CN115404270B - 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, which can output the methylation conversion rate of the library or output the result of whether the library is qualified or not according to the methylation conversion rate of the 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 oxidizing 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5 hmC) to 5-carboxycytosine (5 caC) by a cascade reaction, followed by deamination with deaminase APOBEC to convert cytosine to thymine (T), while 5-hydroxymethylcytosine (5 hmC) and 5-carboxycytosine (5 caC) are not affected. 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.
During the DNA methylation sequencing by the enzymatic conversion method, if unmethylated C can not be converted into T, the methylated C can be misjudged as a methylated state, and then the methylation frequency can be 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 is also a report in the literature on the rapid evaluation of methylation library C-X methylation conversion by means of either qPCR (quantitative PCR) evaluation of the beta-actin promoter region of the reference gene or ddPCR (droplet digital PCR) evaluation of Alu repeats, which is based on the principle of evaluating methylation library C-X methylation conversion using specific region or even single site C-X conversion. 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 the same as the C-T conversion rate trend of the methylation library, presumably because the DNA methylation sequencing by the enzymology conversion method is a reaction in which an enzyme participates, unlike the process of bisulfite sequencing 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 evaluation method of the C-T methylation conversion rate of the methylation library, 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 method 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 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 library conversion rate; by functional fit, it is meant that the site methylation conversion at the reference site and the library methylation conversion can be calculated quantitatively, 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 remaining sites within + -10 bp upstream and downstream of the reference site in the above-described evaluation method is not correlated with the library methylation conversion.
Preferably, the Reference site in the above evaluation method is selected from exogenous lambda DNA, which is incorporated into an exogenous gene as a conventional quality control evaluation, and is selected as an exogenous genome without additional experimental manipulations, and the primers and probes involved are not bound to the human genome, and the specificity of binding of the primers and probes is ensured to some extent, and exogenous completely unmethylated lambda DNA is added at the beginning of the library when selected from exogenous lambda DNA.
Preferably, the reference site in the above evaluation 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 6443 site of the exogenous lambda DNA genome.
Preferably, the correlation between site methylation conversion to control site and library methylation conversion 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 sites C-T sites of corresponding lambda DNA in each methylation conversion rate differential 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 position which can be fitted through the function is the reference position.
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 actual conditions.
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 a library methylation conversion rate based on the site methylation conversion rate of a reference site and a function between the site methylation conversion rate of the reference site and the library methylation conversion rate; 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.
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 enzymology conversion and the application of the method, the methylation conversion rate of the library is evaluated by detecting the conversion rate of lambda DNA 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 of a site in a lambda DNA genome is selected as a reference site, the methylation conversion rate of the site can be fitted with the methylation conversion rate of the library through a function, and the methylation conversion rates of the sites of other sites within +/-10 bp of upstream and 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 downstream sites can be reduced.
Drawings
FIG. 1 shows C-T conversion rates at C-site of lambda DNA in a differential library of C-T conversion rates;
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, wherein 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", "middle", and the like 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 and modifications of the relative relationship between the terms are also regarded as the scope of the present invention without substantial changes in the technical contents.
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 carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
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, "at least one of a, B, and C" explicitly includes only a, only B, only C, 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 as including all such values and ranges. Further, such interpretation should apply regardless of the breadth of the range or the characteristics being described.
Example 1
This example provides selection of a methylation conversion reference site in a methylation library, 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 transformation difference library for all C-T site C-T transformation data (complete transformation 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 the C site of lambda DNA, where the C site of lambda DNA is the C base at 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 the 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 with the increase of the library conversion rate, so that the conversion rate of the library represented by the conversion rate of the single site is not suitable for selection;
(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:
68 cases of methylation libraries with known methylation conversion rates were selected, the C-T conversion rate of the reference site 6443 was detected by ddPCR, the results are shown in Table 1, the results are shown in FIG. 5, and by fitting, the correlation between the C-T conversion rate of the reference site 6443 and the methylation conversion rate of the library was 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, and a final probe concentration of 250nmol/L.
Example 3
This example provides a method for evaluating methylation conversion rate of an enzymatically transformed methylation library, 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 as a Reference site, performing enzymatic methylation conversion treatment on the DNA of the library to be tested, detecting the ratio of the Reference site conversion copy number/total copy number (T/(T + C)) in the transformed lambda DNA by ddPCR, and calculating the methylation conversion rate according to the correlation between the site methylation conversion rate of the Reference site and the methylation conversion rate of the library.
The results of the verification by selecting 12 samples are shown in table 2, the site conversion rate is consistent with the library conversion rate (the calculated library conversion rate/actual conversion rate is more than 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 for enzymatic conversion described in example 3, which is used for rapid quality control of methylation conversion rate of a methylation library in a methylation sequencing process, and after the methylation library is constructed, the method is directly used for evaluating methylation conversion rate of the methylation library, and when the methylation conversion rate of the methylation library is lower than 0.980, the quality control of the methylation library is determined to be unqualified, and the unqualified library does not need to be subjected to subsequent sequencing, thereby reducing waste of sequencing resources on a computer.
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 (7)
1. Evaluation method for methylation conversion rate of DNA methylation sequencing libraryAnd (b) evaluating the methylation conversion of the library by correlating the methylation conversion of the site of a reference site selected from the group consisting of 6443 sites in the lambda DNA genome with the methylation conversion of the library after enzymatic methylation conversion of the DNA of the library to be tested, wherein the correlation between the methylation conversion of the reference site and the methylation conversion of the library 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.
2. The method for evaluating the methylation conversion rate of the DNA methylation sequencing library of claim 1, 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.
3. The method as claimed in claim 2, wherein the template concentration in the ddPCR reaction system is 1 to 10ng/20 μ L, 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.
4. The use of the method for assessing the methylation conversion rate of a DNA methylation sequencing library according to any one of claims 1 to 3, 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.
5. The use of the method of claim 4, 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.
6. 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 3 or implements the method for assessing the methylation conversion rate of a DNA methylation sequencing library according to any one of claims 4 to 5 when the computer program is executed.
7. 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 3 or the method for assessing the methylation conversion rate of a DNA methylation sequencing library according to any one of claims 4 to 5.
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