CN115404270A - Evaluation method of methylation conversion rate of DNA methylation sequencing library, application, terminal equipment and storage medium - Google Patents
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Abstract
The invention discloses an evaluation method of DNA methylation sequencing library methylation conversion rate, application, terminal equipment and a computer readable storage medium, and belongs to the technical field of gene detection. The evaluation method for the methylation conversion rate of the methylation sequencing library selects the site with the methylation conversion rate related to the methylation conversion rate of the methylation library as the reference site, overcomes the problem that the C-T methylation conversion rate of the methylation library is influenced by a base sequence in the construction process of the methylation library by an enzymatic conversion method, can quickly evaluate the methylation conversion rate of the methylation library by utilizing the methylation conversion rate of the reference site, is used for quality control of the methylation conversion rate in DNA methylation detection, is convenient and quick, and solves the problems of long period, time consumption, labor consumption and the like of the traditional method. Also provided are a terminal device and a computer-readable storage medium capable of outputting a methylation conversion rate of a library or outputting a result of whether the library is qualified or not according to the methylation conversion rate of a reference site.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to an evaluation method of DNA methylation sequencing library methylation conversion rate, application, terminal equipment and a computer readable storage medium.
Background
DNA methylation is one of the earliest discovered gene appearance modification modes, playing an important role in maintaining normal cell function, genetic imprinting, embryonic development and tumorigenesis development, and specifically, selectively adding methyl to cytosine (C) under the action of DNA methylation transferase (DNMT) to form 5' -methylcytosine (5 mC).
With the development of high throughput sequencing technology (NGS), genome-wide level analysis of 5' -methylcytosine is possible, i.e. DNA methylation sequencing. Genome-wide bisulfite methylation sequencing (WGBS) can realize accurate detection of the methylation level of all single cytosines in a genome-wide range, and is the gold standard for DNA methylation research, the classical conversion method is to treat DNA with bisulfite, unmethylated cytosine in DNA is converted into uracil (U), while 5' -methylcytosine (5 mC) is not converted, but the conversion process needs high temperature or strong alkali treatment, so that the template is seriously damaged. The enzymatic conversion method is to protect the modified cytosine first with TET2 and an oxidation enhancer, TET2 oxidizes 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5 hmC) to 5-carboxycytosine (5 caC) by a cascade reaction, and then deaminates them with the deaminase APOBEC to convert cytosine to thymine (T) while leaving 5-hydroxymethylcytosine (5 hmC) and 5-carboxycytosine (5 caC) unaffected. The conversion effect of methylation sites C-T by an enzymatic conversion method is the same as that of bisulfite, but the extreme reaction conditions of strong alkali or high temperature and the like to DNA are avoided in the conversion process, and the problem of template damage during bisulfite conversion is perfectly solved. In the DNA methylation sequencing process, no matter which conversion method, the C-X conversion rate (C is converted into U or C is converted into T) can reach 100 percent, so the evaluation of the C-X conversion rate is particularly important for the evaluation of the methylation frequency in the DNA methylation sequencing process.
In the DNA methylation sequencing process by the enzymatic conversion method, if unmethylated C can not be converted into T, the methylation state can be misjudged, and then the methylation frequency is overestimated. The traditional method for evaluating the C-X conversion rate in the methylation library is to add 1% of exogenous completely unmethylated lambda DNA (linear double-stranded completely unmethylated lambda phage DNA obtained by separating methylase deficient Escherichia coli, NCBI Reference Sequence: NC-001416.1) at the beginning of library construction, and evaluate the C-X conversion rate of the methylation library by analyzing the methylation level of the lambda DNA after off-machine, wherein the process needs tedious processes of sequencing, analysis and the like, is time-consuming and labor-consuming, and the data with excessively low methylation conversion rate after off-machine is difficult to be used downstream, thereby causing waste of cost, resources and the like. In addition, there are also reports in the literature that methylation library C-X methylation conversion rate can be rapidly assessed by means of qPCR assessment of the beta-actin promoter region of the reference gene or ddPCR (droplet digital PCR) assessment of Alu repeat sequence, which is based on the principle of assessing methylation library C-X methylation conversion rate by using C-X conversion rate of a specific region or even a single site. However, the inventor found in the research that there are differences in the C-T conversion rate at different sites, and the C-T conversion rate at some sites is not in the same trend as the C-T conversion rate of the methylation library, presumably because the DNA methylation sequencing by the enzymatic conversion method is a reaction in which an enzyme participates, unlike the process of sequencing bisulfite which only participates in chemical substances, the former is related to the base sequence, and the latter is not influenced by the base sequence at all.
In conclusion, the establishment of the quality control standard through a certain gene or a certain class of specific sites/regions of the gene is not necessarily suitable for the quality control of the methylation conversion rate of the methylation library by the enzymology conversion method, and the accurate selection of the specific sites/regions of the gene has important significance for the methylation conversion rate of the methylation library C-X and the further accurate evaluation of the methylation frequency.
Disclosure of Invention
1. Problems to be solved
The invention provides a screening method of a reference site, aiming at the problem that the methylation conversion rate cannot be accurately evaluated by a methylation conversion rate evaluation method established based on a certain gene or a certain gene specific site/region (reference site) because the characteristics (influenced by a base sequence) of a methylation library by an enzymatic conversion method are not considered in the conventional methylation library C-T methylation conversion rate evaluation method, the methylation conversion rate of the reference site is similar to the methylation conversion rate change trend of the methylation library, the methylation conversion rate of the reference site can be used for representing the methylation conversion rate of the methylation library, the methylation conversion rate of the reference site can be used for quickly evaluating the methylation conversion rate of the methylation library, and the methylation conversion rate can be used for quality control of the methylation conversion rate in DNA methylation detection, so that the method is convenient and rapid, and solves the problems of long period, time consumption and labor consumption of the conventional method.
2. Technical scheme
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention provides a method for evaluating the methylation conversion rate of a DNA methylation sequencing library, which comprises the steps of carrying out enzymatic methylation conversion treatment on DNA of a library to be tested, and evaluating the methylation conversion rate of the library through the correlation between the site methylation conversion rate of a reference site and the methylation conversion rate of the library, wherein the site methylation conversion rate of the reference site is related to the methylation conversion rate of the library and can be fitted through a function. The correlation between the site methylation conversion rate of the reference site and the methylation conversion rate of the library means that the site methylation conversion rate of the reference site is lower in the library with lower methylation conversion rate of the library and is increased along with the increase of the methylation conversion rate of the library; by functional fit, it is meant that the site methylation conversion at the reference site can be quantitatively calculated from the library methylation conversion, and thus the site methylation conversion at the reference site is used to calculate the library methylation conversion.
Preferably, the site methylation conversion of the reference site in the above-described assessment method at the remaining sites within. + -.10 bp upstream thereof is not correlated with the library methylation conversion.
Preferably, the Reference site in the above evaluation method is selected from exogenous lambda DNA, and lambda DNA (NCBI Reference Sequence: NC-001416.1) is used as a traditional quality control evaluation to be doped with exogenous gene, the gene is selected without adding experimental operation and is used as an exogenous genome, the related primers and probes do not bind with the human genome, the binding specificity of the primers and probes is ensured to a certain extent, and exogenous completely unmethylated lambda DNA is added at the beginning of the library construction when the gene is selected from exogenous lambda DNA.
Preferably, the reference site in the above assessment method may be selected from endogenous genomic non-CpG sites such as β -actin, GAPDH, ALU gene.
Preferably, the reference site in the above assessment method is selected from the group consisting of the 4625 site or the 6443 site of the foreign lambda DNA genome.
Preferably, the correlation between the site methylation conversion of the control site and the methylation conversion of the library in the above evaluation method is: y = -0.4773x 2 +1.0395x +0.4313, where y is the methylation conversion of the library and x is the methylation conversion of the reference site.
Preferably, the site methylation conversion rate of the reference site is detected by ddPCR.
Preferably, the primer designed on the upstream of the reference site is SEQ ID NO. 1, the primer designed on the downstream of the reference site is SEQ ID NO. 2, the probe for C before transformation is SEQ ID NO. 3, and the probe for T after transformation is SEQ ID NO. 4.
Preferably, the template concentration in the reaction system of ddPCR is 1 to 10 ng/20. Mu.L.
Preferably, the working concentration of the upstream primer and the downstream primer is 600 to 900nmol/L, and the working concentration of the probe is 200 to 300 nmol/L.
Preferably, the annealing temperature of the primer during amplification is 52-58 ℃.
The invention also provides a screening method of the reference site, which utilizes the C-T methylation conversion rate difference library to screen the reference site related to the conversion rate, and firstly constructs libraries with different methylation conversion rates to obtain a methylation conversion rate difference library; then obtaining the conversion rate of all C-T sites of corresponding lambda DNA in each methylation conversion rate difference library; counting the correlation between C-T conversion rate of the C site of lambda DNA and library conversion rate, and selecting site methylation conversion rate to be related to library methylation conversion rate, namely in the library with lower library methylation conversion rate, the site methylation conversion rate of a reference site is lower and is increased along with the increase of the library conversion rate; and the site that can be fitted by the function is the reference site.
The invention also provides application of the method for evaluating the methylation conversion rate of the DNA methylation sequencing library, which is used for evaluating whether the methylation conversion rate of the library meets a set quality control threshold value in the rapid quality control of the methylation conversion rate of the methylation library.
Preferably, the threshold is not lower than 0.980, that is, when the methylation conversion rate of the detected library is lower than 0.980, the quality control of the library is judged to be unqualified, the on-machine sequencing is not required to be arranged, the waste of on-machine resources is reduced, and the specific quality control threshold can be set according to the actual situation.
Preferably, the above application further comprises evaluating the methylation conversion rate of the detected library and then evaluating whether the methylation conversion rate meets the above quality control requirement.
The invention also provides a terminal device, which comprises a memory, a processor and a computer program stored on the memory and capable of running on the processor, wherein the processor runs the computer program to realize the method for evaluating the methylation conversion rate of the DNA methylation sequencing library or the application of the method for evaluating the methylation conversion rate of the DNA methylation sequencing library, namely, the methylation conversion rate of the library is calculated and output according to the site methylation conversion rate of the reference site and the function between the site methylation conversion rate of the reference site and the methylation conversion rate of the library; or further according to a set quality control threshold, when the methylation conversion rate of the library is lower than the set quality control threshold, outputting the result that the library is unqualified in quality control, otherwise, outputting the result that the library is qualified in quality control.
The present invention also provides a computer-readable storage medium storing a computer program capable of calculating and outputting the above-mentioned methylation conversion ratio of a library from the site methylation conversion ratio of a reference site and a function between the site methylation conversion ratio of the reference site and the methylation conversion ratio of the library; or further according to a set quality control threshold, when the methylation conversion rate of the library is lower than the set quality control threshold, outputting a result that the library is unqualified in quality control, otherwise, outputting a result that the library is qualified in quality control.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the method for detecting the methylation conversion rate of the library aiming at the enzymatic conversion and the application thereof, the differences of different base sequences on the C-T conversion rate are comprehensively considered, and the defect of detection of the methylation conversion rate of the enzymatic conversion library is made up.
(2) According to the method for detecting the methylation conversion rate of the library aiming at the enzymatic conversion and the application thereof, the methylation conversion rate of the library is evaluated by detecting the lambda DNA conversion rate through ddPCR, a sample with low conversion rate stops being loaded on the computer, and the waste of sequencing resources on the computer is reduced.
(3) The invention relates to a method for detecting methylation conversion rate of a library aiming at enzymatic conversion and application thereof.A 4625 site or 6443 site in a lambda DNA genome is selected as a reference site, the methylation conversion rate of the site and the methylation conversion rate of the library can be fitted through a function, and the methylation conversion rates of the sites of the rest sites in plus or minus 10 bp in the upstream and the downstream of the site are irrelevant to the methylation conversion rate of the library, so that a specific probe can be designed, and the interference of the upstream and the downstream sites can be reduced.
Drawings
FIG. 1 is C-T conversion at C-site of lambda DNA in a differential library of C-T conversions;
FIG. 2 shows C-T conversion rates at C-site of lambda DNA in a differential library of C-T conversion rates;
FIG. 3 is C-T conversion at C-site of lambda DNA in a differential library of C-T conversions;
FIG. 4 shows C-T conversion rates at C-site of lambda DNA in a differential library of C-T conversion rates;
FIG. 5 is a graph relating lambda DNA site conversion to library conversion, where the X-axis is the C-T conversion of the lambda DNA site and the Y-axis is the C-T conversion of the corresponding library.
Detailed Description
The invention is further described with reference to specific examples.
It should be noted that the terms "upper", "lower", "left", "right" and "middle" used in the present specification are for the sake of clarity, and are not intended to limit the scope of the present invention, and changes or adjustments of the relative relationship thereof may be made without substantial technical changes.
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; as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, measure or value. The degree of flexibility for a particular variable can be readily determined by one skilled in the art.
As used herein, at least one of the terms "is intended to be synonymous with one or more of. For example, "A, B and at least one of C" expressly includes a only, B only, C only, and their respective combinations.
Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limit values of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all of the aforementioned values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or feature being described.
Example 1
This example provides the selection of methylation library methylation conversion reference sites, including:
randomly establishing methylation libraries with different C-T methylation conversion rates by an enzymology conversion method, namely C-T methylation conversion rate difference libraries;
obtaining C-T methylation conversion data for a differential library of C-T methylation conversions by high throughput sequencing (NGS);
obtaining corresponding lambda DNA (NCBI Reference Sequence: NC-001416.1, purchased from Thermo Fisher Scientific) in each C-T methylation conversion difference library for all C-T C-site C-T conversion data (complete conversion site is 100%);
statistically observing the correlation between the C-T conversion rate of the differential library and the C-T conversion rate of all C sites of the lambda DNA in the differential library.
And (4) analyzing results:
the results of the data analysis are shown in FIGS. 1-4, which show the expression of C-T conversion at C-T methylation conversion difference library at C-T site of lambda DNA, where the C position of lambda DNA is the C base at the position of lambda genome (NC _ 001416.1); W/C represents that the C site is located in a double-stranded nucleic acid Watson-Crick, wherein + represents a Watson strand, -represents a Crick strand; c type and sequence represent methylation type and corresponding base sequence; conversion, first column C-T conversion for methylation libraries, the remainder being C-site conversion for lambda DNA. From this, the following can be found:
(1) As shown in FIG. 1, the type 1 sites (such as site 5331) have no correlation between the conversion rate of a single site and the conversion rate of the library, and in the library with low library conversion rate, the conversion rate of the sites is high, and the conversion rate of the sites does not change obviously along with the increase of the library conversion rate, so that the conversion rate of the single site cannot be selected to characterize the conversion rate of the library;
(2) As shown in FIG. 2, for type 2 sites (e.g., site 4367), the single site conversion rate is correlated with the conversion rate of the library, the conversion rate is lower in the library with lower library conversion rate (compared with the C-T conversion rate of the library), and is increased along with the increase of the library conversion rate, but the single site conversion rates of sites upstream and downstream of the site are also correlated with the conversion rate of the library, and the selection is not suitable in consideration of the probe specificity;
(3) As shown in FIGS. 3-4, for type 3 sites (e.g.: sites 4625, 6443), the single site conversion rate is correlated with the conversion rate of the library, the conversion rate is lower in the library with lower library conversion rate (compared with library C-T conversion rate), and is increased with the increase of the library conversion rate, and only the single site conversion rate is correlated with the conversion rate of the library in a certain region, finally, the region where the sites are located is selected to design primers and probes.
Example 2
This example provides a correlation fit between site methylation conversion and library methylation conversion for reference site 6443 in example 1, comprising:
65 methylation libraries with known methylation conversion were selected and tested by ddPCR for C-T conversion at reference site 6443, the primers and probes are shown in Table 1, and the results are shown in FIG. 5As shown, by fitting, the C-T conversion of the reference site 6443 correlates with the methylation conversion of the library as y = -0.4773x 2 +1.0395x +0.4313, where y is the methylation conversion of the library and x is the methylation conversion of the reference site.
TABLE 1
Remarking: final concentration of the primer: 900nmol/L, final probe concentration 250nmol/L.
Example 3
This example provides a method for evaluating methylation conversion rate of methylation library for enzymatic transformation, which comprises adding 1% exogenous completely unmethylated lambda DNA (linear double-stranded completely unmethylated lambda phage DNA isolated from methylase deficient E.coli, NCBI Reference Sequence: NC-001416.1) at the beginning of library construction, selecting lambda DNA 6443 site as Reference site, subjecting the DNA of the library to enzymatic methylation conversion treatment, detecting the ratio of Reference site conversion copy number/total copy number (T/(T + C)) in the transformed lambda DNA by ddPCR method, and calculating the methylation conversion rate of the library according to the correlation between the site methylation conversion rate of the Reference site and the methylation conversion rate of the library.
The 12 samples in example 2 were selected for validation, and the results are shown in table 2, and the site conversion rate and the library conversion rate have high consistency (calculated library conversion rate/actual conversion rate > 99.00%), which indicates that the method can accurately infer the library conversion rate through the site conversion rate.
TABLE 2
Example 4
This example provides a reproducible test of samples, in the same manner as example 3.
The results are shown in table 3, where the results of 2 replicates of the reference site conversion rate are qualitatively and completely consistent with the library conversion rate.
TABLE 3
Example 5
This example provides an application of the method for evaluating methylation conversion rate of a methylation library transformed by enzymology described in example 3, and the method is used for rapid quality control of methylation conversion rate of a methylation library in a methylation sequencing process, after the methylation library is constructed, the methylation conversion rate of the methylation library is directly evaluated by using the above evaluation method, when the methylation conversion rate of the evaluated methylation library is less than 0.980, the methylation library quality control is determined to be unqualified, the unqualified library does not need to be subjected to subsequent sequencing, and waste of sequencing resources on a computer is reduced.
From the above description, it can be seen that the methylation conversion efficiency of the enzymatic human methylation library can be rapidly evaluated by detecting the site conversion rate of the reference site of the present invention using ddPCR. After the library is constructed, direct detection and evaluation are carried out, quality control of the library before the on-machine is carried out, subsequent sequencing is not needed for the unqualified library with low conversion efficiency, and waste of on-machine sequencing resources is reduced.
Example 6
This embodiment provides a terminal device comprising a memory, a processor and a computer program stored in said memory and executable on said processor, said computer program being adapted to obtain a site methylation conversion at a reference site and input it to the terminal device, said computer program being adapted to be executed by the processor to calculate and output a library methylation conversion based on the site methylation conversion at the reference site and a function between the site methylation conversion at the reference site and the library methylation conversion.
Example 7
The embodiment provides a terminal device, which comprises a memory, a processor and a computer program stored on the memory and capable of running on the processor, wherein the computer program is used for obtaining the site methylation conversion rate of a reference site and inputting the site methylation conversion rate into the terminal device, the processor runs the computer program, calculating the library methylation conversion rate according to the site methylation conversion rate of the reference site and a function between the site methylation conversion rate of the reference site and the library methylation conversion rate, and outputting that the methylation library quality control is unqualified when the library methylation conversion rate is lower than 0.980; otherwise, outputting the methylation library with qualified quality control.
Claims (12)
1. A method for evaluating the methylation conversion rate of a DNA methylation sequencing library is characterized in that the method comprises the steps of evaluating the methylation conversion rate of the library through the correlation between the site methylation conversion rate of a reference site and the methylation conversion rate of the library after carrying out enzymatic methylation conversion treatment on DNA of a library to be tested, wherein the site methylation conversion rate of the reference site is correlated with the methylation conversion rate of the library and can be fitted through a function.
2. The method of claim 1, wherein the methylation conversion of the remaining sites within ± 10 bp upstream and downstream of the reference site is unrelated to the methylation conversion of the library.
3. The method for assessing the methylation conversion rate of a DNA methylation sequencing library according to claim 1 or 2, wherein the reference site is selected from exogenous lambda DNA; or selected from endogenous genomic non-CpG sites.
4. The method for evaluating the methylation conversion rate of the DNA methylation sequencing library according to claim 3, wherein the reference site is selected from the 4625 site or 6443 site in the lambda DNA genome.
5. The method for evaluating the methylation conversion rate of the DNA methylation sequencing library according to claim 4, wherein the reference site is selected from 6443 sites in the lambda DNA genome, and the correlation between the methylation conversion rate of the site and the methylation conversion rate of the library is as follows: y = -0.4773x 2 +1.0395x+0.4313, wherein y is the methylation conversion of the library and x is the methylation conversion of the reference site.
6. The method for evaluating the methylation conversion rate of the DNA methylation sequencing library of claim 5, wherein the site methylation conversion rate of the reference site is detected by a ddPCR method, the primer designed at the upstream of the reference site is SEQ ID NO. 1, the primer designed at the downstream of the reference site is SEQ ID NO. 2, the probe for C before transformation is SEQ ID NO. 3, and the probe for T after transformation is SEQ ID NO. 4.
7. The method for evaluating the methylation conversion rate of the DNA methylation sequencing library of claim 6, wherein the template concentration in the reaction system of ddPCR is 1 to 10ng/20 ul, the working concentration of the upstream primer and the downstream primer is 600 to 900nmol/L, and the working concentration of the probe is 200 to 300 nmol/L.
8. The method for screening a reference site in the method for evaluating the methylation conversion rate of a DNA methylation sequencing library of claim 1, wherein the screening method of the reference site is to screen a reference site related to the conversion rate by using a C-T methylation conversion rate difference library, and firstly, a library with different methylation conversion rates is constructed to obtain a methylation conversion rate difference library; then obtaining the conversion rate of all C sites C-T sites in each methylation conversion rate difference library; counting the correlation between the C-T conversion rate of the C site and the conversion rate of the library, and selecting the correlation between the methylation conversion rate of the site and the methylation conversion rate of the library; and the site that can be fitted by the function is the reference site.
9. The use of the method of any one of claims 1 to 7 for assessing the methylation conversion rate of a DNA methylation sequencing library, wherein the method is used for rapid quality control of the methylation conversion rate of a methylation library to assess whether the methylation conversion rate of the library meets a set quality control threshold.
10. The use of the method of claim 9, wherein the quality control threshold is not lower than 0.980, and when the detected methylation conversion rate of the library is lower than 0.980, the quality control of the library is determined to be unqualified.
11. A terminal device comprising a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor implements the method for assessing the methylation conversion rate of a DNA methylation sequencing library according to any one of claims 1 to 7 or implements the method for assessing the methylation conversion rate of a DNA methylation sequencing library according to any one of claims 9 to 10 when the computer program is executed.
12. A computer-readable storage medium, wherein the computer-readable storage medium stores a computer program, which when executed by a processor implements the method for assessing the methylation conversion rate of a DNA methylation sequencing library according to any one of claims 1 to 7 or the method for assessing the methylation conversion rate of a DNA methylation sequencing library according to any one of claims 9 to 10.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002567A (en) * | 2015-06-30 | 2015-10-28 | 北京百迈客生物科技有限公司 | Method for constructing high-throughput simplified methylation sequencing library without reference genome |
| CN107541791A (en) * | 2017-10-26 | 2018-01-05 | 中国科学院北京基因组研究所 | Construction method, kit and the application in plasma DNA DNA methylation assay library |
| CN111020017A (en) * | 2019-12-31 | 2020-04-17 | 北京优迅医学检验实验室有限公司 | Method for evaluating DNA methylation conversion efficiency and application thereof |
| US20220275424A1 (en) * | 2019-07-08 | 2022-09-01 | Ludwig Institute For Cancer Research Ltd | Bisulfite-free, whole genome methylation analysis |
-
2022
- 2022-10-31 CN CN202211343981.0A patent/CN115404270B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002567A (en) * | 2015-06-30 | 2015-10-28 | 北京百迈客生物科技有限公司 | Method for constructing high-throughput simplified methylation sequencing library without reference genome |
| CN107541791A (en) * | 2017-10-26 | 2018-01-05 | 中国科学院北京基因组研究所 | Construction method, kit and the application in plasma DNA DNA methylation assay library |
| US20220275424A1 (en) * | 2019-07-08 | 2022-09-01 | Ludwig Institute For Cancer Research Ltd | Bisulfite-free, whole genome methylation analysis |
| CN111020017A (en) * | 2019-12-31 | 2020-04-17 | 北京优迅医学检验实验室有限公司 | Method for evaluating DNA methylation conversion efficiency and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| ZHOU, JUNHUA等: "BCREval: a computational method to estimate the bisulfite conversion ratio in WGBS", 《BMC BIOINFORMATICS》 * |
| 刘洋洋，等: "DNA甲基化分析中重亚硫酸盐处理DNA转化效率的评估方法", 《遗传》 * |
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