MGMT gene promoter methylation detecting method, sequencing data processing method and processing device

A technology for sequencing data and processing methods, applied in biochemical equipment and methods, genomics, and microbial determination/inspection, etc., can solve problems such as low detection accuracy, and achieve the effect of facilitating evaluation and high accuracy

Active Publication Date: 2019-09-06
GENECAST WUXI PRECISION MEDICAL DIGNOSTIC LAB
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The main purpose of the present invention is to provide a method for detecting methylation of MGMT gene promoter, a meth

Method used

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  • MGMT gene promoter methylation detecting method, sequencing data processing method and processing device
  • MGMT gene promoter methylation detecting method, sequencing data processing method and processing device
  • MGMT gene promoter methylation detecting method, sequencing data processing method and processing device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Detection of MGMT gene promoter methylation level

[0073] The primer sequences used in this example are shown in Table 2 below, synthesized by Jinweizhi Company, and diluted with dewatered to a working concentration of 5-10 μM.

[0074] Table 2:

[0075] name SEQ ID NO: Sequence (5'-3') upstream sequence F 1 TYGYGTTTTGGATATGTTGG downstream sequence R 2 CRAAAAAAAACTCCRCACTC

[0076] Specific steps are as follows:

[0077] 1. Extract the genomic DNA of the sample to be tested.

[0078] 2. Bisulfite transforms genomic DNA.

[0079] 2.1 The initial amount of transformed DNA is 100ng, and the initial volume of the sample is 20μL. If it is less than 20μL, make up with water.

[0080] 2.2 Take 130 μL Lightning Conversion Reagent and add it to the DNA sample, vortex to mix, centrifuge briefly, put it on the PCR machine, and perform the PCR reaction as shown in Table 3:

[0081] table 3:

[0082] temperature time ...

Embodiment 2

[0102] Embodiment 2: Detect the amplification effect of the annealing temperature, working concentration and PCR cycle number of MGMT gene promoter methylation primer

[0103] 1. Extraction of tissue DNA from clinical samples.

[0104] 2. Refer to Example 1 for steps such as bisulfite conversion of genomic DNA and MGMT amplification.

[0105] 3. Choose different primer annealing temperature, working concentration and number of PCR cycles.

[0106] 3.1 Choice of primer annealing temperature: 40°C, 45°C, 50°C, 55°C, 60°C.

[0107] 3.2 Choice of primer working concentration: 4 μM, 5 μM, 10 μM, 15 μM, 16 μM.

[0108] 3.3 Selection of the number of PCR cycles: 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles.

[0109] 4. Test results:

[0110] 4.1 The detection results of primer annealing temperature are shown in Table 6:

[0111] Table 6:

[0112] Annealing temperature Test results 40℃ More non-specific amplification 45℃ Amplify the correct targe...

Embodiment 3

[0120] Example 3: Processing method of MGMT gene methylation sequencing data

[0121] 1. Comparison

[0122] Call bismark to compare each pair of fastq files to the MGMT human reference genome sequence as paired reads, generate an initial bam file, and set the parameter "--phred33-quals".

[0123] 2. Sorting

[0124] Call the sort module of SAM tools to sort the initial bam file according to the chromosome position, with default parameters.

[0125] 3. Add Read Group information

[0126] Call the Add Or Replace Read Groups module of Picard to add ReadGroup information to the sorted bam file, and set the parameter "VALIDATION_STRINGENCY=LENIENT".

[0127] 4. Remove the overlapping interval between the paired-end sequences

[0128] Call the clip Overlap module of Bam Util to remove the overlapping sequences between the paired-end sequences in the bam file after alignment. In the subsequent analysis, this part of the overlapping sequences will not be filtered, which will affe...

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Abstract

The invention provides an MGMT gene promoter methylation detecting method, a sequencing data processing method and a processing device. The processing method comprises the steps of acquiring methylation sequencing data from the MGMT gene promoter, wherein the methylation sequencing data are a double-end sequencing sequence; comparing the methylation sequencing data with a human reference genome sequence for obtaining a comparing result, wherein a comparing result comprises a first-end first matching area, a first-end second matching area, a second-end first matching area and a second-end second matching area, the first-end second matching area is overlapped with the second-end second matching area; eliminating the first-end second matching area or the second-end second matching area in a comparing result for obtaining to-be-analyzed data; and performing methylation site identification on to-be-analyzed data for obtaining a methylation result of the MGMT gene promoter. The methylation site which is detected by the processing method has relatively high accuracy and relatively high flux, thereby facilitating evaluation of methylation level in an integral manner.

Description

technical field [0001] The present invention relates to the field of gene detection, in particular to a method for detecting methylation of MGMT gene promoter, a method for processing sequencing data and a processing device. Background technique [0002] MGMT is a DNA repair protein ubiquitous in cells, which can convert O 6 The guanine complex is removed from DNA, restoring damaged guanine and protecting chromosomes from damage by alkylating agents. In this process, MGMT acts as both a methyltransferase and a methyl acceptor protein to complete the transfer reaction alone. [0003] The methylation status of MGMT gene promoter has a certain correlation with the sensitivity of alkylating agents. The alkylating agents temozolomide (TMZ), pyrimidine nitrosourea (ACNU) and bischloroethyl nitrosourea (BCNU) are widely used as chemotherapeutic drugs in the treatment of human tumors. An important site of action for these alkylating agents is the O 6 guanine, while MGMT can rapi...

Claims

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Application Information

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IPC IPC(8): G16B20/30C12Q1/6886
CPCC12Q1/6886C12Q2600/154G16B20/30
Inventor 闫慧婷洪媛媛于佳宁李彩琴李鑫宋小凤陈维之何骥
Owner GENECAST WUXI PRECISION MEDICAL DIGNOSTIC LAB
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