Age detection method based on kidney tissue DNA methylation sequencing

Abstract

The invention relates to an age detection method based on kidney tissue DNA methylation. The age detection method mainly comprises the following steps: (a) extracting genome DNA from kidney tissue; (b) carrying out MspI enzyme digestion on the genome DNA; (c) filling the enzyme-digested product in the step (b), adding A at the tail end, and adding a linker; (d) subjecting the product of step (c) to bisulfite conversion; (e) amplifying the product in the step (d), and recovering the target DNA fragment to obtain a methylation sequencing library; (f) performing next-generation sequencing and data analysis on the methylation sequencing library; and (g) substituting a data analysis result into a 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 specifically, to an age detection method based on methylation sequencing. Background Art

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

[0003] The commonly used methods for detecting mouse age now have obvious disadvantages. For example, the weight method is easily affected by factors such as the stomach content and reproductive status of mice, and there are many overlaps between the values ​​of each age group, so there may be certain errors in the age division; the body length method is easily affected by the proficiency of the measurer and the body condition of the mouse, which may cause human errors; the crystal dry weight method has a large workload, strict requirements, and is difficult to operate; the molar wear method is affected by food ratio, molar growth, etc. and has certain limitations. 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 determine the age of mice.

[0004] Based on the existing detection methods, there is an urgent need for a highly accurate and easy-to-operate age detection method in aging research. Summary of the invention

[0005] The purpose of the present invention is to establish an age detection method based on renal tissue DNA methylation sequencing, which is suitable for renal tissue DNA methylation library construction, second-generation sequencing and age detection of mice.

[0006] This application relates to an age detection method based on renal tissue DNA methylation sequencing, and the specific steps are as follows:

[0007] (a) Genomic DNA was extracted from mouse kidney tissue;

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

[0009] (c) filling the digested product in step (b), adding A at the end, and adding a linker;

[0010] (d) subjecting the product of step (c) to bisulfite conversion;

[0011] (e) amplifying the product in step (d), recovering the target DNA fragment, and obtaining a methylation sequencing library;

[0012] (f) performing next-generation sequencing and data analysis on the methylation sequencing library;

[0013] (g) Substituting the result of data analysis into the methylation age detection model formula to calculate the age of the mouse, wherein the formula of the methylation age detection model formula is: x is the methylation degree of the site, and n is the end value of the cumulative formula, 446, which means there are 446 methylation sites in total. Figure 6 CpG site and corresponding parameter table, i is the index of the current item in the accumulation process, β i See Figure 6 Table of CpG sites and corresponding parameters.

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

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

[0016] Furthermore, in step (b), the specific steps of MspI digestion are as follows: 100 ng of genomic DNA is taken from each sample and digested with 10 Units MspI (R0106S, NEB) at 37°C for more than 16 hours. The reason for using MspI enzyme is that the 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.

[0017] In some embodiments, the sequence of the linker in step (c) contains 5-methylcytosine, and the sequences are SEQ ID NO: 1 and SEQ ID NO: 2. The inclusion of 5-methylcytosine is to ensure that the base does not change during the subsequent bisulfite conversion. If this treatment is not performed, the base will change during the subsequent bisulfite conversion, which will cause sequencing failure.

[0018] Furthermore, in step (c), the adapter needs to be pre-annealed. The synthesized P5 (sequence shown in SEQ ID NO: 1) and P7 (sequence shown in SEQ ID NO: 2) 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 adapter through the pre-annealing treatment, wherein the annealing conditions are 10ul P5 (100uM), 10ul P7 (100uM), 3ul Tris-HCl (10mM) pH 8.0, and 77ulddH2O. The illumina adapter is used to match the subsequent sequencing with an illumina instrument. The advantage of adding the adapter first and then doing the bisulfite is that the initial dosage requirement is low.

[0019] Further, the digested product of step (b) was filled in a 50ul reaction system, wherein the 50ul reaction system contained 10U of Klenow fragmentase (3'→5' exosome, NEB), 40uM dGTP, 40uM 5' methylated dCTP (Roche), 400uM dATP and 1×NEB buffer 2, and the filling step included: incubating the digested 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).

[0020] Furthermore, the 50 ul reaction system also contained 2000 U of T4 ligase (NEB), 10 ul of adenylated DNA and 0.5 to 1.0 uM of adapter, and the ligation was carried out at 16° C. for more than 16 hours.

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

[0022] In some embodiments, the specific steps of amplifying the product in step (d) are: taking 20ul of the converted DNA obtained in step (d), adding 25ulKAPA HiFi HotStart ReadyMix (2X) (Roche), 5ulLibraryAmplification Primer Mix (10X) (Roche), mixing and performing PCR reaction, after 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.

[0023] Furthermore, 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.

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

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

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) Easy to operate. This method only requires methylation sequencing of the mouse kidney tissue DNA for age detection, and the sequencing results can be calculated using a formula to obtain the mouse's age.

[0028] (2) High accuracy. The methylation age detection model established by this method is based on a large number of samples. After verification by a large number of samples, the correlation between the calculated age and the chronological age is as high as 0.98 (see Figure 4 ).

[0029] (3) Less affected by external factors. Conventional age detection methods are mostly based on the measurement of physiological morphology, which is affected by food, growth environment, individual differences of mice, etc. This method is based on the DNA methylation level of mouse kidney tissue to determine the age of mice at the molecular level and is less affected by external factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Combined with the following Figure 1 When read together, the above and other features of the present application will be more fully described. It is understood that these drawings only depict several implementations of the present application and should not be considered as limiting the scope of the present application. By using the drawings, the present application will be more clearly and detailed.

[0031] Figure 1 Schematic diagram of the process of building a DNA methylation library for liver tissue.

[0032] Figure 2 This is a graph showing the quality inspection results of the kidney DNA methylation library 2100 using the Agilent 2100 Bioanalyzer in Example 1.

[0033] Figure 3 This is a graph showing the comparison between the prediction results and chronological age of the samples used to construct the methylation age detection model in Example 1.

[0034] Figure 4 This is a graph showing the comparison between the age calculated using the methylation age detection model in Example 2 and the chronological age.

[0035] Figure 5 This is a display diagram comparing the three ages obtained by calculation and the chronological age in Example 3.

[0036] Figure 6 The attached table of CpG sites and corresponding parameters of Example 1 is shown in FIG. DETAILED DESCRIPTION

[0037] The following examples are described to assist the understanding of the present application, and the examples are not and should not be interpreted in any way as limiting the scope of protection of the present application.

[0038] The experimental methods in the following examples where specific conditions are not specified are based on conventional experimental conditions, such as the conditions described in Sambrook et al. Molecular Cloning Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise specified, the materials used in the examples are all commercially available products.

[0039] Example 1: Establishment of a methylation age detection model based on renal tissue DNA methylation sequencing

[0040] (1) Six groups of mice of different ages (2 months, 4 months, 6 months, 10 months, 16 months, and 20 months) were selected, each group containing 10 mice, for a total of 60 mice. 5 mg of kidney tissue was obtained from each mouse, and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (50) (69504, Qiagen) according to its operating procedures;

[0041] (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 h;

[0042] (3) P5 (sequence shown in SEQ ID NO: 1) and P7 (sequence shown in SEQ ID NO: 2) containing 5-methylcytosine were annealed to synthesize Illumina adapters. The annealing conditions were 10ul P5 (100uM), 10ul P7 (100uM), 3ul Tris-HCl (10mM) pH 8.0, 77ul ddH2O, 95°C for 10min, then cooled to 15°C at a rate of 0.1°C / s, and then cooled to 4°C after 5min at 15°C;

[0043] (4) The digested product of step (2) was filled in a 50ul reaction system, which contained 10U of Klenow fragmentase (3'→5' exo, NEB), 40uM dGTP, 40uM 5' methylated dCTP (Roche), 400uM dATP and 1×NEB buffer 2, and the reaction was incubated at 30°C for 20 minutes, and then incubated at 37°C for 20 minutes. The filled DNA fragment was ligated with the Illumina adapter obtained in step (3) in a 50ul reaction system, which contained 2000U of T4 ligase (NEB), 10ul of adenylated DNA and 0.5 to 1.0uM of adapter, and the ligation was carried out at 16°C for more than 16 hours;

[0044] (5) using EpiTect Bisulfite Kit (Qiagen) according to its operating procedures to convert the product of step (4) into bisulfite;

[0045] (6) Take 20ul of the converted DNA obtained in step (5), add 25ul KAPA HiFi HotStart ReadyMix (2X) (Roche), 5ul Library Amplification Primer Mix (10X) (Roche), mix well and perform PCR reaction: pre-denaturation at 98°C for 45s, denaturation at 98°C for 15s, annealing at 60°C for 30s, extension at 72°C for 30s, perform 16 cycles, extend at 72°C for 1min, and store at 4°C; the obtained PCR product is subjected to 6% polyacrylamide gel electrophoresis at a voltage of 120V for 1h, stained with 1 / 10,000 Gelred dye for 5min, then observed and photographed under ultraviolet light, and the band between 180 and 260bp is cut and recovered. The fragment length range of the above methylation sequencing library is detected using Agilent 2100 Bioanalyzer. The detection results are as follows: Figure 2 As shown in FIG. , the prepared methylation sequencing library is obtained. The methylation sequencing library construction flow chart is shown in FIG. Figure 1 ;

[0046] (7) The obtained methylation sequencing library is subjected to the following data analysis to obtain genome-wide methylation information:

[0047] (7.1) The methylation sequencing library was sent to the Illumina NextSeq 500 sequencing platform for sequencing, with a sequencing read length of 150 bp and a paired-end sequencing mode, and the offline data was obtained;

[0048] (7.2) For the offline data, FastQC, Cutadapt and Trimmomatic were used for data quality control and preprocessing (using default parameters) to obtain valid data after removing low-quality sequences and sequencing adapters;

[0049] (7.3) Use Bismark, a methylation alignment software, to align the valid data to the mouse reference genome (mm10), remove PCR duplicates, identify methylation sites, and then determine the methylation levels and methylation types of these sites;

[0050] (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.

[0051] (7.5) Based on the methylation statistics of all samples, we screened a total of 102,845 candidate CpG sites for the methylation age detection model.

[0052] (8) We first used the lasso regression algorithm on 102,845 CpG methylation sites to predict sites that contribute significantly to age detection. Lasso regression minimizes the loss function and adds an L1 norm penalty term to reduce the parameters of insignificant methylation sites to 0, thereby selecting contributing sites to build the model. After this process, we successfully screened out 446 CpG sites that contribute to the model and the corresponding parameter values ​​of each site (see Figure 6 CpG sites and corresponding parameters table).

[0053] We then used the methylation data of the training group mice to evaluate the performance of the trained model, mainly through the scatter plot (Scatter Plot) and correlation coefficient (Correlation Coefficient) of age. The results showed that our model showed good performance in predicting age (see Figure 3 ).

[0054] The lasso regression formula is as follows:

[0055]

[0056] That is, y = β0 + β1x1 + β2x2 + ... + β 446 x 446

[0057] y is the predicted age;

[0058] β0 is the intercept of the model (value is -5.74923675687395, see Figure 6 Table 1 CpG sites and corresponding parameters table);

[0059] x is the methylation degree of the site;

[0060] n is the terminal value of the cumulative formula, 446, which means there are 446 methylation sites in total;

[0061] i is the index of the current item in the accumulation process.

[0062] Substitute the parameters corresponding to the 446 methylation sites in the attached table into the formula and add them up to calculate the predicted age.

[0063] Example 2: Verifying the accuracy of the methylation age detection model

[0064] (1) Six groups of mice of different ages (2 months, 4 months, 6 months, 10 months, 16 months, and 20 months) were selected, each group containing 3-5 mice, for a total of 26 mice. 5 mg of kidney tissue was obtained from each mouse, and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (50) (69504, Qiagen) according to its operating procedures;

[0065] (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; (3) P5 (sequence shown in SEQ ID NO: 1) and P7 (sequence shown in SEQ ID NO: 2) containing 5-methylcytosine were annealed to synthesize Illumina adapters. The annealing conditions were 10ul P5 (100uM), 10ul P7 (100uM), 3ul Tris-HCl (10mM) pH 8.0, 77ul ddH2O, 95°C for 10min, then cooled to 15°C at a rate of 0.1°C / s, and then cooled to 4°C after 5min at 15°C;

[0066] (4) The digested product of step (2) was filled in a 50ul reaction system, which contained 10U of Klenow fragmentase (3'→5' exo, NEB), 40uM dGTP, 40uM 5' methylated dCTP (Roche), 400uM dATP and 1×NEB buffer 2, and the reaction was incubated at 30°C for 20 minutes, and then incubated at 37°C for 20 minutes. The filled DNA fragment was ligated with the Illumina adapter obtained in step (3) in a 50ul reaction system, which contained 2000U of T4 ligase (NEB), 10ul of adenylated DNA and 0.5 to 1.0uM of adapter, and the ligation was carried out at 16°C for more than 16 hours;

[0067] (5) using EpiTect Bisulfite Kit (Qiagen) according to its operating procedures to convert the product of step (4) into bisulfite;

[0068] (6) Take 20ul of the converted DNA obtained in step (5), add 25ul KAPA HiFi HotStart ReadyMix (2X) (Roche), 5ul Library Amplification Primer Mix (10X) (Roche), mix well and perform PCR reaction: pre-denaturation at 98°C for 45s, denaturation at 98°C for 15s, annealing at 60°C for 30s, extension at 72°C for 30s, perform 16 cycles, extend at 72°C for 1min, and store at 4°C; the obtained PCR product is subjected to 6% polyacrylamide gel electrophoresis at a voltage of 120V for 1h, stained with 1 / 10,000 Gelred dye for 5min, then observed and photographed under ultraviolet light, and the band between 180 and 260bp is cut and recovered to obtain the prepared methylation sequencing library;

[0069] (7) The obtained methylation sequencing library is subjected to the following data analysis to obtain genome-wide methylation information:

[0070] (7.1) The methylation sequencing library was sent to the Illumina NextSeq 500 sequencing platform for sequencing, with a sequencing read length of 150 bp and a paired-end sequencing mode, and the offline data was obtained;

[0071] (7.2) For the offline data, FastQC, Cutadapt and Trimmomatic were used for data quality control and preprocessing (using default parameters) to obtain valid data after removing low-quality sequences and sequencing adapters;

[0072] (7.3) Use Bismark, a methylation alignment software, to align the valid data to the mouse reference genome (mm10), remove PCR duplicates, and determine the methylation levels and methylation types of these sites;

[0073] (8) Substituting the obtained methylation site information into the methylation age detection model formula, the age of the mouse was calculated, and the correlation between the predicted age and the chronological age was statistically analyzed. The results showed that our model showed good performance in predicting age (see Figure 4 ).

[0074] Example 3: Comparison of the method of the present application with the prior art

[0075] In order to verify that the age detection method based on renal tissue DNA methylation sequencing of the present application can produce reliable results, it is compared with the currently commonly used weight method and body length method.

[0076] The body weight and body length of the 26 mice in Example 2 were measured, and the corresponding ages were calculated based on the measured data.

[0077] The scatter plot shows that the correlations between the age calculated in Example 2 and the age calculated by weight and body length are 0.98, 0.95 and 0.96 respectively (see Figure 5 ), and it can also be seen in the figure that the calculation method in Example 2 is more accurate.

[0078] Although the present application has disclosed multiple aspects and embodiments, other aspects and embodiments will be obvious to those skilled in the art, and several modifications and improvements may be made without departing from the concept of the present application, all of which belong to the protection scope of the present application. The multiple aspects and embodiments disclosed in the present application are only for illustration and are not intended to limit the present application. The actual protection scope of the present application shall be subject to the claims.

Claims

1. An age detection method based on renal tissue DNA methylation sequencing, characterized in that: The specific steps are as follows: (a) Genomic DNA was extracted from mouse kidney 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 446, which means there are 446 methylation sites in total, i is the index of the current item in the accumulation process, β i See Figure 6 for a table of CpG sites and corresponding parameters.

2. The age detection method based on renal tissue DNA methylation sequencing according to claim 1, characterized in that: In step (b), the specific steps of MspI digestion are as follows: 100 ng of genomic DNA is taken from each sample and digested with 10 Units of MspI (R0106S, NEB) 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 renal 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 renal tissue DNA methylation sequencing according to claim 1, characterized in that: In step (c), the connector needs to be pre-annealed. The synthesized P5 (sequence shown in SEQ ID NO: 1) and P7 (sequence shown in SEQ ID NO: 2) 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 (100uM), 10ul P7 (100uM), 3ul Tris-HCl (10mM) pH8.0, and 77ulddH2O.

5. The age detection method based on renal tissue DNA methylation sequencing according to claim 1, characterized in that: The enzyme digestion product of step (b) is filled in a 50ul reaction system, wherein the 50ul reaction system contains 10U of Klenow fragmentase (3'→5' exoclease, NEB), 40uM dGTP, 40uM 5' methylated dCTP (Roche), 400uM dATP and 1×NEB buffer 2. The filling step comprises: incubating the enzyme digestion 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 renal tissue DNA methylation sequencing according to claim 1, characterized in that: The 50ul reaction system also contains 2000U of T4 ligase (NEB), 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 renal 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 EpiTect Bisulfite Kit (Qiagen) according to its protocol.

8. The age detection method based on renal 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) (Roche) and 5ulLibrary Amplification PrimerMix (10X) (Roche), 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 renal 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.

10. A marker for an age detection method, the marker being derived from a DNA methylation site in mouse kidney tissue, characterized in that: The methylation sites include chr1:9943983, chr1:16105703, chr1:33814413, chr1:37540916, chr1:37540919, chr1:43445731, chr1:43445758, chr1:52232911, chr1:52726856, chr1:55088458, chr1:56972766, chr1:56972834, chr1:60098657, chr1:64617622, ​​chr1:74855718, chr1:84695715, chr1:86526914, c hr1:91494730、chr1:91494784、chr1:93479066、chr1:97661263、chr1:97661269、chr1:106759210、chr1:118311175、chr1:118311269、chr1:11983 6890, chr1:119997135, chr1:128244408, chr1:128359618, chr1:1527659 32. chr1:155159203, chr1:155558252, chr1:156420742, chr1:165764152 , chr1:166409971, chr1:167001320, chr1:170867635, chr1:172377165, chr1:179546784, chr1:181841978, chr1:182282550, chr1:182341343, chr 1:189343824, chr10:11343551, chr10:34206926, chr10:34207056, chr10 :36975054、chr10:39370267、chr10:39926925、chr10:42502390、chr10:4 3479301, chr10:43578577, chr10:45067518, chr10:45067559, chr10:629 80267, chr10:67535665, chr10:67548625, chr10:67548937, chr10:75560 070, chr10:75560129, chr10:77166101, chr10:77622384, chr10:7767788 5. chr10:77677917, chr10:77902493, chr10:78069547, chr10:80589999,chr10:80843965、chr10:83337521、chr10:84534095、chr10:84622528、chr10:85872227、chr10:88379609、chr10:93310291、chr10:99263988、chr10:110745728、chr10:116473948、chr10:116581880、chr10:120978682、chr10:121311060、chr10:128590379、chr11:3964259、chr11:5065089、chr11:6065331、chr11:8664550、chr11:20332405、chr11:20830952、chr11:21370763、chr11:26593286、chr11:30771556、chr11:30771651、chr11:30772117、chr11:32533150、chr11:32533162、chr11:35121849、chr11:51651000、chr11:52360623、chr11:53770156、chr11:58938484、chr11:58938607、chr11:59290029、chr11:60538360、chr11:61854231、chr11:62458020、chr11:68901703、chr11:69605243、chr11:73160425、chr11:75194944、chr11:75588034、chr11:75588047、chr11:75588057、chr11:76399410、chr11:78183373、chr11:78183387、chr11:78751476、chr11:79711332、chr11:82908395、chr11:83298733、chr11:83440963、chr11:86993713、chr11:87359195、chr11:94149046、chr11:96850163、chr11:98982248、chr11:100472443、chr11:100939352、chr11:100939360、chr11:102309160、chr11:102318834、chr11:106788196、chr11:107547506、chr11:107548368、chr11:114668982、chr11:116412897、chr11:117076888、chr11:117654388、chr11:120086499、chr11:120237099、chr11:121388076、chr12:3309185、chr12:8674609、chr12:26414867、chr12:26415112、chr12:31654916、chr12:34906890、chr12:54862519、chr12:59131017、chr12:75818668、chr12:82169865、chr12:84317583、chr12:87200148、chr12:98259143、chr12:99392680、chr12:102283289、chr12:103388938、chr12:105337200、chr12:108792299、chr12:108793895、chr12:110738761、chr12:111574652、chr12:113000525、chr13:3918161、chr13:12565863、chr13:13954799、chr13:17944267、chr13:23369059、chr13:24281310、chr13:31626255、chr13:31807264、chr13:32338511、chr13:35740709、chr13:38036889、chr13:38036939、chr13:38659018、chr13:45389792、chr13:45389838、chr13:47122706、chr13:48967436、chr13:55464810、chr13:55570834、chr13:56222126、chr13:56703444、chr13:59675890、chr13:64274401、chr13:69810316、chr13:77135476、chr13:77135479、chr13:91461219、chr13:93771739、chr13:98263039、chr13:100125038、chr13:100730808、chr13:107022015、chr13:111807809、chr13:112800914、chr13:112800924、chr13:114458659、chr13:114818470、chr13:118386811、chr14:11553691、chr14:14120826、chr14:18331387、chr14:25460526、chr14:34673634、chr14:34820103、chr14:45657675、chr14:54517319、chr14:54877441、chr14:66297427、chr14:70075052、chr14:76010716、chr15:5244058、chr15:11995559、chr15:51991242、chr15:57892418、chr15:59373994、chr15:75566693、chr15:75969360、chr15:75969377、chr15:75969392、chr15:76327723、chr15:76343713、chr15:79774381、chr15:79774464、chr15:92397361、chr15:93335997、chr15:98789804、chr15:99126063、chr15:99126584、chr15:100422597、chr15:100422626、chr15:100422911、chr15:100761715、chr16:3872309、chr16:3872577、chr16:5050528、chr16:13256983、chr16:16358554、chr16:18626535、chr16:20611709、chr16:22162657、chr16:31200932、chr16:31200970、chr16:35939110、chr16:35939143、chr16:78576304、chr16:85173244、chr17:3115682、chr17:5492749、chr17:5492794、chr17:12992095、chr17:13760694、chr17:14203824、chr17:24352098、chr17:25016791、chr17:25184577、chr17:26113668、chr17:28328671、chr17:32284455、chr17:35135324、chr17:56626610、chr17:57062133、chr17:66101563、chr17:71552423、chr17:78834913、chr17:84466052、chr18:6135861、chr18:12305723、chr18:12741299、chr18:20746449、chr18:31609619、chr18:31609637、chr18:31634502、chr18:32837500、chr18:32837531、chr18:34006546、chr18:34931726、chr18:35703137、chr18:36018259、chr18:36018271、chr18:36320243、chr18:38211984、chr18:38297010、chr18:42275853、chr18:52465793、chr18:52768579、chr18:53245607、chr18:57354726、chr18:61398869、chr18:64661049、chr18:67449037、chr18:70053147、chr18:70568533、chr18:77713711、chr18:77714504、chr18:80986372、chr19:3708470、chr19:3768331、chr19:4712099、chr19:5117768、chr19:6364188、chr19:7217520、chr19:32711979、chr19:36926514、chr19:37207141、chr19:41830084、chr19:45006369、chr19:45783441、chr19:53600772、chr19:59346208、chr19:60756095、chr19:60756290、chr2:3512556、chr2:4882069、chr2:12418849、chr2:14604394、chr2:18027432、chr2:18998164、chr2:25054639、chr2:25272014、chr2:31950267、chr2:32982041、chr2:36136286、chr2:49452607、chr2:69822742、chr2:73529246、chr2:73774936、chr2:76647535、chr2:83812981、chr2:84840650、chr2:91922165、chr2:116975648、chr2:117121459、chr2:120027606、chr2:122028766、chr2:122631511、chr2:122631549、chr2:122631621、chr2:130178831、chr2:130424037、chr2:140066526、chr2:143546205、chr2:147195416、chr2:163644960、chr2:165112365、chr2:167689377、chr2:167689380、chr2:173000442、chr2:179975863、chr2:180589548、chr2:181135140、chr2:181319911、chr3:27938703、chr3:40540794、chr3:40540815、chr3:41565309、chr3:58414998、chr3:84259334、chr3:86920876、chr3:87796960、chr3:88022971、chr3:90254671、chr3:92080948、chr3:97658602、chr3:98013924、chr3:100162640、chr3:100488930、chr3:102013086、chr3:104638906、chr3:108415778、chr3:108591631、chr3:110250142、chr3:119783297、chr3:121262947、chr3:121262970、chr3:121814642、chr3:122246105、chr3:122274355、chr3:122293279、chr3:122729961、chr3:127895840、chr3:129533932、chr3:129901651、chr3:137623783、chr3:148988861、chr4:21931442、chr4:21931460、chr4:24850902、chr4:24850928、chr4:32239144、chr4:32239182、chr4:33310503、chr4:40722841、chr4:42999953、chr4:55531629、chr4:58911705、chr4:58911875、chr4:62360352、chr4:82859314、chr4:89310735、chr4:93335610、chr4:94603459、chr4:95050809、chr4:98383152、chr4:98395880、chr4:99120987、chr4:100777085、chr4:101264874、chr4:103119692、chr4:111415360、chr4:118409501、chr4:119233146、chr4:120405679、chr4:122961000、chr4:123665027、chr4:124657672、chr4:130663533、chr4:135573466、chr4:136835763、chr4:139092892、chr4:150236920、chr4:152131292、chr4:152339014、chr4:155761082、chr4:155992412、chr5:30667212、chr5:30960164、chr5:34660701、chr5:36748221、chr5:124477885、chr5:135107144、chr6:30390967、chr6:53287443、chr6:87850569、chr6:116264155、chr6:119480542、chr7:4789193、chr7:19382365、chr7:141131165、chr8:46471435、chr8:46471445、chr8:56551382、chr8:61223787、chr8:111933488、chr9:15357584、chr9:70012589。、

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