to all methylation haplotype sequences in the target DNA interval. In an even more preferred embodiment, the probes designed by the present invention are specific for methylation haplotypes associated with diseases, particularly methylation haplotypes associated with genetic diseases related to cancer or non-invasive prenatal diagnosis (NIPT).

[0019] In some embodiments, in the method of the present invention, the methylation haplotype sequence characteristics include one or more of the following: the coordinates of the target DNA interval where the methylation haplotype is located, the interval length of the methylation haplotype in the target DNA interval, the CpG site distribution of the methylation haplotype and the number of CpG sites, and the methylation pattern.

[0020] In some embodiments, the method of the present invention further comprises calculating the Gibbs free energy change (DeltaG) value of the probe, and selecting the probe according to the Gibbs free energy change (DeltaG) value.

[0021] In a preferred embodiment, based on the Gibbs free energy change (DeltaG) value, a probe having a Gibbs free energy change (DeltaG) value in the range of -10 kcal / mol to -35 kcal / mol is selected. In a more preferred embodiment, a probe having a Gibbs free energy change (DeltaG) value in the range of -15 kcal / mol to -25 kcal / mol is selected.

[0022] In a more preferred embodiment, the design temperature for calculating the Gibbs free energy change (DeltaG) value of the probe in the method of the present invention is from 20-70°C and the salt concentration is 0.05-1M.

[0023] In some embodiments, the method of the present invention further comprises calculating the physicochemical properties of the probe, wherein the physicochemical properties include GC content, melting temperature, and risk of secondary structure formation, thereby selecting a probe having a GC content of 5%-95%, more preferably 10%-90%, a melting temperature higher than the design temperature, and a secondary structure melting temperature lower than 45 degrees Celsius;

[0024] The risk of secondary structure formation is determined by probe secondary structure detection which calculates the energy and melting temperature of the sequence itself forming a hairpin structure and a self-dimer, wherein primer3-py is preferably used to calculate the energy and melting temperature of the secondary structure.

[0025] In some embodiments, the method of the present invention further comprises performing off-target detection on the probes and removing probes with off-target risks.

[0026] In a preferred embodiment, the BLAST algorithm is used for off-target detection in the method of the present invention. In a more preferred embodiment, the BLAST algorithm is optimized for specific parameters, including, for example, 1. turning off dust database filtering; 2. turning off softmasking filtering; 3. using a short sequence mode for searching. In other preferred embodiments, the BLAST algorithm is used to count the number and quality of hits to determine off-target risk.

[0027] In some embodiments, the method of the present invention further comprises performing a probe interaction test on the probes to remove probes with interaction risks, such as probes that easily form probe dimers.

[0028] In a preferred embodiment, the probe interaction detection performed in the method of the present invention includes calculating the energy and melting temperature after sequence interaction to determine the interaction risk. In a more preferred embodiment, using, for example, primer3-py to calculate the energy and melting temperature after sequence interaction.

[0029] In some embodiments, probes designed and prepared according to the methods of the present invention can be used to specifically enrich for methylation haplotypes associated with a disease, such as one or more diseases including cancer and genetic diseases associated with non-invasive prenatal diagnosis (NIPT).

[0030] In a further embodiment, the probes designed and prepared according to the method of the present invention can be used to specifically enrich tumor-associated methylation haplotypes in body fluids or tissue samples. In a preferred embodiment, the body fluid can include plasma, and the body fluid sample can include a blood cfDNA sample.

[0031] In another aspect, the present invention provides a probe capable of specifically enriching for methylated haplotype regions, particularly regions associated with diseases such as cancer and genetic diseases associated with non-invasive prenatal diagnosis (NIPT). The probe is designed and / or prepared according to the above-described method of the present invention.

[0032] In some embodiments, the probes involved in and prepared by the present invention are oligonucleotides selected from single-stranded DNA, single-stranded RNA, double-stranded complementary DNA, double-stranded complementary RNA, double-stranded incomplete complementary DNA and double-stranded incomplete complementary RNA, as well as other oligonucleotides containing base and phosphate-sugar backbone modifications, including locked nucleic acids.

[0033] In some embodiments, the probe of the present invention is an oligonucleotide, and the bases included in the oligonucleotide are canonical bases or non-canonical bases, including xanthine or hypoxanthine.

[0034] In some embodiments, the probe of the present invention may include a modification, wherein the position of the modification is selected from the 5' end, the 3' end and the middle of the probe; the modification includes biotin, digoxigenin, phosphorylation and a fluorescent group; preferably, the modification is biotin.

[0035] In another aspect, the present invention provides a method for specifically enriching methylation signals, comprising:

[0036] (a) obtaining a test sample;

[0037] (b) processing the test sample to obtain a plurality of C / mC converted test fragments; and

[0038] (c) contacting the plurality of C / mC-converted test fragments with the probes obtained according to the method of the present invention, thereby enriching the test fragments by hybridization capture.

[0039] In some embodiments, the treatment of the test sample in the methods of the present invention is chemical treatment (e.g., bisulfite), enzymatic treatment (e.g., cytosine deaminase (APOBEC)), or chemical-enzymatic hybrid treatment (e.g., TET-assisted pyridine borane sequencing (TAPS)).

[0040] In some embodiments, the method for specifically enriching methylation signals according to the present invention may further comprise the steps of:

[0041] (d) sequencing the enriched test fragments to obtain a set of multiple sequence reads, thereby performing methylation analysis.

[0042] In a more preferred embodiment, the sequencing used in the above method of the present invention includes high-throughput sequencing, such as next-generation sequencing.

[0043] In some embodiments, the test sample used in the method of the present invention is a nucleic acid sample, including but not limited to body fluid and tissue DNA samples, such as cell-free DNA (cfDNA), more preferably blood cfDNA or other body fluid cfDNA (including urine, cerebrospinal fluid, etc.). In a preferred embodiment, the test sample is blood cfDNA.

[0044] In some embodiments, the methods of the present invention include performing multiple rounds of hybridization capture. In a preferred embodiment, the methods of the present invention perform two rounds of hybridization capture, and further preferably, an enrichment is performed between the two rounds of hybridization capture. Preferably, the same probe is used in multiple rounds of hybridization capture.

[0045] In some embodiments, in the method of the present invention, the temperature of hybridization capture is gradually decreased from 95°C to 20-70°C, preferably to 40-60°C.

[0046] The technical solution provided by the present invention can be used to design probes that specifically enrich disease-related methylation signals. It can be used to specifically enrich methylation signals in low-content gene samples, especially to specifically enrich extremely low-abundance methylation haplotype regions, thereby:

[0047] 1) It can improve the sensitivity of capturing extremely low abundance methylation haplotype regions,

[0048] 2) A smaller amount of sequencing data can be used to obtain the same amount of effective information. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1: Schematic diagram of the technical process of short probes for tumor haplotype capture.

[0050] Figure 2: Shift in methylation levels across methylated reference samples.

[0051] Figure 3: Increase in the proportion of target methylation haplotypes over the methylation reference.

[0052] Figure 4: Specific enrichment of target methylation haplotypes by short probes in real samples (colon cancer).

[0053] Figure 5: Specific enrichment of target methylation haplotypes by short probes in real samples (lung cancer).

[0054] Figure 6: Specific enrichment of target methylation haplotypes by short probes in real samples (placenta). Specific implementation plan

[0055] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, synthetic biology, and the like, which are within the skill of the art. Such techniques are fully explained in the literature: "Molecular Cloning: A Laboratory Manual," 2nd ed. (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FM Ausubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction," (Mullis et al., eds., 1994); Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994) and March's Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th ed., John Wiley & Sons (New York, NY 1992), provide those skilled in the art with a general guide to many of the terms used in this application.

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the purposes of the present invention, the following terms are defined below.

[0057] The articles "a / an" and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. The term "and / or" should be understood to mean any one or both of the alternatives.

[0058] As used herein, the term "about" or "approximately" refers to a quantity, level, value, amount, frequency, percentage, dimension, size, amount, weight or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0059] Throughout this specification, unless the context requires otherwise, the terms "comprises," "includes," "contains," and "has" should be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In certain embodiments, the terms "comprises," "includes," "contains," and "have" are used synonymously.

[0060] “Consisting of means including, but not limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and no other elements may be present.

[0061] “Consisting essentially of is meant to include any elements listed after the phrase “consisting essentially of” and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase “consisting essentially of is intended to indicate that the listed elements are required or mandatory, but no other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0062] Throughout this specification, references to "one embodiment," "some embodiments," "a specific embodiment," and similar expressions mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0063] As used herein, term " base ", also known as core base, nitrogenous base, is the nitrogenous compound that forms nucleoside, constitutes the basic building block of nucleic acid. Common base has 5 kinds in organism, is respectively adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), also referred to as classical base. They are the basic units that form genetic code, and wherein base A, G, C and T are present in DNA, and A, G, C and U are present in RNA. Additionally, base can also be non-classical base, and these bases are mostly the derivatives that are formed by methylating at the different positions of above-mentioned purine or pyrimidine base or carrying out other chemical modification, including for example hypoxanthine and xanthine.

[0064] The term "methylation" as used herein refers to the addition of a methyl group to a DNA molecule. A "CpG" site is the most common methylation site, but methylation sites are not limited to CpG sites. For example, DNA methylation may occur in the cytosine of CHG and CHH, where H is adenine, cytosine, or thymine. The term "CpG site" as used herein refers to a region in a DNA molecule where a cytosine nucleotide is followed by a guanine nucleotide in a linear sequence of bases along its 5' to 3' direction. "CpG" is an abbreviation for 5'-C-phosphate-G-3', which is a cytosine and guanine separated by only one phosphate group. Multiple cytosines in a CpG dinucleotide can be methylated to form 5-methylcytosine.

[0065] Methylation haplotype blocks (MHBs) are regions of tightly coupled CpG sites in the human genome. Biologically, MHBs are regions of the genome that are often tightly co-regulated in terms of epigenetic state.

[0066] "Biological sample" refers to any sample taken from an individual (e.g., a human, such as a cancer patient or a person suspected of having cancer) and containing one or more nucleic acid molecules of interest. A biological sample can be a body fluid, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal washings, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, etc. Stool samples can also be used. In various embodiments, a majority of the DNA in a biological sample enriched for cell-free DNA (e.g., a plasma sample obtained by a centrifugation protocol) can be cell-free (as opposed to cells), e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the DNA can be cell-free.

[0067] The terms "cell-free nucleic acid," "cell-free DNA," or "cfDNA" refer to a collection of nucleic acid fragments circulating in an individual's body (e.g., bloodstream) that originate from one or more healthy cells and / or one or more cancer cells. In addition, cfDNA may come from other sources, such as viruses and fetal DNA.

[0068] The term "circulating tumor DNA" or "ctDNA" refers to nucleic acid fragments originating from tumor cells that are released into an individual's blood as a result of biological processes such as apoptosis or necrosis of dying cells or active release by living tumor cells.

[0069] The methods and probes of the present invention can be used to diagnose diseases, particularly cancers, which can be melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gum cancer, tongue cancer, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, sarcoma, or bladder cancer. Cancer can include tumors composed of tumor cells.

[0070] In the probe design of the present invention, the method used for calculating the Gibbs free energy change (DeltaG) value of the probe can be the thermodynamic calculation performed by the DNA / DNA thermodynamics parameters of Santa Lucia in 2004. According to the existing knowledge of probe design, the length of the optimal probe can be determined based on the Gibbs free energy change and the physicochemical properties of the probe. In the present invention, the Gibbs free energy change range is set according to theoretical planning and a large amount of experience. Taking into account the stability of the experimental system and the inclusion of as many candidate probe sequences as possible under feasible calculation amount, the length of the designed probe can be 20-100nt, and more preferably 30-60nt. As an example and not limitation, for example, for a DNA sequence rich in GC, the length of the designed probe can be shorter (GC binds more strongly, three hydrogen bonds), while for a DNA sequence rich in AT (AT binds two hydrogen bonds, which is weaker), the length of the designed probe will be longer.

[0071] The probe designed in the present invention can select the target DNA interval for which it is targeted according to actual needs, especially for one or more CpG sites therein. For example, CpG sites with significantly higher / lower methylation levels in tumor samples (compared with the methylation levels in healthy samples) can be screened out through preliminary calculations. For example, probes can be designed for CpG sites that are close to each other, and the number of CpG sites that can be covered by one probe ranges from 1 to 30. Additionally, a sequence that does not contain other non-target CpG sites within 50nt before and after the CpG site can be selected as the target DNA interval for designing probes. And after screening by the present invention, an optimal probe is retained for each methylation pattern on each target region. In a preferred embodiment, the designed probe is a group of probes, which may include but are not limited to 1 to 100, 1 to 1000, 1 to 10000, or even 1 to 10 9 probes, wherein it will be appreciated that the number of probes depends on the number of target DNA regions and the actual design method.

[0072] In the present invention, the short probe is a probe having a length of less than about 120 nt, for example, less than about 119, 118, 117, 116, 115, 114, 113, 112, 111 or 110 nt, less than about 105 nt, less than about 100 nt, less than about 90 nt, less than about 80 nt, less than about 70 nt, less than about 60 nt, less than about 50 nt, less than about 40 nt or less than about 30 nt. In some embodiments, the length of the probe is, for example, about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 to about 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 nt, and any lengths therebetween. For example, the length of the short probes of the present invention can be about 5-120 nt, 5-115 nt, 5-110 nt, 5-105 nt, 5-100 nt, 5-90 nt, 5-80 nt, 5-70 nt, 5-60 nt, 5-50 nt, 10-120 nt, 10-115 nt, 10-110 nt, 10-105 nt, 10-100 nt, 10-90 nt, 10-80 nt, 10-70 nt, 10-60 nt, 10-50 nt, 15-120 nt, 15-115 nt, 15-110 nt, 15-105 nt, 15-100 nt, 15-90 nt, 15-80 nt, 15-70 nt, 15-60 nt, 15-50 nt, 0nt, 20-120nt, 20-115nt, 20-110nt, 20-105nt, 20-100nt, 20-90nt, 20-80nt, 20-70nt, 20-60nt, 20-50nt, 25-120nt, 25-115nt, 25-110nt, 25-105nt, 25-100nt, 25-90nt, 25-80nt, 25-70nt, 25-60nt, 25-50nt, 30-120nt, 30-115nt, 30-110nt, 30-105nt, 30-100nt, 30-90nt, 30-80nt, 30-70nt, 30-60nt or 30-50nt.

[0073] In the present invention, the probe is selected to have a Gibbs free energy change (DeltaG) value in the range of about -10 kcal / mol to -35 kcal / mol, such as about -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20 kcal / mol to about -25, -26, -27, -28, -29, -30, -31, -32, -33, -34 or 35 kcal / mol, and ranges between any values ​​therein. For example, the probe may be selected to have a Gibbs free energy change (DeltaG) value in the range of approximately -10 kcal / mol to -25, -26, -27, -28, 29, -30, -31, -32, -33, -34, or -35 kcal / mol, -11 kcal / mol to -25, -26, -27, -28, 29, -30, -31, -32, -33, -34, or -35 kcal / mol, -12 kcal / mol to -25, -26, -27, -28, 29, -30, -31, -32, -33, -34, or -35 kcal / mol, -13 kcal / mol to -25, -26, -27, -28, 29, -30, -31, -32, -33, -34, or -35 kcal / mol, or -20 kcal / mol to -25, -26, -27, -28, 29, -30, -31, -32, -33, -34 or -35 kcal / mol; or -20 kcal / mol to -25, -26, -27, -28, 29, -30, -31, -32, -33, -34 or -35 kcal / mol.

[0074] In the present invention, the design temperature for calculating the Gibbs free energy change (DeltaG) value of the probe can be approximately from 20-70°C, for example, about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 to about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70°C, and ranges between any values ​​therein. For example, the design temperature may be approximately 20-70°C, 25-65°C, 30-60°C, 35-55°C, 25-70°C, 30-65°C, 35-60°C, 40-55°C, 30-70°C, 35-65°C, 40-60°C, 45-55°C, 35-70°C, 40-65°C, 45-60°C or 50-55°C.

[0075] In the present invention, the salt concentration for calculating the Gibbs free energy change (DeltaG) value of the probe can be about 0.05-1 M, for example, about 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 94, 0.95, 0.96, 0.97, 0.98, 0.99 or 1 M, and ranges between any values ​​therein. For example, the salt concentration can be about 0.05-1M, 0.05-0.95M, 0.05-0.9M, 0.05-0.85M, 0.05-0.8M, 0.05-0.75M, 0.05-0.7M, 0.05-0.65M, 0.05-0.6M, 0.05-0.55M, 0.05-0.5M, 0.1-1M, 0.1-0.9M, 0.1-0.8M, 0.1-0.7M, 0.1-0.6M, 0.1-0.5M, 0.2-1M, 0.2 -0.9M, 0.2-0.8M, 0.2-0.7M, 0.2-0.6M, 0.2-0.5M, 0.3-1M, 0.3-0.9M, 0.3-0.8M, 0.3-0.7M, 0.3-0.6M, 0.3-0.5M, 0.4-1M, 0.4-0.9M, 0.4-0.8M, 0.4-0.7M, 0.4-0.6M, 0.4-0.6M, 0.5-1M, 0.5-0.9M, 0.5-0.8M, 0.5-0.7M or 0.5-0.6M.

[0076] In the present invention, the GC content of the selected probe can be about 5%-95%, for example, in the range of about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45% to about 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%, and ranges between any values ​​therein. For example, the GC content may be about 5%-95%, 5%-90%, 5%-85%, 5%-80%, 5%-75%, 5%-70%, 5%-65%, 5%-60%, 5%-55%, 5%-50%, 10%-90%, 10%-90%, 10%-80%, 10%-80%, 10%-70%, 10%-70%, 10%-60%, 10%-60%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-50%, 10%-90%, 10%-90%, 10%-80%, 10%-80%, 10%-80%, 10%-70%, 10%-70%, 10%-6 ... 0%, 15%-95%, 15%-90%, 15%-85%, 15%-80%, 15%-75%, 15%-70%, 15%-65%, 15%-60%, 15%-55%, 15%-50%, 20%-90%, 20%-90%, 20%-80%, 20%-80%, 20%-70%, 20%-70%, 20%-60%, 20%-60%, 20%-50%, 20%-50 %, 25%-95%, 25%-90%, 25%-85%, 25%-80%, 25%-75%, 25%-70%, 25%-65%, 25%-60%, 25%-55%, 25%-50%, 30%-930%, 30%-90%, 30%-80%, 30%-80%, 30%-70%, 30%-70%, 30%-60%, 30%-60%, 30%-50%, 30%-50 %, 35%-95%, 35%-90%, 35%-85%, 35%-80%, 35%-75%, 35%-70%, 35%-65%, 35%-60%, 35%-55%, 35%-50%, 40%-90%, 40%-90%, 40%-80%, 40%-80%, 40%-70%, 40%-70%, 40%-60%, 40%-60%, 40%-50% or 40%-50%.

[0077] In the present invention, the melting temperature of the secondary structure of the probe can be about less than 45 degrees Celsius, for example, about less than 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20 degrees Celsius.

[0078] In the present invention, the temperature of hybridization capture using illumination can be gradually decreased from about 95°C (e.g., about 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81 or 80°C) to about 20-70°C, such as about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 to about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70°C, and ranges therebetween. For example, the temperature of the hybrid capture can be gradually decreased to about 20-70°C, 20-65°C, 20-60°C, 20-55°C, 20-50°C, 25-70°C, 25-65°C, 25-60°C, 25-55°C, 25-50°C, 30-70°C, 30-65°C, 30-60°C, 30-55°C, 30-50°C, 35-70°C, 35-65°C, 35-60°C, 35-55°C, 35-50°C, 40-70°C, 40-65°C, 40-60°C, 40-55°C, 40-50°C, 45-70°C, 45-65°C, 45-60°C, 45-55°C or 45-50°C.

[0079] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0080] To effectively amplify and accurately detect the methylation levels of corresponding sites, the embodiments of the present invention provide corresponding probe and primer design principles. Based on the primer design principles or principles disclosed in the embodiments of the present invention, those skilled in the art may adjust, replace, or combine them as needed in actual situations, and the synthesized probes are all within the scope of protection of this application.

[0081] Example

[0082] Hereinafter, the present invention will be described in detail by way of examples. However, the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

[0083] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0084] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0085] Example 1. Design of short probes for colorectal cancer-related methylation haplotype regions

[0086] Colorectal cancer-related regions were selected as targets, and probes were designed according to the method provided by the present invention.

[0087] 1. We obtained tumor-specific methylation haplotype sequences and their characteristics in colorectal cancer from the upstream methylation haplotype algorithm. The specific input format is: methylation haplotype coordinates, methylation haplotype interval length, and the pattern of all CpG sites included in the methylation haplotype.

[0088] 2. For the haplotypes obtained in each haplotype interval, in order to meet the requirements of length and coverage in probe design, we extended the haplotype sequence to both ends. The extended sequence does not contain CpG sites. Considering the computational complexity, we extended the maximum length of one side to 50nt.

[0089] 3. The original sequence is annotated according to the methylation haplotypes of healthy and tumor tissues. The sequence is then methylated, converting demethylated Cs to Ts while leaving methylated Cs unchanged. This constructs a target DNA interval encompassing the methylation haplotype pattern.

[0090] 4. Slide the window of 20-100nt to generate all possible candidate probe sequences

[0091] 5. Calculate physicochemical properties and thermodynamic parameters. After preliminary screening of each probe based on indicators such as Gibbs free energy change (DeltaG), physicochemical properties, and secondary structure risk, use BLAST to compare the corresponding methylation haplotype patterns, calculate the number and quality of hits, and determine the off-target risk.

[0092] 6. Perform pairwise probe interaction calculations and evaluation on candidate probes that meet the requirements in 5. For each methylation pattern in each target region with multiple candidate probes, we will select and retain one probe based on the degree of overlap and physicochemical properties.

[0093] The probe finally selected was named "SP-PoC-1" as shown in Table 1:

[0094] Example 2. Specific enrichment of methylation standards using probe SP-PoC-1

[0095] We used the short probe SP-PoC-1 designed in Example 1 to conduct enrichment experiments on the methylation reference HCT116 at different ratios, and verified that the short probe can specifically enrich the targeted target region.

[0096] 1. Sample preparation

[0097] Commercial human colon cancer cell line HCT116, three methylation level references: 100% methylation, 0% methylation, and a proportional mixture of 50% methylation.

[0098] 2. Hybridization capture and sequencing

[0099] The SP-PoC-1 probe set was used to perform library construction, hybridization capture, and NGS sequencing on three samples according to the optimal conditions.

[0100] 3. Data processing and analysis

[0101] Sequencing data were aligned using BWA-meth, and site methylation levels were calculated using Methy-Dackel. The average methylation level within each target haplotype interval was calculated, as well as the frequency of the target haplotype in the targeted region.

[0102] i. Shift in methylation levels:

[0103] For the same sample, a traditional 120nt four-stranded long probe (left) and an enriched short probe (right) were used for hybridization capture. The results are shown in Figure 2: the Y-axis represents the average methylation level (Average Beta) of the capture region for the long and short probes, respectively, and the X-axis represents the average methylation level at the same position in the corresponding WGBS sample.

[0104] As can be seen in the left figure, using the 120nt four-stranded probe, there is no significant shift in methylation levels before and after hybridization capture (no enrichment, no shift), while the right figure shows a significant shift in methylation levels before and after hybridization capture using the short probe. After capture with the hypermethylated (Hyper) probe, the average methylation level in the region is significantly higher than that of the WGBS. Conversely, the average methylation level in the region captured with the demethylated (Hypo) probe is significantly lower than that of the WGBS. Therefore, the short probe specifically enriches for the methylated molecule type it targets.

[0105] ii. Increase in the proportion of methylated haplotypes:

[0106] Figure 3 shows the proportion of all targeted methylation haplotypes in the region in the three reference samples. Among them, the X-axis is the proportion of the target tumor haplotype in WGBS (the number of tumor haplotype molecules divided by the number of all molecules in the region), and the Y-axis is the proportion of the target tumor haplotype after short probe capture. The left picture shows the region targeted by the hypermethylation probe; the right picture shows the region targeted by the demethylation. If the probe is not enriched, the coordinate of the X-axis of the same site should be close to the coordinate of the y-axis, and a large number of points should be near y=x. When the probe is enriched, the proportion of the target tumor haplotype after capture (Y-axis) should be higher than the proportion of the target tumor haplotype in WGBS (X-axis), that is, a large number of points should be in the upper left of y=x. The shading in the figure is the density of the points.

[0107] We can see that in both high-methylation and low-methylation areas, the frequencies of the tumor haplotypes we targeted increased significantly compared with the baseline WGBS, indicating that the target methylation haplotypes were specifically enriched at the molecular level.

[0108] Example 3. Specific enrichment of colorectal cancer samples using probe SP-PoC-1

[0109] We used the short probe SP-PoC-1 designed in Example 1 to perform hybridization capture experiments in real samples (colon cancer tissue and adjacent tissue samples), and verified that the short probe can specifically enrich the targeted target region.

[0110] Figure 4 shows the enrichment of target methylation haplotypes in real colorectal cancer tissue and paired adjacent normal tissue samples. As described in the methylation haplotype ratio section in Example 2, we also observe that the short probe significantly enriches for the target methylation haplotype in the tumor tissue sample. Compared to unbiased WGBS, the short probe technology significantly increases the frequency of the target methylation haplotype in the data, improving the signal-to-noise ratio. Furthermore, the difference in enrichment frequency between cancer tissue and paired adjacent normal tissue demonstrates the ability of the short probe technology to effectively distinguish cancer tissues, confirming its clinical utility.

[0111] Example 4. Specific enrichment of lung cancer samples using probe SP-PoC-1-1

[0112] We used a subset of the short probe SP-PoC-1 designed in Example 1 to perform hybridization capture experiments in real samples (lung cancer tissue and adjacent tissue samples), and verified that the short probes can specifically enrich the targeted target region.

[0113] Table 2: Subset of short probe SP-PoC-1

[0114] Figure 5 shows real lung cancer tissue and adjacent adjacent samples. As described in the methylation haplotype ratio section in Example 3, we can also observe that the short probe has a significant enrichment effect on the target methylation haplotype in the tumor tissue sample: compared with the unbiased WGBS, the short probe technology greatly increases the frequency of the target methylation haplotype in the data and improves the signal-to-noise ratio.

[0115] Example 5. Specific enrichment on placental DNA samples

[0116] In the field of non-invasive prenatal testing (NIPT), short probes can also specifically enrich targets. We selected potential methylation markers for NIPT as target sites and designed the short probe SP-PoC-NIPT. We performed hybridization capture experiments in placental DNA samples and verified that the short probe can specifically enrich the targeted target sites.

[0117] The probe design method in Example 5 is the same as that in Example 1, except that a different target is selected from Example 1, that is, the NIPT-related region is selected as the target, and the probe is designed according to the method provided by the present invention. Specifically:

[0118] The placenta-specific methylation haplotype sequences and their characteristics in NIPT were obtained from the existing technology. The specific input format is: the coordinates of the methylation haplotype, the interval length of the methylation haplotype, and the pattern of all CpG sites included in the methylation haplotype.

[0119] Table 3: Short Probe SP-PoC-NIPT

[0120] Figure 6 shows the specific enrichment results of real placenta samples. As described in the methylation haplotype ratio section in Examples 3 and 4, we can also observe that the short probe has a significant enrichment effect on the target methylation haplotype in the placenta sample: compared with the unbiased WGBS, the short probe technology greatly increases the frequency of the target methylation haplotype in the data and improves the signal-to-noise ratio.

[0121] While various implementations of the present disclosure have been described above, the foregoing description is intended to be illustrative, not exhaustive, and not limited to the disclosed implementations. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described implementations. The terminology used herein is selected to best explain the principles of the implementations, their practical applications, or improvements to existing technologies, or to enable others skilled in the art to understand the various implementations disclosed herein.

Claims

1. A method for preparing a probe for enriching methylation haplotypes, comprising: 1) constructing a target DNA interval containing a specific methylation haplotype sequence according to the sequence; 2) According to the methylation haplotype sequence characteristics, one or more short probes are prepared for the target DNA interval, wherein the short probe is a probe with a length of less than 120 nt.

2. The method according to claim 1, wherein the probe is 20-100 nt in length; The probe is configured to hybridize to a methylated sequence in a target DNA interval; Preferably, the probe hybridizes to a region including one or more CpG sites; Preferably, the probe hybridizes as a whole to all methylation haplotype signature sequences in the target DNA interval, and preferably the methylation haplotype is associated with the disease.

3. The method according to claim 1, wherein the methylation haplotype sequence characteristics include one or more of the following: the coordinates of the target DNA interval where the methylation haplotype is located, the interval length of the methylation haplotype in the target DNA interval, the CpG site distribution of the methylation haplotype and the number of CpG sites, and the methylation pattern.

4. The method according to claim 1 further comprises calculating the Gibbs free energy change (DeltaG) value of the probe and selecting the probe according to the Gibbs free energy change (DeltaG) value, preferably the selected probe has a Gibbs free energy change (DeltaG) value in the range of -10 kcal / mol to -35 kcal / mol, more preferably a Gibbs free energy change (DeltaG) value in the range of -15 kcal / mol to -25 kcal / mol.

5. The method according to claim 4, wherein the design temperature for calculating the Gibbs free energy change (DeltaG) value of the probe is from 20-70°C and the salt concentration is 0.05-1M.

6. The method according to claim 1, further comprising calculating the physicochemical properties of the probe, wherein the physicochemical properties include GC content, melting temperature, and risk of secondary structure formation, so as to select a probe having a GC content of 5%-95%, more preferably 10%-90%, a melting temperature higher than the design temperature, and a melting temperature of the secondary structure lower than 45 degrees Celsius; The risk of secondary structure formation is determined by probe secondary structure detection which calculates the energy and melting temperature of the sequence itself forming a hairpin structure and a self-dimer, wherein primer3-py is preferably used to calculate the energy and melting temperature of the secondary structure.

7. The method according to claim 1 further comprises performing off-target detection on the probes to remove probes with off-target risks.

8. The method according to claim 7, wherein off-target detection is performed using a BLAST algorithm, Preferably, specific parameters of BLAST are optimized, including 1. turning off dust database filtering; 2. turning off softmasking filtering; 3. using short sequence mode for searching; And count the number and quality of hits to determine off-target risks. 9 . The method according to claim 1 , further comprising performing a probe interaction test on the probes to remove probes that have a risk of interaction and are prone to forming probe dimers.

10. The method according to claim 9, wherein the probe interaction detection comprises calculating the energy and melting temperature after sequence interaction to determine the interaction risk, wherein primer3-py is preferably used to calculate the energy and melting temperature after sequence interaction.

11. The method according to any one of claims 1-10, wherein the probe is used to specifically enrich for methylation haplotypes associated with diseases, including cancer and genetic diseases associated with non-invasive prenatal diagnosis (NIPT).

12. The method according to any one of claims 1-10, wherein the probe is used to specifically enrich disease-related methylation haplotypes in body fluids or tissue samples, the body fluids comprising plasma, and the body fluid samples comprising blood cfDNA samples.

13. A probe prepared according to the method of claims 1-10, which specifically enriches for methylated haplotype regions, wherein the probe is an oligonucleotide selected from single-stranded DNA, single-stranded RNA, double-stranded complementary DNA, double-stranded complementary RNA, double-stranded incomplete complementary DNA and double-stranded incomplete complementary RNA and other oligonucleotides containing bases and phosphate-sugar backbone modifications, including locked nucleic acids, and the bases included in the oligonucleotides are classical bases or non-classical bases, including xanthine or hypoxanthine.

14. The probe according to claim 13, wherein the probe comprises a modification, wherein the position of the modification is selected from the 5' end, the 3' end and the middle of the probe; the modification comprises biotin, digoxigenin, phosphorylation and a fluorescent group; preferably, the modification is biotin.

15. A method for specifically enriching methylation signals, the method comprising: (a) obtaining a test sample; (b) processing the test sample to obtain a plurality of C / mC converted test fragments; and (c) contacting the plurality of C / mC-converted test fragments with the probes obtained according to the method of claims 1 to 12, thereby enriching the test fragments by hybridization capture.

16. The method according to claim 15, wherein the treatment comprises chemical treatment, enzyme treatment and chemical-enzyme hybrid treatment, wherein the chemical treatment comprises the use of bisulfite, the enzyme treatment comprises the use of cytosine deaminase (APOBEC), and the chemical-enzyme hybrid treatment comprises the use of TET-assisted pyridine borane sequencing (TAPS).

17. The method according to claim 15, further comprising: (d) sequencing the enriched test fragments to obtain a set of multiple sequence reads, thereby performing methylation analysis, The sequencing includes high-throughput sequencing, preferably next-generation sequencing.

18. The method according to claim 15, wherein the test sample is a nucleic acid sample, including body fluid and tissue DNA samples, preferably cell-free DNA (cfDNA), more preferably blood cfDNA or other body fluid cfDNA, wherein the other body fluids include urine or cerebrospinal fluid.

19. The method according to claim 15, comprising performing multiple rounds of hybridization capture, preferably performing two rounds of hybridization capture, and further preferably performing an enrichment between the two rounds of hybridization capture, wherein preferably the multiple rounds of hybridization capture use the same probe.

20. The method according to claim 15, wherein the temperature of the hybridization capture is gradually decreased from 95°C to 20-70°C, preferably to 40-60°C.

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