CN110211633B

CN110211633B - Detection method for MGMT gene promoter methylation, processing method for sequencing data and processing device

Info

Publication number: CN110211633B
Application number: CN201910373154.8A
Authority: CN
Inventors: 闫慧婷; 洪媛媛; 于佳宁; 李彩琴; 李鑫; 宋小凤; 陈维之; 何骥
Original assignee: Wuxi Precision Medical Laboratory Co ltd
Current assignee: Wuxi Precision Medical Laboratory Co ltd
Priority date: 2019-05-06
Filing date: 2019-05-06
Publication date: 2021-08-31
Anticipated expiration: 2039-05-06
Also published as: CN110211633A

Abstract

The invention provides a detection method for MGMT gene promoter methylation, a processing method and a processing device for sequencing data. The processing method comprises the steps of obtaining methylation sequencing data from an MGMT gene promoter, wherein the methylation sequencing data are double-ended sequencing sequences; comparing the methylation sequencing data with a human reference genome sequence to obtain a comparison result, wherein the comparison result comprises a first end first matching region, a first end second matching region, a second end first matching region and a second end second matching region, and the first end second matching region is overlapped with the second end second matching region; removing the first end second matching region or the second end second matching region in the comparison result to obtain data to be analyzed; and identifying methylation sites in the data to be analyzed to obtain a methylation result of the MGMT gene promoter. The methylation site detected by the processing method is high in accuracy and high in flux, and the methylation level can be evaluated conveniently from the whole level.

Description

Detection method for MGMT gene promoter methylation, processing method for sequencing data and processing device

Technical Field

The invention relates to the field of gene detection, in particular to a detection method for MGMT gene promoter methylation, a processing method for sequencing data and a processing device.

Background

MGMT is a DNA repair protein ubiquitous in cells and capable of binding O⁶The guanine complex is removed from the DNA, restoring damaged guanine and protecting chromosomes from the damage of alkylating agents. In this process, MGMT acts as both a methyltransferase and a methyl acceptor protein, completing the transfer reaction separately.

The methylation state of the MGMT gene promoter has certain correlation with the sensitivity of an alkylating agent medicament. Alkylating agents, such as Temozolomide (TMZ), pyrimidine nitrosourea (ACNU) and dichloroethyl nitrosourea (BCNU), are widely used in the treatment of human tumors as chemotherapeutic drugs. An important site of action for these alkylating agents is O⁶Guanine, and MGMT is capable of rapidly removing O⁶Alkyl compounds on guanine, thereby reducing the curative effect of the alkylating agent in killing tumors and causing tumor drug resistance.

Therefore, the detection of the methylation state of the MGMT gene promoter is helpful for predicting the sensitivity of the tumor to chemotherapeutic drugs of alkylating agents, and is further helpful for guiding the formulation of a chemotherapeutic scheme and avoiding drug resistance.

At present, the common methods for detecting the methylation of the MGMT promoter comprise the following steps: bisulfite Sequencing PCR (BSP), Methylation Specific PCR (MSP), fluorometry, and methylation sensitive high resolution melting curve analysis (MS-HRM).

Among them, the Bisulfite Sequencing PCR (BSP) method mainly detects methylation state by PCR combined sanger sequencing technology, but is not suitable for large-scale detection due to complex operation and long detection period. The number of clones picked at the same time may cause false positives in the results, so BSP can only be counted as a semi-quantitative method.

Methylation-specific PCR (msp) methods, which use PCR amplification to determine the presence of methylation in a sample, are practical and widely used, but do not allow quantitative detection and have a high risk of false positives.

The fluorescence quantitative method is a technology developed based on MSP, mainly adds a TaqMan probe in the detection process, thereby ensuring higher sensitivity and accuracy, but can only realize integrated analysis if more methylation sites are detected, and simultaneously has higher probe cost, so the method is not suitable for the detection of a large number of samples and more sites.

Methylation-sensitive high-resolution melting curve analysis (MS-HRM) is to judge whether methylation exists by converting the difference of single base sequences into the difference of melting curves, but the method has high requirements on instruments, a fluorescent quantitative PCR instrument with an HRM module is required, and the method can only analyze the methylation state of the whole fragment and cannot clearly determine the methylation state of each CpG site.

Therefore, there is still a need to provide an efficient and accurate detection scheme for detecting the methylation state of the MGMT gene.

Disclosure of Invention

The invention mainly aims to provide a detection method for MGMT gene promoter methylation, a processing method and a processing device for sequencing data, so as to solve the problem of low detection accuracy in the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided a processing method of MGMT gene promoter methylation sequencing data, the processing method comprising: obtaining methylation sequencing data from an MGMT gene promoter, wherein the methylation sequencing data is a double-end sequencing sequence; comparing the methylation sequencing data with a human reference genome sequence to obtain a comparison result, wherein the comparison result comprises a first end first matching region, a first end second matching region, a second end first matching region and a second end second matching region, and the first end second matching region is overlapped with the second end second matching region; removing the first end second matching area or the second end second matching area in the comparison result to obtain data to be analyzed; and (3) carrying out methylation site recognition on data to be analyzed to obtain a methylation result of the MGMT gene promoter.

Further, prior to aligning the methylation sequencing data to the human reference genomic sequence, the processing method further comprises: performing C-to-T conversion pretreatment on the human reference genome sequence; and performing C-to-T conversion pretreatment on the double-ended sequencing sequence.

Further, after obtaining the data to be analyzed and before performing methylation site recognition on the data to be analyzed, the processing method further comprises the step of correcting the data to be analyzed, and the step of correcting the data to be analyzed comprises the following steps: and correcting the data to be analyzed by using the human reference genome sequence, the position information of the human reference genome sequence and the crowd high-frequency SNP locus.

Further, the step of identifying methylation sites in the data to be analyzed to obtain the methylation result of the MGMT gene promoter comprises the following steps: carrying out primary identification on methylation sites in data to be analyzed to obtain primary identification sites; carrying out credibility screening on the initial identification site to obtain a methylation result of the MGMT gene promoter; preferably, the parameter setting conditions of the credibility screening are as follows: the coverage is less than 3000000, the probability ratio standard of the best genotype to the next best genotype is more than or equal to 20, and the comparison quality is more than 5.

In order to achieve the above object, according to one aspect of the present invention, there is provided a processing device of MGMT gene promoter methylation sequencing data, the processing device comprising: the acquisition module is used for acquiring methylation sequencing data from an MGMT gene promoter, wherein the methylation sequencing data is a double-end sequencing sequence; the system comprises a comparison module, a comparison module and a comparison module, wherein the comparison module is used for comparing methylation sequencing data with a human reference genome sequence to obtain a comparison result, and the comparison result comprises a first end first matching region, a first end second matching region, a second end first matching region and a second end second matching region, wherein the first end second matching region is overlapped with the second end second matching region; the removing module is used for removing the first end second matching area or the second end second matching area in the comparison result to obtain data to be analyzed; and the methylation recognition module is used for recognizing methylation sites in the data to be analyzed to obtain a methylation result of the MGMT gene promoter.

Further, the processing apparatus further includes: the first pretreatment module is used for carrying out C-to-T conversion pretreatment on the human reference genome sequence; and a second pretreatment module for performing C-to-T conversion pretreatment on the paired-end sequencing sequence.

Furthermore, the processing device also comprises a correction module for correcting the data to be analyzed, and the correction module is used for correcting the data to be analyzed by utilizing the human reference genome sequence, the position information of the human reference genome sequence and the crowd high-frequency SNP locus.

Further, the methylation identification module comprises: the preliminary identification module is used for carrying out preliminary identification on the methylation sites in the data to be analyzed to obtain preliminary identification sites; the credibility screening module is used for carrying out credibility screening on the primary identified sites to obtain a methylation result of the MGMT gene promoter; preferably, the parameter setting conditions of the credibility screening are as follows: the coverage is less than 3000000, the probability ratio standard of the best genotype to the next best genotype is more than or equal to 20, and the comparison quality is more than 5.

According to another aspect of the present invention, there is provided a method for detecting methylation of a promoter of an MGMT gene, the method comprising: carrying out bisulfite conversion on gDNA of a sample to be detected to obtain converted DNA; constructing an amplicon library for the converted DNA to obtain the amplicon library; sequencing the amplicon library to obtain sequencing data; and carrying out methylation analysis on the sequencing data by adopting any processing method or any processing device to obtain a methylation result of the MGMT gene promoter.

Further, an amplicon library construction is carried out on the converted DNA by adopting an amplification primer to obtain the amplicon library, wherein the amplification primer comprises an upstream sequence and a downstream sequence, the upstream sequence is SEQ ID NO. 1, and the downstream sequence is SEQ ID NO. 2; preferably, the working concentration of the amplification primer is 5-15 mu M, preferably 10 mu M; preferably, the annealing temperature of the amplification primer is 45-55 ℃, and preferably 50 ℃; preferably, in the process of constructing the amplicon library for the transformation DNA by amplification of the amplification primer, the transformation DNA is amplified for 30-40 cycles, preferably 35 cycles, to obtain the amplicon library.

By applying the technical scheme of the invention, the methylation analysis process of the methylation sequencing data is improved, so that the accuracy of the finally detected methylation sites is higher, and the flux of the sites capable of being detected is correspondingly higher, thereby being beneficial to evaluating the methylation level from the whole level.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows a schematic flow diagram of a processing method of MGMT gene promoter methylation sequencing data according to an embodiment of the present invention;

FIG. 2 shows a schematic diagram of a processing device for MGMT gene promoter methylation sequencing data in a preferred embodiment of the present application;

FIG. 3 shows the methylation levels of individual CpG sites detected by the pyrophosphate detection method in example 6;

FIG. 4 shows the methylation level at each CpG site and the methylation level at each DNA template molecule as detected in example 6 using the methods of the present application.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data so used may be interchanged under appropriate circumstances such that embodiments of the application described herein may be used. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

For convenience of description, some terms or expressions referred to in the embodiments of the present application are explained below:

plus and minus strands of DNA: it refers to two strands that are complementary in opposite directions. The strand given by the reference genome is the so-called forward strand (for), and the other strand is the reverse strand (reverse).

And sense strand (sense strand) and antisense strand (antisense strand): refers to the set of two complementary DNA strands carrying the numbered protein information, called the sense strand, also called the coding strand, which is identical to the RNA sequence. The other complementary strand, called the antisense strand, is the strand that gives the RNA as the template, although it is complementary to the RNA in the reverse direction, and is therefore also called the template strand.

In a double stranded DNA molecule comprising several genes, the sense strands of the respective genes are not all on the same strand. That is, some genes have their sense strand positive (forward strand) and some genes have their sense strand reverse (reverse strand), i.e., one of the two DNA strands is the sense strand for some genes and the other is the antisense strand for other genes.

As mentioned in the background art, the methylation detection method of MGMT gene promoter in the prior art has the defects of low efficiency or low accuracy, and in order to improve the situation, the inventor carries out comparative analysis on the existing methylation detection method of MGMT gene promoter, and finds that when the existing Bisulfite Sequencing PCR (BSP) method is used for designing a primer, partial C base of a DNA sequence is converted into T after sulfite treatment, so that the CG content and the TM value in the sequence region are greatly changed, and further the ideal primer sequence obtained by conventional primer design software on the sequence is influenced. In order to provide an amplification primer with better specificity and higher amplification efficiency, the inventor designs dozens of pairs of primers aiming at the promoter site of the gene, fully considers the characteristics of DNA treated by sulfite, screens candidate target primers by simulating GC content and TM value after C base is converted into T, and finally determines a pair of primers with the best amplification efficiency and specificity through experimental verification. Methylation detection is tried by an NGS method on the basis of the primer amplification product, and sequencing data is found by an improved methylation analysis process, so that the accuracy of the finally detected methylation site is higher, and the flux of the site capable of being detected is correspondingly higher, thereby facilitating the evaluation of the methylation level by combining the integral methylation site information.

On the basis of the above research results, the applicant proposed the technical solution of the present application. In an exemplary embodiment, a method for processing MGMT gene promoter methylation sequencing data is provided, and fig. 1 is a flowchart illustrating a method for processing MGMT gene promoter methylation sequencing data in the examples of the present application. As shown in fig. 1, the processing method includes:

step S10, obtaining methylation sequencing data from an MGMT gene promoter, wherein the methylation sequencing data are double-ended sequencing sequences;

step S30, comparing the methylation sequencing data with the human reference genome sequence to obtain a comparison result, wherein the comparison result comprises a first end first matching region, a first end second matching region, a second end first matching region and a second end second matching region, and the first end second matching region and the second end second matching region are overlapped;

step S50, removing the first end second matching area or the second end second matching area in the comparison result to obtain the data to be analyzed;

and step S70, carrying out methylation site recognition on the data to be analyzed to obtain a methylation result of the MGMT gene promoter.

According to the processing method of methylation sequencing data aiming at the MGMT gene promoter, the sequence of the overlapping region in the comparison of the sequencing data at the two ends in the comparison result is subjected to duplication elimination, so that the result is more accurate in subsequent identification and methylation level statistics.

In the above comparison step, the existing methylation comparison strategy can be adopted. In a preferred embodiment, before aligning the methylation sequencing data to the human reference genomic sequence, the processing further comprises: performing C-to-T conversion pretreatment on the human reference genome sequence; and performing C-to-T conversion pretreatment on the double-ended sequencing sequence.

Specifically, depending on the amplification source of the methylation sequencing data to be processed (whether derived from the positive or negative strand of the genome), the sense and antisense strands corresponding to the positive or negative strand of the corresponding human reference genomic sequence are pretreated for C to T (or G to a) conversion, respectively, to serve as reference alignment sequences. Accordingly, the sequencing sequences at each end of the paired-end sequencing sequence were pretreated for C to T (or G to A) transformation, respectively.

Before alignment, it is not clear whether the paired-end sequenced sequence belongs to the positive or negative strand of the human reference genomic sequence, and only after alignment can it be determined according to the alignment position.

In order to make the methylation level of each subsequent site relatively more accurate, in a preferred embodiment, after obtaining the data to be analyzed and before identifying the methylation site in the data to be analyzed, the processing method further comprises the step of correcting the data to be analyzed, and the step of correcting the data to be analyzed comprises the steps of: and correcting the data to be analyzed by using the human reference genome sequence, the position information of the human reference genome sequence and the crowd high-frequency SNP locus.

The correction step can remove some low quality sites, the so-called quality including sequencing quality or alignment quality. The specific correction software can adopt a Bisulfit Count Covariates module and a Bisulfit Table Recarrangement module in the BisSNP software to correct. The correction steps are carried out to improve the identification accuracy.

In order to further improve the reliability of each methylation site, in a preferred embodiment, the step of identifying the methylation site in the data to be analyzed to obtain the methylation result information of the promoter of the MGMT gene comprises: carrying out primary identification on methylation sites in data to be analyzed to obtain primary identification sites; carrying out credibility screening on the primary identified sites to obtain methylation result information of the MGMT gene promoter; preferably, the parameter setting conditions of the credibility screening are as follows: the coverage is less than 3000000, the probability ratio standard of the best genotype to the next best genotype is more than or equal to 20, and the comparison quality is more than 5.

Specifically, in the initial identification step, the bisfilt Genotyper module of BisSNP can be adopted to simultaneously identify SNP/methylation sites, so as to obtain initial vcf files of SNP and CpG methylation respectively. Then, the primarily identified methylated VCF files are sorted according to the genome positions through a sort By Ref And Add Cor module of BisSNP, And then the low-credibility methylated sites in the sorted methylated VCF files are filtered By a VCF post process module of BisSNP. The specific filtering condition can be the default value of the software module.

It should be noted that the steps illustrated in the above-described flow diagrams may be performed in a computer system, such as a set of computer-executable instructions, and that, although a logical order is illustrated in the flow diagrams, in some cases, the steps illustrated or described may be performed in an order different than presented herein.

The embodiment of the present application further provides a processing apparatus for MGMT gene promoter methylation sequencing data, and it should be noted that the processing apparatus of the embodiment of the present application can be used for executing the processing method for MGMT gene promoter methylation sequencing data provided in the embodiment of the present application. The processing apparatus will be described below.

Fig. 2 is a schematic diagram showing a processing device for MGMT gene promoter methylation sequencing data in the example of the present application. As shown in fig. 2, the processing apparatus includes: an acquisition module 20, an alignment module 40, a removal module 60, and a methylation identification module 80.

An obtaining module 20, configured to obtain methylation sequencing data derived from an MGMT gene promoter, where the methylation sequencing data is a double-ended sequencing sequence;

a comparison module 40, configured to compare the methylation sequencing data with a human reference genome sequence to obtain a comparison result, where the comparison result includes a first end first matching region, a first end second matching region, a second end first matching region, and a second end second matching region, and the first end second matching region overlaps with the second end second matching region;

a removing module 60, configured to remove the first end second matching region or the second end second matching region in the comparison result, so as to obtain data to be analyzed;

and the methylation recognition module 80 is used for recognizing methylation sites in the data to be analyzed to obtain a methylation result of the MGMT gene promoter.

According to the processing device, the methylation sequencing data of the target fragment are obtained through the obtaining module, then the comparison module is executed to obtain the comparison result, then the removing module is executed to remove the duplication of the sequence of the overlapping region in the comparison of the sequencing data at the two ends in the comparison result, and the methylation identification module is enabled to be more accurate in the result of the identified and counted methylation level.

The alignment module can be an existing methylated alignment module. In a preferred embodiment, the processing apparatus further includes: the first pretreatment module is used for carrying out C-to-T conversion pretreatment on the human reference genome sequence; and a second pretreatment module for performing C-to-T conversion pretreatment on the paired-end sequencing sequence.

In order to make the methylation level of each subsequent site relatively more accurate, in a preferred embodiment, the processing device further comprises a correction module for correcting the data to be analyzed, wherein the correction module is used for correcting the data to be analyzed by using the human reference genome sequence, the position information of the human reference genome sequence and the high-frequency SNP sites of the human population.

The correction module can remove some low quality sites, so-called quality including sequencing quality or alignment quality. The specific correction software can adopt a Bisulfit Count Covariates module and a Bisulfit Table Recarrangement module in the BisSNP software to correct. The correction module is beneficial to improving the identification accuracy.

To further increase the confidence of each methylation site, in a preferred embodiment, the methylation recognition module comprises: the preliminary identification module is used for carrying out preliminary identification on the methylation sites in the data to be analyzed to obtain preliminary identification sites; the credibility screening module is used for carrying out credibility screening on the primary identified sites to obtain a methylation result of the MGMT gene promoter; preferably, the parameter setting conditions of the credibility screening are as follows: the coverage is less than 3000000, the probability ratio standard of the best genotype to the next best genotype is more than or equal to 20, and the comparison quality is more than 5.

In a third exemplary embodiment, there is provided a method for detecting methylation of a promoter of an MGMT gene, the method comprising: carrying out bisulfite conversion on gDNA of a sample to be detected to obtain converted DNA; constructing an amplicon library for the converted DNA to obtain the amplicon library; sequencing the amplicon library to obtain sequencing data; and carrying out methylation analysis on the sequencing data by adopting any one of the processing methods or the processing devices to obtain methylation result information of the MGMT gene promoter.

According to the detection method, the methylation detection result of the MGMT gene promoter is more accurate by adopting the processing flow of the methylation sequencing data.

On the basis that the amplification primers of the target gene promoter are improved to ensure better amplification efficiency and specificity, the detection method further comprises an improved amplicon library construction scheme. In a preferred embodiment, the amplicon library is constructed by using an amplification primer to the converted DNA, and the amplicon library is obtained, wherein the amplification primer comprises an upstream sequence and a downstream sequence, the upstream sequence is SEQ ID NO. 1, and the downstream sequence is SEQ ID NO. 2.

According to the detection method provided by the application, the improved primer is adopted to amplify the target region, so that the amplification efficiency is high, the specificity is high, and the DNA condition of the obtained target region is relatively more accurate. And then, the amplified target region is further constructed into an amplicon library, and the methylation state is detected by a high-throughput sequencing method, so that the number of the methylation sites of the MGMT gene promoter is increased, namely, the detection throughput and efficiency are improved.

In order to more effectively amplify the promoter region of the target gene, the inventors also optimized the working concentration and annealing temperature of the designed primer, thereby improving amplification efficiency and specificity. Thus, in a preferred embodiment, the working concentration of the primer is 5-15. mu.M, preferably 10. mu.M; in another preferred embodiment, the annealing temperature of the primer during amplification is 45 ℃ to 55 ℃, preferably 50 ℃. In other preferred embodiments, in the process of constructing the amplicon library for the transforming DNA by amplifying the amplification primers, the transforming DNA is amplified for 30-40 cycles, preferably 35 cycles, to obtain the amplicon library.

The following will further illustrate the benefits of the present application in conjunction with specific examples.

The reagents and manufacturers required in the following examples are as in table 1 below:

table 1:

example 1: detection of methylation level of MGMT gene promoter

The primer sequences used in this example are shown in Table 2 below, synthesized by Kingchi corporation and diluted with water to a working concentration of 5-10. mu.M.

Table 2:

name (R)	SEQ ID NO:	Sequence (5 '-3')
			Upstream sequence F	1	TYGYGTTTTGGATATGTTGG
Downstream sequence R	2	CRAAAAAAAACTCCRCACTC

The method comprises the following specific steps:

firstly, extracting the genome DNA of a sample to be detected.

Secondly, transforming genome DNA by bisulfite.

2.1 initial amount of 100ng of the transforming DNA, initial volume of the sample was 20. mu.L, and when it was less than 20. mu.L, the sample was made up with water.

2.2 Add 130. mu.L of Lightning Conversion Reagent to the DNA sample, shake and mix, centrifuge briefly, place on the PCR instrument, perform the PCR reaction according to the following Table 3:

table 3:

temperature of	Time
		98℃	8min
54℃	60min
		4℃	20h

2.3 to Zymo-Spin ^TM600 mu L M-Binding Buffer was added to IC Column, and the product of the 2.2 step reaction was added to Zymo-Spin containing M-Binding Buffer^TMIn IC Column, blow and beat with a gun, and stand for 2 min. Centrifuge at 12000rpm for 1 min.

And 2.4, adding the liquid in the collecting pipe back to the adsorption column again, standing for 2min, centrifuging at 12000rpm for 1min, and discarding the waste liquid.

2.5 Add 100 u L M-Wash Buffer, 12000rpm centrifugal 1min, discard waste liquid.

2.6 adding 200 mu L L-Desulphosphorylation Buffer to incubate for 15-20min at room temperature (20-30 ℃), centrifuging at 12000rpm for 1min after incubation is finished, and discarding waste liquid.

2.7 Add 200 u L M-Wash Buffer, 12000rpm centrifugal 1min, discard waste liquid.

2.8 repeat 2.7 steps, add 200. mu. L M-Wash Buffer, centrifuge at 12000rpm for 1min, discard waste.

2.9 the column was returned to the collection tube, centrifuged at 12,000rpm for 2min and the waste liquid was decanted. And (4) opening the adsorption column, placing at room temperature for 2-5min to thoroughly dry the residual rinsing liquid in the adsorption material.

2.10 transferring the adsorption column into a clean centrifuge tube, suspending and dripping 20 μ L of elution buffer TE preheated at 50 deg.C into the middle part of the adsorption membrane for elution, standing at room temperature for 2-5min, and centrifuging at 12000rpm for 1 min.

2.11 adding the liquid in the collecting tube back to the adsorption column again, standing at room temperature for 2-5min, centrifuging at 12000rpm for 1min, and storing the centrifugal tube with the collected transformed DNA at-20 ℃.

Third, MGMT gene promoter amplification

3.1 prepare the mixture (Mix) according to table 4 below, Mix with shaking:

table 4:

reagent	Volume of
		KAPA HiFi HS Uracil+RM	12.5μL
MGMT-F(SEQ ID NO:1)	1μL
		MGMT-R(SEQ ID NO:2)	1μL
DNA after transformation	5μL
		Water (W)	5.5μL
Total volume	25μL

3.2 adding the DNA transformed in the last step into Mix prepared according to the table 3, shaking and mixing evenly.

3.3 short centrifugation, placing on a PCR instrument, and performing PCR reaction according to the following Table 5:

table 5:

3.4 magnetic bead purification

The PCR products obtained in the above step were subjected to library construction and sequencing in accordance with the DNA NGS library construction method (the preparation step of KAPA Hyper Prep kit of KAPA Co.).

Example 2: detection of the amplification effects of the MGMT gene promoter methylation primers on annealing temperature, working concentration and PCR cycle number

Firstly, extracting the DNA of a clinical sample tissue.

Second, procedures of bisulfite conversion of genomic DNA, MGMT amplification, and the like were performed in reference to example 1.

And thirdly, selecting different primer annealing temperatures, working concentrations and PCR cycle numbers.

3.1 selection of primer annealing temperature: 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃.

3.2 selection of working concentration of primer: 4. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 16. mu.M.

3.3 selection of PCR cycle number: 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles.

Fourthly, detection results:

4.1 the results of the detection of the annealing temperature of the primers are shown in Table 6:

table 6:

annealing temperature	The result of the detection
		40℃	The amplification is more nonspecific
45℃	Amplifying the correct target band
		50℃	Amplifying the correct target band
55℃	Correct amplification resultTo the purpose belt
		60℃	Non-amplified band

4.2 the results of the primer working concentration measurements are shown in Table 7:

table 7:

working concentration	The result of the detection
		4μM	Non-amplified band
5μM	Amplifying the correct target band
		10μM	Amplifying the correct target band
15μM	Amplifying the correct target band
		16μM	More primer dimer

4.3 results of PCR cycle number measurements are shown in Table 8:

table 8:

example 3: processing method of MGMT gene methylation sequencing data

First, comparison

And calling bismark to align each pair of fastq files as paired reads to an MGMT human reference genome sequence to generate an initial bam file, and setting parameters to be-phred 33-squares.

Second, sorting

And calling a sort module of the SAM tools, sorting the initial bam files according to the chromosome positions, and defaulting parameters.

Adding Read Group information

And calling an Add Or Replace Group module of Picard to Add Read Group information to the sorted bam file, and setting 'VALIDATION _ STRINGENCY ═ LENIENT' as a parameter.

Fourthly, removing the overlapping interval between the two end sequences

And calling a clip overlay module of the Bam Util to remove the overlapping sequence between the double-end sequences in the compared Bam file, and during subsequent analysis, the overlapping sequence cannot be filtered, so that the calculation of the Beta value is influenced.

Fifthly, establishing indexes

And calling an index module of SAMtools to establish an index for the finally generated bam file, and generating a bai file matched with the removed and repeated bam file.

Sixthly, correcting data

And (2) correcting the processed bam file, the processed bed file (manually input file, which records the position information of the human reference genome sequence), the fasta file of the human reference genome sequence and the human high-frequency generated vcf file by using a Bisfite Count Covariates module and a Bisfite Table Recalbration module of the BisSNP in sequence to remove low-quality (including sequencing quality and/or comparison quality) sites, so that the identification accuracy is improved.

Seven, SNP/methylation site joint identification

The initial vcf files for SNPs (non-interesting sites, which part of the data may not be used) and methylation (i.e. CpG sites) were obtained using the bissulforite Genotyper module of BisSNP to identify SNP/methylation sites simultaneously.

Eight, methylation site ordering

The preliminary identified methylated vcf files were ranked By genomic position using the sort By Ref And Cor modules of BisSNP.

Nine, methylation site filtration

The VCF post process of BisSNP was used to filter sequenced subsequent methylated VCF files.

Ten, data arrangement

The filtered methylated vcf files are arranged into a readable file format to obtain methylation detection results, which are shown in table 9.

Table 9:

attached: the positive criteria in the above table are that the methylation level is 10% or more, and the determination is positive.

Example 4: repetitive evaluation of MGMT gene methylation detection

First, sample preparation

Preparing 3 batches of MGMT standard substances with the same mutation frequency (the theoretical mutation frequency is 10.00%, 15% and 20%), performing repeated detection on the 3 batches of samples, and counting the methylation frequency of the 3 batches of sample detection.

Secondly, amplifying the target region and constructing an amplicon library for sequencing detection, wherein the specific steps of bisulfite transforming genome DNA, MGMT amplification and the like refer to example 1, and the analysis flow of sequencing data refers to example 3.

Thirdly, detecting results: the results of the methylation frequency of the 3 batches are shown in Table 10.

Table 10:

as can be seen from Table 10, the test results showed small CV (coefficient of variation) values among 3 batches and good reproducibility.

Implementation 5: consistency of clinical sample MGMT gene methylation detection and pyrophosphate detection

Firstly, extracting the DNA of a clinical sample tissue.

Second, procedures such as bisulfite conversion of genomic DNA and MGMT amplification were performed in reference to example 1, and the procedure for analyzing sequencing data was performed in reference to example 3. Pyrosequencing was used for validation and control comparisons.

Third, the results of detecting and determining the methylation level of the clinical samples are shown in Table 11.

Table 11:

attached: the methylation level of each sample was measured as the average methylation level of four sites in the pyrophosphorylation test, and was determined to be positive when 10% or more was reached.

As can be seen from Table 11, the results of the clinical samples were compared with the pyrosequencing detection method and the results of the tests were verified, and the results of the MGMT NGS detection using the primers of the present application were consistent with the results of the pyrosequencing detection, indicating that the methylation state of the MGMT gene promoter detected by the amplicons amplified by the primers of the present application through high-throughput sequencing and an improved methylation analysis process did not decrease the accuracy due to the improvement of the sequencing throughput.

Example 6: MGMT gene methylation detection versus pyrosequencing advantages

Firstly, the methylation sites detected by two different methods are counted, and the statistical results are shown in Table 12.

Table 12: MGMT NGS detection site and pyrophosphate detection site

As can be seen from table 12, the number of methylated sites detected by the amplicon library constructed using the primers of the present application and the improved sequencing data analysis protocol is significantly greater than the number of sites detected by current pyrophosphate detection methods.

Second, the dimensionality of methylation detected by the two different methods was compared, and the results are shown in FIGS. 3 and 4.

FIG. 3 shows the methylation levels of each CpG site detected by the pyrophosphate detection method, and FIG. 4 shows the methylation levels of each CpG site detected by the method of the present application (comparison in the vertical direction of the same site) and the methylation levels of each DNA template molecule (comparison in the horizontal direction of the same sequence). As can be seen from FIGS. 3 and 4, the methylation detection of the present application can embody more haplotype site information than pyrosequencing.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: by adopting the improved primers to amplify the target region, the specificity and the amplification efficiency are high, the amplified target region can be conveniently constructed into an amplicon library, and the methylation state is detected through an improved analysis process, so that the number of the MGMT gene promoter methylation sites is increased, the detection flux and efficiency are increased, the detection accuracy is improved, and a more reliable basis is provided for guiding medication.

As will be appreciated by one skilled in the art, embodiments of the present application may be provided as a method, system, or computer program product. Accordingly, the present application may take the form of an entirely hardware embodiment, an entirely software embodiment or an embodiment combining software and hardware aspects. Furthermore, the present application may take the form of a computer program product embodied on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, and the like) having computer-usable program code embodied therein.

The present application is described with reference to flowchart illustrations and/or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the application. It will be understood that each flow and/or block of the flow diagrams and/or block diagrams, and combinations of flows and/or blocks in the flow diagrams and/or block diagrams, can be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general purpose computer, special purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions specified in the flowchart flow or flows and/or block diagram block or blocks.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A processing method of MGMT gene promoter methylation sequencing data is characterized by comprising the following steps:

obtaining methylation sequencing data derived from an MGMT gene promoter, wherein the methylation sequencing data is a double-ended sequencing sequence;

comparing the methylation sequencing data with a human reference genome sequence to obtain a comparison result, wherein the comparison result comprises a first end first matching region, a first end second matching region, a second end first matching region and a second end second matching region, and the first end second matching region and the second end second matching region are overlapped;

removing the first end second matching region or the second end second matching region in the comparison result to obtain data to be analyzed;

carrying out methylation site recognition on the data to be analyzed to obtain a methylation result of the MGMT gene promoter;

the step of identifying methylation sites in the data to be analyzed to obtain the methylation result of the MGMT gene promoter comprises the following steps:

carrying out primary identification on the methylation sites in the data to be analyzed to obtain primary identification sites;

carrying out credibility screening on the initial identification site to obtain a methylation result of the MGMT gene promoter;

the parameter setting conditions of the credibility screening are as follows: the coverage is less than 3000000, the probability ratio standard of the best genotype to the next best genotype is more than or equal to 20, and the comparison quality is more than 5.

2. The process of claim 1, wherein prior to aligning the methylation sequencing data to the human reference genomic sequence, the process further comprises:

performing C-to-T transformation pretreatment on the human reference genomic sequence; and

and C-to-T conversion pretreatment is carried out on the double-ended sequencing sequence.

3. The process of claim 1, wherein after obtaining the data to be analyzed and before performing methylation site recognition on the data to be analyzed, the process further comprises the step of correcting the data to be analyzed, wherein the step of correcting the data to be analyzed comprises:

and correcting the data to be analyzed by using the human reference genome sequence, the position information of the human reference genome sequence and the high-frequency SNP locus of the crowd.

4. A processing device for MGMT gene promoter methylation sequencing data, the processing device comprising:

the acquisition module is used for acquiring methylation sequencing data from an MGMT gene promoter, wherein the methylation sequencing data is a double-ended sequencing sequence;

an alignment module, configured to compare the methylation sequencing data with a human reference genome sequence to obtain an alignment result, where the alignment result includes a first end first matching region, a first end second matching region, a second end first matching region, and a second end second matching region, and the first end second matching region overlaps with the second end second matching region;

the removing module is used for removing the first end second matching area or the second end second matching area in the comparison result to obtain data to be analyzed;

the methylation recognition module is used for carrying out methylation site recognition on the data to be analyzed to obtain a methylation result of the MGMT gene promoter;

the methylation identification module comprises:

the initial identification module is used for carrying out initial identification on the methylation sites in the data to be analyzed to obtain initial identification sites;

the credibility screening module is used for screening the credibility of the initial identification site to obtain a methylation result of the MGMT gene promoter;

5. The processing apparatus according to claim 4, characterized in that the processing apparatus further comprises:

a first pre-processing module for performing C-to-T conversion pre-processing on the human reference genome sequence; and

and the second pretreatment module is used for performing C-to-T conversion pretreatment on the double-ended sequencing sequence.

6. The processing apparatus as claimed in claim 4, wherein the processing apparatus further comprises a correction module for correcting the data to be analyzed, the correction module being configured to correct the data to be analyzed using the human reference genomic sequence, the position information of the human reference genomic sequence, and the crowd high frequency SNP sites.

7. A method for detecting methylation of an MGMT gene promoter, the method comprising:

carrying out bisulfite conversion on gDNA of a sample to be detected to obtain converted DNA;

constructing an amplicon library for the converted DNA to obtain the amplicon library;

sequencing the amplicon library to obtain sequencing data;

subjecting the sequencing data to methylation analysis using the processing method of any one of claims 1 to 3 or the processing apparatus of any one of claims 4 to 6 to obtain methylation results of the MGMT gene promoter.

8. The detection method of claim 7, wherein an amplicon library is constructed from the transformed DNA using an amplification primer, wherein the amplification primer comprises an upstream sequence and a downstream sequence, the upstream sequence is SEQ ID NO. 1, and the downstream sequence is SEQ ID NO. 2.

9. The detection method according to claim 8, wherein the working concentration of the amplification primer is 5-15 μ M.

10. The detection method according to claim 8, wherein the working concentration of the amplification primer is 10 μ M.

11. The detection method according to claim 8, wherein the annealing temperature of the amplification primer is 45-55 ℃.

12. The method of claim 8, wherein the amplification primers anneal to 50 ℃.

13. The detection method according to claim 8, wherein in the process of constructing the amplicon library from the transformed DNA by amplification of the amplification primers, the transformed DNA is amplified for 30-40 cycles to obtain the amplicon library.

14. The detection method according to claim 8, wherein the amplification primer is used for amplifying the transforming DNA for 35 cycles during the process of constructing the amplicon library of the transforming DNA, so as to obtain the amplicon library.