Age detection method based on DNA methylation sequencing of liver tissue

Abstract

This application relates to an age detection method based on DNA methylation of liver tissue, mainly including the steps of: (a) extracting genomic DNA from liver tissue; (b) digesting the genomic DNA with MspI; (c) filling in the ends of the digestion products in step (b), adding A at the ends, and adding adapters; (d) performing bisulfite conversion on the products in step (c); (e) amplifying the products in step (d), recovering the target DNA fragments, and obtaining a methylation sequencing library; (f) performing second-generation sequencing and data analysis on the methylation sequencing library; (g) substituting the results of the data analysis into the methylation age detection model formula to calculate the age of the mouse.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly, to a method for detecting age based on methylation sequencing. Background Art

[0002] In the research of the field of aging, mice, as one of the species closest to humans, are often used as model species to explore the aging mechanism. In the research of the aging mechanism, especially in the research of intervening in the aging process, it is very important to accurately detect the age of mice. Common methods for detecting the age of mice are mainly based on physiology and behavior, such as body weight method, molar wear method, crystal dry weight method, body length method, head bone dry weight method, behavior test, etc.

[0003] The commonly used methods for detecting the age of mice at present all have obvious disadvantages. For example, the body weight method is greatly affected by factors such as the gastric contents and reproductive status of the mice, and there is a lot of overlap in the values between different age groups, and there may be certain errors in age division; the body length method is prone to human error due to the influence of the proficiency of the measurer and the state of the mouse body; the crystal dry weight method has a large workload, strict requirements and high operation difficulty; the molar wear method has certain limitations due to the influence of food ratio, molar growth, etc. In recent years, there are also methods for determining the age of mice based on the expression of molecular markers, but the accuracy is not high and generally needs to be combined with physiological characteristics to judge the age of mice.

[0004] Based on the existing detection methods, there is an urgent need for an age detection method with high accuracy and easy operation in aging research. Summary of the Invention

[0005] The purpose of the present invention is to establish a method for detecting age based on DNA methylation sequencing of liver tissue, which is applicable to the construction of DNA methylation libraries of liver tissue, second-generation sequencing and age detection of mice.

[0006] The present application relates to a method for detecting age based on DNA methylation sequencing of liver tissue, and the specific steps are as follows:

[0007] (a) Extract genomic DNA from mouse liver tissue;

[0008] (b) Digest the genomic DNA with MspI;

[0009] (c) Fill in the digested product in step (b), add A at the end, and add adapters;

[0010] (d) Perform bisulfite conversion on the product in step (c);

[0011] (e) Amplify the product in step (d), recover the target DNA fragment, and obtain a methylation sequencing library;

[0012] (f) Subject the methylated sequencing library to second-generation sequencing and data analysis;

[0013] (g) Substitute the results of data analysis into the methylation age detection model formula to calculate the age of the mouse. The formula of the methylation age detection model formula is: x is the methylation degree of the site, n is the termination value 371 of the accumulation formula, representing a total of 371 methylation sites. The methylation sites are shown in the CpG site and corresponding parameter table in Table 1. i is the index of the current item in the accumulation process, and β i See the CpG site and corresponding parameter table in Table 1.

[0014] In some embodiments, the amount of liver tissue in step (a) is 1 mg - 25 mg.

[0015] Furthermore, in step (b), the amount of genomic DNA is 100 ng - 1 μg.

[0016] Furthermore, the specific steps of MspI digestion in step (b) are: Take 100 ng of genomic DNA from each sample and digest it with 10 Units of MspI (R0106S, NEB) at 37°C for more than 16 hours. The reason for using MspI is that this endonuclease is not sensitive to methylation sites and the digestion is not affected by methylation sites. Other endonucleases are sensitive to methylation sites and cannot perform digestion when encountering methylation sites.

[0017] In some embodiments, the sequences of the adapters in step (c) contain 5-methylcytosine, and the sequences are SEQ ID NO:1 and SEQ ID NO:2. Containing 5-methylcytosine is to ensure that the bases do not change during subsequent bisulfite conversion. If this treatment is not done, the bases will change during subsequent bisulfite conversion, resulting in sequencing failure.

[0018] Furthermore, in step (c), the adapters need to be pre-annealed. Anneal the synthesized P5 containing 5-methylcytosine (sequence shown in SEQ ID NO:1) with P7 (sequence shown in SEQ ID NO:2) at 95°C for 10 min and then cool it at a rate of 0.1°C / s to 15°C. After 5 min at 15°C, cool it to 4°C. Through pre-annealing, anneal P5 containing 5-methylcytosine with P7 to synthesize the Illumina adapter. Among them, the annealing conditions are 10 ul of P5 (100 uM), 10 ul of P7 (100 uM), 3 ul of Tris-HCl (10 mM) pH 8.0, and 77 ul of ddH2O. Using the illumina adapter is to match the subsequent sequencing with the illumina instrument. The advantage of adding the adapter first and then performing bisulfite conversion is that the starting dosage requirement is low.

[0019] Furthermore, the digested product in step (b) is filled in a 50 μl reaction system, and the 50 μl reaction system contains 10 U of Klenow fragment enzyme (3'→5' exonuclease, NEB), 40 μM of dGTP, 40 μM of 5'-methylated dCTP (Roche), 400 μM of dATP, and 1× NEB buffer 2. The filling step includes: incubating the digested product in step (b) in the 50 μl reaction system at 30 °C for 20 minutes, and then incubating at 37 °C for 20 minutes. The filled DNA fragment is ligated to the Illumina adapter obtained in step (3).

[0020] Furthermore, the 50 μl reaction system further contains 2000 U of T4 ligase (NEB), 10 μl of adenylated DNA, and 0.5 to 1.0 μM of adapter, and is ligated at 16 °C for more than 16 hours.

[0021] In some embodiments, in step (d), the product in step (c) is subjected to bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen) according to its operating steps.

[0022] In some embodiments, the specific steps for amplifying the product in step (d) are as follows: Take 20 μl of the converted DNA obtained in step (d), add 25 μl of KAPA HiFi HotStart ReadyMix (2X) (Roche), 5 μl of Library Amplification Primer Mix (10X) (Roche), mix well and then perform a PCR reaction. After 16 cycles, extend at 72 °C for 1 min and store at 4 °C; wherein, the conditions of the PCR reaction are: pre-denaturation at 98 °C for 45 s, denaturation at 98 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s.

[0023] Furthermore, in step (e), the steps for recovering the target DNA fragment from the obtained PCR product are as follows: Electrophorese the obtained PCR product on a 6% polyacrylamide gel at a voltage of 120 V for 1 h. Subsequently, stain with Gelred dye at a concentration of one ten-thousandth for 5 minutes, then observe and photograph under an ultraviolet lamp, and cut and recover the band between 180 - 260 bp.

[0024] Furthermore, in step (e), the Agilent 2100 Bioanalyzer is used to detect the fragment length range of the above methylation sequencing library to obtain the prepared methylation sequencing library.

[0025] Furthermore, the recovered target DNA fragment in step (e) is 180 - 260 bp.

[0026] A marker for the age detection method based on liver tissue DNA methylation sequencing described above, the marker is derived from DNA methylation sites in mouse liver tissue, and is characterized in that the methylation sites include: chr1:15312761, chr1:33736808, chr1:33745240, chr1:34198870, chr1:53457896, chr1:71603645, chr1:72858391, chr1:74579588, chr1:75469776, chr1:89145594, chr1:131289544, chr1:131292549, chr1:132360639, chr1:133088376, chr1:133185854, chr1:136122008, chr1:155717621, chr1:161020252, chr1:165310605, chr1:170599741, chr1:186083651, chr1:186968288, chr1:189836555, chr1:192741266, chr10:6830136, chr10:38599832, chr10:42197867, chr10:43690682, chr10:59467730, chr10:59467736, chr10:61167017, chr10:61201662, chr10:62393775, chr10:68097490, chr10:69973651, chr10:69989530, chr10:75969927, chr10:76388972, chr10:76614625, chr10:76614665, chr10:77042820, chr10:80638086, chr10:81370697, chr10:81514368, chr10:81514379, chr10:117076866, chr10:119108813, chr10:119490954, chr10:121496157, chr10:121607037, chr10:121607055, chr10:121973100, chr10:127556923, chr10:127689489, chr11:3127214, chr11:4149986, chr11:6798051, chr11:20020791, chr11:36023996, chr11:46265946, chr11:55062348, chr11:57501620,chr11:59331130, chr11:60205837, chr11:60721791, chr11:69406372, chr11:72913128, chr11:72913156, chr11:74636006, chr11:75446014, chr11:77104440, chr11:78011692, chr11:78171155, chr11:83212918, chr11:87862824, chr11:96772861, chr11:97351621, chr11:100802519, chr11:101579036, chr11:106073325, chr11:109559799, chr11:113624670, chr11:118063887, chr11:119264605, chr11:120272249, chr11:120274030, chr11:120284505, chr11:120284523, chr11:120284531, chr11:121138571, chr12:29832377, chr12:38805046, chr12:39875500, chr12:51909894, chr12:78468284, chr12:78468290, chr12:79268766, chr12:79276390, chr12:80989871, chr12:81314921, chr12:81357039, chr12:83350859, chr12:85816703, chr12:91647550, chr12:99903559, chr12:100940089, chr12:109973506, chr12:110551097, chr12:110640246, chr12:118458777, chr13:12861299, chr13:13964924, chr13:42183697, chr13:42183756, chr13:42768983, chr13:46758810, chr13:47049329, chr13:49194957, chr13:49194999, chr13:49195006, chr13:49379830, chr13:55638189, chr13:56750481, chr13:58672986, chr13:75810626, chr13:84247021, chr13:92682831chr13:99007192, chr13:105846560, chr14:19768531, chr14:20727577, chr14:30569075, chr14:30901250, chr14:51920496, chr14:55863549, chr14:56116055, chr14:59586457, chr14:75331930, chr14:79619032, chr14:101929333, chr15:8280995, chr15:10146661, chr15:11391683, chr15:25926894, chr15:25990556, chr15:27774029, chr15:27774036, chr15:27774056, chr15:37279502, chr15:37279596, chr15:37492678, chr15:50057443, chr15:55676371, chr15:73563072, chr15:76175116, chr15:76177403, chr15:76177730, chr15:76180621, chr15:76698849, chr15:77763325, chr15:79225530, chr15:79308756, chr15:79317768, chr15:79717654, chr15:81674934, chr15:82678899, chr15:82678907, chr15:82937410, chr15:83500032, chr15:83502255, chr15:83502269, chr15:83502330, chr15:84556583, chr15:85141638, chr15:85212597, chr15:86031232, chr15:86031301, chr15:87675497, chr15:93500719, chr15:99426537, chr15:100241443, chr16:4089605, chr16:5241312, chr16:14319506, chr16:18166051, chr16:21934841, chr16:21934876, chr16:30956175, chr16:31396280, chr16:33049899, chr16:34996067, chr16:34996080, chr16:36931704chr16:48310108, chr16:49224270, chr16:55054573, chr16:82048237, chr16:89884772, chr16:89884785, chr16:90831679, chr16:91036606, chr16:91169724, chr16:92614767, chr16:93738555, chr16:95719003, chr17:13490869, chr17:16997185, chr17:17167887, chr17:23760975, chr17:24923819, chr17:25965417, chr17:26279989, chr17:26280013, chr17:26280055, chr17:26655620, chr17:27091324, chr17:27091330, chr17:27271084, chr17:28806325, chr17:28806369, chr17:30517182, chr17:30759620, chr17:32316554, chr17:32819144, chr17:35831142, chr17:45514556, chr17:45576099, chr17:50293159, chr17:56318156, chr17:56419443, chr17:56419449, chr17:57081110, chr17:70929173, chr17:74213804, chr17:74213822, chr17:80242085, chr17:91572990, chr18:31896738, chr18:31990223, chr18:34822372, chr18:61697711, chr18:64426046, chr18:66061561, chr18:67219979, chr18:67401914, chr18:68079554, chr18:84246195, chr19:4289682, chr19:5667111, chr19:14957788, chr19:14957801, chr19:14957843, chr19:24111472, chr19:28240301, chr19:31918094, chr19:43854199, chr19:55400758, chr19:56350261, chr19:56433312, chr2:5975895chr2:26090144, chr2:26372702, chr2:26372719, chr2:27286869, chr2:30232137, chr2:30989861, chr2:32026500, chr2:32694887, chr2:39931557, chr2:56845824, chr2:64055379, chr2:72482996, chr2:73566761, chr2:83930495, chr2:85070658, chr2:91273892, chr2:92973702, chr2:103702335, chr2:104168207, chr2:120039017, chr2:121332339, chr2:122097243, chr2:126831243, chr2:128794537, chr2:129108596, chr2:132219269, chr2:145602686, chr2:155578491, chr2:155578495, chr2:158117897, chr2:164164697, chr2:164164705, chr2:164832005, chr2:166583159, chr2:168925959, chr2:168993403, chr2:170429372, chr2:173804055, chr3:6864559, chr3:24654707, chr3:32341648, chr3:40708588, chr3:48608873, chr3:51669813, chr3:87535758, chr3:87927612, chr3:89235264, chr3:94152686, chr3:95309157, chr3:95415388, chr3:95422927, chr3:95559642, chr3:148817482, chr3:148817486, chr4:5799343, chr4:33085725, chr4:35431787, chr4:43641663, chr4:47027174, chr4:47277827, chr4:53271207, chr4:56740843, chr4:56740858, chr4:61919146, chr4:62808790, chr4:62808792, chr4:63377118, chr4:88355459, chr4:88376348chr4:106638986, chr4:116312975, chr4:118082342, chr4:118223800, chr4:118376694, chr4:118647024, chr4:123297847, chr4:125071341, chr4:131900066, chr4:131900371, chr4:132364864, chr4:132721629, chr4:133850754, chr4:134148661, chr4:135034381, chr4:137555249, chr4:148149486, chr4:151028868, chr4:151708213, chr4:153210652, chr4:154586373, chr4:154970990, chr5:34616835, chr5:123531840, chr6:92080586, chr7:24105615, chr7:30262670, chr7:35381429, chr7:44882844, chr7:64337651, chr7:127582850, chr7:131138550, chr8:45040542, chr8:85044866, chr8:104015728, chr8:108951105, chr8:121560869, chr8:123304361, chr9:32259547, chr9:78198735, chr9:103151237, chrX:76598996.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) Easy to operate. For age detection by this method, only methylation sequencing of mouse liver tissue DNA is required, and the age of the mouse can be obtained by calculating the sequencing results through a formula.

[0029] (2) High accuracy. The methylation age detection model established by this method through a large number of samples has been verified by a large number of samples, and the correlation between the calculated age and the chronological age is as high as 0.98 (see Figure 4 ).

[0030] (3) Less affected by external factors. Conventional age detection methods are mostly based on the determination of physiological morphology and are affected by factors such as food, growth environment, and individual differences of mice. However, this method is based on the DNA methylation level of mouse liver tissue to determine the age of mice at the molecular level and is less affected by external factors. Description of the Drawings

[0031] In conjunction with the following appended Figure 1 figures, the above and other features of the present application will be more fully described when reading. It can be understood that these drawings only depict several embodiments of the present application content, and thus should not be considered as a limitation on the scope of the present application content. By using the drawings, the present application content will be more clearly and detailedly described.

[0032] Figure 1 It is a schematic flow chart for constructing a DNA methylation library of liver tissue.

[0033] Figure 2 It is the quality inspection result diagram of the liver DNA methylation library by Agilent 2100 Bioanalyzer in Example 1.

[0034] Figure 3 It is a display diagram comparing the predicted results of the samples used to construct the methylation age detection model with the chronological age in Example 1.

[0035] Figure 4 It is a display diagram comparing the age calculated by the methylation age detection model with the chronological age in Example 2.

[0036] Figure 5 It is a display diagram comparing the three ages obtained by calculation with the chronological age in Example 3.

[0037] Figure 6 It is the table of CpG sites and corresponding parameters in Table 1 of Example 1. Detailed implementation manners

[0038] The following examples are described to assist in understanding the present application. The examples are not and should not in any way be construed as limiting the scope of protection of the present application.

[0039] For the experimental methods without specific conditions in the following examples, they are carried out according to conventional experimental conditions, such as the conditions described in the Molecular Cloning Laboratory Manual by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise specified, the materials used in the examples are all commercially available products.

[0040] Example 1: Establishing a methylation age detection model based on DNA methylation sequencing of liver tissue

[0041] (1) Six groups of mice of different ages (2 months, 4 months, 6 months, 10 months, 16 months, 20 months) were taken, with 10 mice in each group, for a total of 60 mice. 5 mg of liver tissue was taken from each mouse, and genomic DNA was extracted using the DNeasy Blood&Tissue Kit (50) (69504, qiagen) according to its operating procedure;

[0042] (2) 100 ng of genomic DNA was taken from each sample and digested with 10 Units of MspI (R0106S, NEB) at 37 °C for more than 16 hours;

[0043] (3) P5 (sequence shown in SEQ ID NO:1) containing 5-methylcytosine and P7 (sequence shown in SEQ ID NO:2) were annealed to synthesize the Illumina adapter. The annealing conditions were 10 ul of P5 (100 uM), 10 ul of P7 (100 uM), 3 ul of Tris-HCl (10 mM) pH 8.0, 77 ul of ddH2O. After 10 min at 95 °C, the temperature was decreased to 15 °C at a rate of 0.1 °C / s, and after 5 min at 15 °C, it was decreased to 4 °C;

[0044] (4) The digested product in step (2) was blunted in a 50 ul reaction system, which contained 10 U of Klenow fragment enzyme (3'→5' exonuclease, NEB), 40 uM of dGTP, 40 uM of 5'-methylated dCTP (Roche), 400 uM of dATP, and 1×NEB buffer 2. The reaction was incubated at 30 °C for 20 minutes and then at 37 °C for 20 minutes. The blunted DNA fragment was ligated to the Illumina adapter obtained in step (3). In a 50 ul reaction system, it contained 2000 U of T4 ligase (NEB), 10 ul of adenylated DNA, and 0.5 to 1.0 uM of the adapter, and was ligated at 16 °C for more than 16 hours;

[0045] (5) The product in step (4) was subjected to bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen) according to its operating procedure;

[0046] (6) Take 20 μl of the transformed DNA obtained in step (5), add 25 μl of KAPA HiFi HotStart ReadyMix (2X) (Roche), 5 μl of Library Amplification Primer Mix (10X) (Roche), mix well and perform PCR reaction: pre-denature at 98 °C for 45 s, denature at 98 °C for 15 s, anneal at 60 °C for 30 s, extend at 72 °C for 30 s. After performing 16 cycles, extend at 72 °C for 1 min and store at 4 °C; The obtained PCR product is subjected to 6% polyacrylamide gel electrophoresis at a voltage of 120 V for 1 h, stained with Gelred dye at a concentration of one ten-thousandth for 5 minutes, then observed and photographed under an ultraviolet lamp, cut and recovered the band between 180 - 260 bp, and use Agilent 2100 Bioanalyzer to detect the fragment length range of the above methylation sequencing library. The detection result is as Figure 2 shown, and the prepared methylation sequencing library is obtained. The flow chart for constructing the methylation sequencing library is shown in Figure 1 ;

[0047] (7) Analyze the obtained methylation sequencing library through the following data analysis to obtain genome-wide methylation information:

[0048] (7.1) Send the methylation sequencing library to the Illumina NextSeq 500 sequencing platform for sequencing. The sequencing read length is 150 bp, and the sequencing mode is paired-end sequencing, and the off-machine data is obtained;

[0049] (7.2) For the off-machine data, use FastQC, Cutadapt, and Trimmomatic for data quality control and preprocessing (using default parameters) to obtain valid data after removing low-quality sequences and sequencing adapters;

[0050] (7.3) Use the methylation alignment-specific software Bismark to align the valid data to the mouse reference genome (mm10), remove PCR duplicates at the same time, and identify methylation sites, and then determine the methylation level and methylation type of these sites;

[0051] (7.4) To ensure the reliability of the results, we removed samples with less than 500,000 CpG sites. Finally, we obtained a total of 50 samples, including 10 samples with a chronological age of 2 months, 10 samples with a chronological age of 4 months, 8 samples with a chronological age of 6 months, 8 samples with a chronological age of 10 months, 7 samples with a chronological age of 16 months, and 7 samples with a chronological age of 20 months;

[0052] (7.5) Based on the methylation statistical results of all samples, a total of 121,885 candidate CpG sites for the methylation age detection model were screened by us.

[0053] (8) We first used the lasso regression algorithm for the 121,885 CpG methylation sites to predict the sites that contribute significantly to age detection. Lasso regression minimizes the loss function and adds an L1 norm penalty term to shrink the parameters of insignificant methylation sites to 0, thereby selecting the contributing sites to construct the model. Through this process, we successfully screened out 371 CpG sites that contribute to the model and the corresponding parameter values for each site (see Figure 6 Appendix 1 CpG Site and Corresponding Parameter Table).

[0054] After that, we used the methylation data of the training group mice to evaluate the performance of the trained model, mainly through the scatter plot and correlation coefficient of age. The results show that our model shows good performance in predicting age (see Figure 3 ).

[0055] The lasso regression formula is as follows:

[0056]

[0057] That is, y = β0 + β1x1 + β2x2 +... + β 371 x 371

[0058] y is the predicted age;

[0059] β0 is the intercept of the model (the value is -2.53504470088752, see Figure 6 Table 1 CpG Site and Corresponding Parameter Table);

[0060] x is the methylation degree of the site;

[0061] n is the termination value of the summation formula, 371, representing a total of 371 methylation sites;

[0062] i is the index of the current term in the summation process.

[0063] Bringing the parameters corresponding to the 371 methylation sites in the appendix into the formula and summing them up can calculate the predicted age.

[0064] Example 2: Verifying the Accuracy of the Methylation Age Detection Model

[0065] (1) Six groups of mice of different ages (2 months, 4 months, 6 months, 10 months, 16 months, 20 months) were taken, with 3 - 5 mice in each group, for a total of 24 mice. 5 mg of liver tissue was taken from each mouse, and genomic DNA was extracted using the DNeasy Blood&Tissue Kit (50) (69504, qiagen) according to its operating procedures;

[0066] (2) 100 ng of genomic DNA from each sample was digested with 10 Units of MspI (R0106S, NEB) at 37 °C for more than 16 hours; (3) P5 containing 5 - methylcytosine (sequence shown in SEQ ID NO:1) and P7 (sequence shown in SEQ ID NO:2) were annealed to synthesize the Illumina adapter. The annealing conditions were 10 ul of P5 (100 uM), 10 ul of P7 (100 uM), 3 ul of Tris - HCl (10 mM) pH 8.0, 77 ul of ddH2O. After 10 min at 95 °C, the temperature was decreased to 15 °C at a rate of 0.1 °C / s, and after 5 min at 15 °C, it was decreased to 4 °C;

[0067] (4) The digested product in step (2) was blunted in a 50 - ul reaction system, which contained 10 U of Klenow fragment enzyme (3'→5' exonuclease, NEB), 40 uM of dGTP, 40 uM of 5' - methylated dCTP (Roche), 400 uM of dATP, and 1×NEB buffer 2. The reaction was incubated at 30 °C for 20 minutes and then at 37 °C for 20 minutes. The blunted DNA fragment was ligated to the Illumina adapter obtained in step (3). In a 50 - ul reaction system, it contained 2000 U of T4 ligase (NEB), 10 ul of adenylated DNA, and 0.5 to 1.0 uM of the adapter, and was ligated at 16 °C for more than 16 hours;

[0068] (5) The product in step (4) was subjected to bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen) according to its operating procedures;

[0069] (6) Take 20 μl of the transformed DNA obtained in step (5), add 25 μl of KAPA HiFi HotStart ReadyMix (2X) (Roche), and 5 μl of Library Amplification Primer Mix (10X) (Roche). After mixing, perform PCR reaction: pre-denature at 98 °C for 45 s, denature at 98 °C for 15 s, anneal at 60 °C for 30 s, extend at 72 °C for 30 s. After 16 cycles, extend at 72 °C for 1 min and store at 4 °C; subject the obtained PCR product to 6% polyacrylamide gel electrophoresis at 120 V for 1 h, stain with Gelred staining solution (one ten-thousandth) for 5 minutes, then observe and photograph under ultraviolet light, cut and recover the band between 180 - 260 bp to obtain the prepared methylation sequencing library;

[0070] (7) Analyze the obtained methylation sequencing library through the following data analysis to obtain the whole-genome methylation information:

[0071] (7.1) Send the methylation sequencing library to the Illumina NextSeq 500 sequencing platform for sequencing with a sequencing read length of 150 bp and a paired-end sequencing mode, and obtain the off-machine data;

[0072] (7.2) For the off-machine data, use FastQC, Cutadapt, and Trimmomatic for data quality control and preprocessing (using default parameters) to obtain the effective data after removing low-quality sequences and sequencing adapters;

[0073] (7.3) Use the methylation-specific alignment software Bismark to align the effective data to the mouse reference genome (mm10), and at the same time remove PCR duplicates to determine the methylation level and methylation type of these sites;

[0074] (8) Substitute the obtained methylation site information into the methylation age detection model formula, calculate the age of the mice, and statistically analyze the correlation between the predicted age and the chronological age. The results show that our model exhibits good performance in predicting age (see Figure 4 ).

[0075] Example 3: Comparison of the method of the present application with the prior art

[0076] In order to verify that the age detection method based on liver tissue DNA methylation sequencing of the present application can produce reliable results, a comparison is made here with the commonly used body weight method and body length method.

[0077] Detect the body weight and body length of the 24 mice in Example 2, and calculate the corresponding age according to the measured data. Compare the ages calculated in Example 2.

[0078] Scatter plots were drawn for the ages calculated in Example 2 and the ages calculated from body weight and body length, with correlation coefficients of 0.98, 0.95, and 0.96 respectively (see Figure 5 ), and it can also be seen from the figure that the calculation method in Example 2 is more accurate.

[0079] Although multiple aspects and embodiments of the present application have been disclosed, other aspects and embodiments will be apparent to those skilled in the art. Without departing from the concept of the present application, several modifications and improvements can be made, and these all fall within the protection scope of the present application. The multiple aspects and embodiments disclosed in the present application are only for illustrative purposes and are not intended to limit the present application. The actual protection scope of the present application is subject to the claims.

Claims

1. An age detection method based on DNA methylation sequencing of liver tissue, characterized in that: The specific steps are as follows: (a) Extracting genomic DNA from mouse liver tissue; (b) digesting the genomic DNA with MspI; (c) filling the digested product in step (b), adding A at the end, and adding a linker; (d) subjecting the product of step (c) to bisulfite conversion; (e) amplifying the product in step (d), recovering the target DNA fragment, and obtaining a methylation sequencing library; (f) performing next-generation sequencing and data analysis on the methylation sequencing library; (g) Substituting the results of data analysis into the methylation age detection model formula to calculate the age of the mouse, where: The formula of the methylation age detection model formula is: x is the methylation degree of the site, n is the terminal value of the accumulation formula 371, which means there are 371 methylation sites in total, i is the index of the current item in the accumulation process, β i See Appendix 1 for a table of CpG sites and corresponding parameters; Among them, the 371 methylation sites are: chr1:15312761, chr1:33736808, chr1:33745240, chr1:34198870, chr1:53457896, chr1:71603645, chr1:72858391, chr1:74579588, chr1:75469776, chr1:89145594, chr1:131289544, chr1:131292549, chr1:132360639, chr1:133088376, chr1:133185854, chr1:136122008, chr1 : 155717621, chr1: 161020252, chr1: 165310605, chr1: 170599741, chr1: 1 86083651, chr1: 186968288, chr1: 189836555, chr1: 192741266, chr10: 683 0136, chr10: 38599832, chr10: 42197867, chr10: 43690682, chr10: 594677 30. chr10: 59467736, chr10: 61167017, chr10: 61201662, chr10: 62393775, chr10: 68097490, chr10: 69973651, chr10: 69989530, chr10: 75969927, chr10: 76388972, chr10: 76614625, chr10: 76614665, chr10: 77042820, chr10 :80638086, chr10: 81370697, chr10: 81514368, chr10: 81514379, chr10: 1 17076866, chr10: 119108813, chr10: 119490954, chr10: 121496157, chr10: 121607037, chr10: 121607055, chr10: 121973100, chr10: 127556923, chr1 0: 127689489, chr11: 3127214, chr11: 4149986, chr11: 6798051, chr11: 200 20791, chr11: 36023996, chr11: 46265946, chr11: 55062348, chr11: 575016 20. chr11: 59331130, chr11: 60205837, chr11: 60721791, chr11: 69406372,chr11:72913128、chr11:72913156、chr11:74636006、chr11:75446014、chr11:77104440、chr11:78011692、chr11:78171155、chr11:83212918、chr11:87862824、chr11:96772861、chr11:97351621、chr11:100802519、chr11:101579036、chr11:106073325、chr11:109559799、chr11:113624670、chr11:118063887、chr11:119264605、chr11:120272249、chr11:120274030、chr11:120284505、chr11:120284523、chr11:120284531、chr11:121138571、chr12:29832377、chr12:38805046、chr12:39875500、chr12:51909894、chr12:78468284、chr12:78468290、chr12:79268766、chr12:79276390、chr12:80989871、chr12:81314921、chr12:81357039、chr12:83350859、chr12:85816703、chr12:91647550、chr12:99903559、chr12:100940089、chr12:109973506、chr12:110551097、chr12:110640246、chr12:118458777、chr13:12861299、chr13:13964924、chr13:42183697、chr13:42183756、chr13:42768983、chr13:46758810、chr13:47049329、chr13:49194957、chr13:49194999、chr13:49195006、chr13:49379830、chr13:55638189、chr13:56750481、chr13:58672986、chr13:75810626、chr13:84247021、chr13:92682831、chr13:99007192、chr13:105846560、chr14:19768531、chr14:20727577、chr14:30569075、chr14:30901250、chr14:51920496、chr14:55863549、c hr14:56116055、chr14:59586457、chr14:75331930、chr14:79619032、chr 14: 101929333, chr15: 8280995, chr15: 10146661, chr15: 11391683, chr15: 25926894, chr15: 25990556, chr15: 27774029, chr15: 27774036, chr15: 2 7774056, chr15: 37279502, chr15: 37279596, chr15: 37492678, chr15: 50057443, chr15: 55676371, chr15: 73563072, chr15: 76175116, chr15: 7617 7403, chr15: 76177730, chr15: 76180621, chr15: 76698849, chr15: 77763325, chr15: 79225530, chr15: 79308756, chr15: 79317768, chr15: 79717654 、chr15:81674934、chr15:82678899、chr15:82678907、chr15:82937410、chr15:83500032、chr15:83502255、chr15:83502269、chr15:83502330、ch r15:84556583、chr15:85141638、chr15:85212597、chr15:86031232、chr15:86031301、chr15:87675497、chr15:93500719、chr15:99426537、chr15: 100241443、chr16:4089605、chr16:5241312、chr16:14319506、chr16:181 66051、chr16:21934841、chr16:21934876、chr16:30956175、chr16:31396 280、chr16:33049899、chr16:34996067、chr16:34996080、chr16:3693170 4、chr16:48310108、chr16:49224270、chr16:55054573、chr16:82048237、chr16:89884772、chr16:89884785、chr16:90831679、chr16:91036606、chr16:91169724、chr16:92614767、chr16:93738555、chr16:95719003、chr17:13490869、chr17:16997185、chr17:17167887、chr17:23760975、chr17:24923819、chr17:25965417、chr17:26279989、chr17:26280013、chr17:26280055、chr17:26655620、chr17:27091324、chr17:27091330、chr17:27271084、chr17:28806325、chr17:28806369、chr17:30517182、chr17:30759620、chr17:32316554、chr17:32819144、chr17:35831142、chr17:45514556、chr17:45576099、chr17:50293159、chr17:56318156、chr17:56419443、chr17:56419449、chr17:57081110、chr17:70929173、chr17:74213804、chr17:74213822、chr17:80242085、chr17:91572990、chr18:31896738、chr18:31990223、chr18:34822372、chr18:61697711、chr18:64426046、chr18:66061561、chr18:67219979、chr18:67401914、chr18:68079554、chr18:84246195、chr19:4289682、chr19:5667111、chr19:14957788、chr19:14957801、chr19:14957843、chr19:24111472、chr19:28240301、chr19:31918094、chr19:43854199、chr19:55400758、chr19:56350261、chr19:56433312、chr2:5975895、chr2:26090144、chr2:26372702、chr2:26372719、chr2:27286869、chr2:30232137、chr2:30989861、chr2:32026500、chr2:32694887、chr2:39931557、chr2:56845824、chr2:64055379、chr2:72482996、chr2:73566761、chr2:83930495、chr2:85070658、chr2:91273892、chr2:92973702、chr2:103702335、chr2:104168207、chr2:120039017、chr2:121332339、chr2:122097243、chr2:126831243、chr2:128794537、chr2:129108596、chr2:132219269、chr2:145602686、chr2:155578491、chr2:155578495、chr2:158117897、chr2:164164697、chr2:164164705、chr2:164832005、chr2:166583159、chr2:168925959、chr2:168993403、chr2:170429372、chr2:173804055、chr3:6864559、chr3:24654707、chr3:32341648、chr3:40708588、chr3:48608873、chr3:51669813、chr3:87535758、chr3:87927612、chr3:89235264、chr3:94152686、chr3:95309157、chr3:95415388、chr3:95422927、chr3:95559642、chr3:148817482、chr3:148817486、chr4:5799343、chr4:33085725、chr4:35431787、chr4:43641663、chr4:47027174、chr4:47277827、chr4:53271207、chr4:56740843、chr4:56740858、chr4:61919146、chr4:62808790、chr4:62808792、chr4:63377118、chr4:88355459、chr4:88376348、chr4:106638986、chr4:116312975、chr4:118082342、chr4:118223800、chr4:118376694、chr4:118647024、chr4:123297847、chr4:125071341、chr4:131900066、chr4:131900371、chr4:132364864、chr4:132721629、chr4:133850754、chr4:134148661、chr4:135034381、chr4:137555249、chr4:148149486、chr4:151028868、chr4:151708213、chr4:153210652、chr4:154586373、chr4:154970990、chr5:34616835、chr5:123531840、chr6:92080586、chr7:24105615、chr7:30262670、chr7:35381429、chr7:44882844、chr7:64337651、chr7:127582850、chr7:131138550、chr8:45040542、chr8:85044866、chr8:104015728、chr8:108951105、chr8:121560869、chr8:123304361、chr9:32259547、chr9:78198735、chr9:103151237、chrX:76598996。、 2. The age detection method based on liver tissue DNA methylation sequencing according to claim 1, characterized in that: In step (b), the specific steps of MspI digestion are as follows: 100 rig of genomic DNA is taken from each sample and digested with 10 Units of MspI at 37°C for more than 16 hours. The reason for using MspI enzyme is that this endonuclease is insensitive to methylation sites and the digestion is not affected by methylation sites. Other endonucleases are sensitive to methylation sites and cannot perform digestion when encountering methylation sites.

3. The age detection method based on liver tissue DNA methylation sequencing according to claim 1, characterized in that: The sequence of the linker in step (c) contains 5-methylcytosine, and the sequences are SEQ ID NO: 1 and SEQ ID NO:

2.

4. The age detection method based on liver tissue DNA methylation sequencing according to claim 1, characterized in that: In step (c), the connector needs to be pre-annealed. The synthesized P5 and P7 containing 5-methylcytosine are cooled to 15°C at a rate of 0.1°C / s after 10 minutes at 95°C, and then cooled to 4°C after 5 minutes at 15°C. The P5 and P7 containing 5-methylcytosine are annealed to synthesize the Illumina connector through the pre-annealing treatment, wherein the annealing conditions are 10ul P5, 10ul P7, 3ul Tris-HCl pH 8.0, and 77ul ddH2O.

5. The age detection method based on liver tissue DNA methylation sequencing according to claim 1, characterized in that: The enzyme cleavage product of step (b) is filled in a 50ul reaction system, wherein the 50ul reaction system contains 10U of Klenow fragmentase, 40uM of dGTP, 40uM of 5'methylated dCTP, 400uM of dATP and 1×NEB buffer 2, and the filling step comprises: incubating the enzyme cleavage product of step (b) in the 50ul reaction system at 30°C for 20 minutes, and then incubating at 37°C for 20 minutes, and connecting the filled DNA fragment to the Illumina adapter obtained in step (3).

6. The age detection method based on liver tissue DNA methylation sequencing according to claim 5, characterized in that: The 50ul reaction system also contains 2000U of T4 ligase, 10ul of adenylated DNA and 0.5 to 1.0uM of adapter, and the ligation is carried out at 16°C for more than 16 hours.

7. The age detection method based on liver tissue DNA methylation sequencing according to claim 1, characterized in that: In step (d), the product of step (c) is subjected to bisulfite conversion using an EpiTect Bisulfite Kit according to its protocol.

8. The age detection method based on liver tissue DNA methylation sequencing according to claim 1, characterized in that: The specific steps of amplifying the product in step (d) are as follows: taking 20ul of the converted DNA obtained in step (d), adding 25ulKAPA HiFi HotStart ReadyMix 2X and 5ulLibrary Amplification Primer Mix 10X, mixing and performing PCR reaction, performing 16 cycles, extending at 72°C for 1min, and storing at 4°C; wherein the conditions of the PCR reaction are: pre-denaturation at 98°C for 45s, denaturation at 98°C for 15s, annealing at 60°C for 30s, and extension at 72°C for 30s.

9. The age detection method based on liver tissue DNA methylation sequencing according to claim 1, characterized in that: In step (e), the step of recovering the target DNA fragment from the obtained PCR product is as follows: the obtained PCR product is subjected to 6% polyacrylamide gel electrophoresis at a voltage of 120 V for 1 h, then stained with 1 / 10,000 Gelred dye for 5 minutes, then observed and photographed under ultraviolet light, and the band between 180 and 260 bp is cut and recovered.

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