CN118389510A - A method and detection kit for detecting SHOX2 gene promoter methylation based on nanopore targeted sequencing technology - Google Patents

Abstract

A method and a detection kit for detecting SHOX2 gene promoter methylation based on a nano Kong Ba-way sequencing technology belong to the technical field of molecular medicine. The methylation state of the methylation site of the SHOX2 gene promoter is detected by combining a nanopore long-reading long-sequencing technology and a Cas9 in-vitro cutting technology, four crRNA (ribonucleic acid) interval sequences for Cas9 targeted cutting of the SHOX2 gene promoter are designed for the SHOX2 promoter region, and the target region is cut twice at the upstream and downstream sides respectively and comprises the SHOX2 promoter region and flanking regions thereof. The method can rapidly and efficiently obtain the most original methylation state of the target DNA of the sample without treating the sample by the bisulfite, and can evaluate the methylation level from the whole level and obtain the methylation state of a single methylation site.

Description

A method and detection kit for detecting SHOX2 gene promoter methylation based on nanopore targeted sequencing technology

Technical Field

The present invention belongs to the field of in vitro nucleic acid detection, and in particular relates to a method and a detection kit for detecting SHOX2 gene promoter methylation based on nanopore targeted sequencing technology.

Background technique

Epigenetic changes are one of the important pathogenesis of cancer, and abnormal changes in methylation patterns are extremely common in all cancers. Compared with normal cells, the genome of cancer cells is generally hypomethylated, while key gene regulatory elements such as gene promoters remain highly methylated. Cancer cells drive the occurrence and development of cancer through genetic and epigenetic changes, and use epigenetic reprogramming to change signaling pathways to ensure their own survival. Abnormal changes in methylation patterns often precede the malignant proliferation of tumors, so methylation biomarkers have received widespread attention in application scenarios such as early screening, clinical testing, and prediction of treatment outcomes.

Human SHOX2 is a homolog of the human SHOX gene and is located on chromosome 3. It is mainly involved in transcriptional regulation during the embryogenesis period of vertebrates. Studies have shown that SHOX2 plays an important role in the occurrence and development of tumors. Its CpG island is frequently hypermethylated and its expression is elevated in tumor tissues and cells such as lung cancer, breast cancer, esophageal squamous cell carcinoma (ESCC) and hepatocellular carcinoma (HCC). Therefore, SHOX2 methylation can be used for early screening, occurrence and development of the disease, and prognosis judgment, and may provide potential therapeutic targets.

Currently, the commonly used methods for detecting SHOX2 gene promoter methylation include: methylation-specific PCR (MSP), methylation-specific quantitative PCR method (qMSP), bisulfite sequencing PCR (BSP) and methylation-sensitive high-resolution melting curve analysis (MS-HRM).

The methylation-specific PCR (MSP) method designs two different primers for methylation and non-methylation for the DNA sequence after sulfite conversion, and performs polymerase chain reaction (PCR) amplification respectively. Finally, the presence or absence of product bands is analyzed by agarose gel electrophoresis to determine whether the target region is methylated. This method does not require special instruments and is widely used, but the quality of the test results is heavily dependent on primer design, quantitative detection cannot be achieved, and there is a high risk of false positives.

The methylation-specific quantitative PCR method (qMSP) is a technology developed based on MSP. It mainly adds TaqMan probes during the detection process to ensure high sensitivity and accuracy. However, if more methylation sites are detected, only overall analysis can be achieved. Therefore, this method is not suitable for the detection of more CG sites and large-scale samples.

The bisulfite sequencing PCR (BSP) method mainly detects methylation status by combining PCR with Sanger sequencing technology. The BSP method designs primers that do not contain CG sites for PCR based on the DNA sequence after sulfite conversion, and then connects the product to the vector, transforms it into Escherichia coli for culture, selects single clones from the positive clones for sequencing, and calculates the methylation level of the promoter CG site based on the sequencing results. However, since this method requires a large number of single clones to be sequenced, the operation is cumbersome, and the detection cycle is long, it is not suitable for large-scale detection, and incomplete sulfite treatment is prone to false positive results.

Methylation-sensitive high-resolution melting curve analysis (MS-HRM) mainly determines whether methylation exists by converting the difference in base sequence into the difference in melting curve based on the difference in sequence length and GC content. However, this method has high requirements for instruments and fluorescent dyes, requiring a fluorescent quantitative PCR instrument with an HRM module, and this method can only analyze the overall methylation status of the fragment, but cannot clarify the methylation status of each CpG site.

There is still a need to provide an efficient and accurate detection scheme for the detection of the methylation status of the SHOX2 gene.

Summary of the invention

The purpose of the present invention is to provide a methylation detection kit and a method for detecting methylation of the SHOX2 gene promoter for quickly and efficiently obtaining the most original methylation state of sample DNA in view of the deficiencies in the prior art. The methylation state of the methylation site of the SHOX2 gene promoter is detected by combining nanopore long-read sequencing technology with Cas9 in vitro cutting technology, and four Cas9 targeted cutting probe crRNAs are designed for the SHOX2 promoter region, cutting twice upstream and downstream of the target region, including the SHOX2 gene promoter region and its flanking regions.

In order to achieve the above-mentioned invention object, the present invention adopts the following technical means:

The present invention provides a group of crRNA spacer sequences targeting the SHOX2 gene promoter, the nucleotide sequences of which are shown in the sequence table SEQ ID No.1 to No.4, specifically as follows:

SEQ ID No.1:UGGGUCUCCGAAACUCGCUU;

SEQ ID No.2:UCUGCUAUUUAGUACAACAG;

SEQ ID No.3:AGCGUUCUAUACCAAUUCAA;

SEQ ID No. 4: AUGCGAAGCUGAGCAUUGCG.

The present invention provides a methylation detection kit containing the crRNA spacer sequence targeting the SHOX2 gene promoter.

The detection kit includes: RNPs formed by incubating crRNA·tracRNAmix (10μM) with Cas9 enzyme at room temperature, dATP, NEB Taq polymerase, Adapter Mix (AMX), Ligation Buffer (LNB), NEBNext QuickT4 DNA Ligase, Flush Tether (FLT), Flush Buffer (FB), Sequencing Buffer (SQB), and Loading Beads (LB).

The RNPs formed by incubating the crRNA·tracRNAmix with the Cas9 enzyme at room temperature are prepared by the following method:

1) Mixing equal volumes of crRNA spacer sequences targeting the SHOX2 gene promoter as shown in SEQ ID No. 1 to No. 4 in the sequence listing to form a crRNA pool;

2) Mix equal amounts of crRNApool and tracRNA, add buffer, heat in a thermal cycler, and then cool to room temperature to anneal to form crRNA·tracRNAmix;

3) Incubate crRNA·tracRNAmix with Cas9 enzyme at room temperature to form RNPs.

In step 2), the heating temperature in the thermal cycler is 95° C. and the heating time is 5 min.

In step 3), the incubation time at room temperature is 30 minutes.

The present invention provides the crRNA spacer sequence targeting the SHOX2 gene promoter or the use of the detection kit in detecting methylation of the SHOX2 gene promoter.

A method for detecting SHOX2 gene promoter methylation based on nanopore targeted sequencing technology comprises the following steps:

(1) Design of crRNA for target gene

The SHOX2 gene promoter (which contains CpG dinucleotides around the transcription start site) was used as the target region, and two crRNAs were designed on each side of the two ends of the region to evaluate the effect of the shearing strategy on the targeted enrichment efficiency;

(2) Preparation of Cas9 RNP complex

The crRNA spacer sequences targeting the SHOX2 gene promoter (SEQ ID No. 1 to No. 4) were mixed in equal volumes to form a crRNA pool (100 μM), and equal amounts of crRNA pool (100 μM) and tracRNA (100 μM) were mixed, and then Buffer was added. The mixture was heated at 95° C. for 5 min in a thermal cycler and then cooled at room temperature for annealing to form crRNA·tracRNAmix (10 μM). The crRNA·tracRNAmix (10 μM) was incubated with Cas9 enzyme at room temperature for 30 min to form RNPs.

(3) Cas9 targeted cleavage of genomic DNA

The crRNA guides the RNP complex to target and cut the dephosphorylated genomic DNA. The DNA end at the Cas9 cutting site generates a 5′ phosphate group. The target DNA end is then ligated to the adapter sequence after adding an A tail.

(4) Preparation of sequencing library

Use ONT's ligation kit (SQK-LSK109) to prepare the library; in a 1.5mL EP tube, mix the prepared targeted genomic DNA, Adapter Mix (AMX), Ligation Buffer (LNB), and NEBNextQuick T4 DNA Ligase, centrifuge, and incubate at room temperature for 15-20 minutes; then use AMPure XP magnetic beads to purify the library to remove excess adapter sequences and DNA fragments without adapters to reduce sequencing background; use 0.3X AMPureXP to purify the library, and finally elute the library with 12μL of elution buffer to obtain a purified DNA library;

(5) Loading samples for sequencing;

(6) Result analysis: Nanopore sequencing equipment MinION/GridION/PromethION and software MinKNOW were used to obtain the fast5 file of the original electrical signal; the fast5 file was base-identified and converted into a FASTQ file containing the base sequence and quality value. The FASTQ file was compared with the reference genome to generate a Bam file; the sequencing depth and coverage in the target region, as well as the on-target and off-target rates of the targeted probe crRNA were calculated based on the Bam file.

Methylation sites were identified based on the raw electrical signals and the reference genome, and the methylation level and sequencing depth, methylation and unmethylation depth, and methylation rate of the methylation sites were calculated.

Compared with the existing methylation detection technology, the outstanding advantages and technical effects of the present invention are:

1. The method of the present invention does not require DNA interruption and can achieve long-read sequencing, thereby obtaining methylation information of longer DNA fragments.

2. The library construction step of the present invention does not require PCR amplification and has no GC preference.

3. DNA can be directly sequenced to obtain the original methylation modification information of the DNA double helix without chemical or enzymatic treatment.

4. The present invention provides a set of crRNA spacer sequences targeting the SHOX2 gene promoter. The method of the present invention does not require the sample to be treated with bisulfite to detect the methylation site, and can quickly and efficiently obtain the most original methylation state of the sample DNA. It can not only evaluate the methylation level from the overall level, but also obtain the methylation state of a single methylation site. It provides a basis for early screening, risk assessment, diagnosis, prognosis and treatment monitoring of tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram showing the scope of the SHOX2 gene promoter targeting region;

FIG2 is a diagram showing the quality control results of methylation in the targeted region of the SHOX2 gene promoter;

FIG3 is a diagram showing the methylation status of the targeted region of the SHOX2 gene promoter.

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention clearer, the following examples will further illustrate the present invention in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

A method for detecting SHOX2 gene promoter methylation based on nanopore targeted sequencing technology comprises the following steps:

(1) Use the corresponding nucleic acid extraction kit to extract DNA from the sample to be tested (blood or tissue sample), and perform quality control on the extracted DNA.

For the method of detecting SHOX2 gene promoter methylation based on nanopore targeted sequencing technology, 3 to 8 μg of starting DNA is required.

The DNA purity was evaluated using NanoDrop2000 spectrophotometer (Thermo Scientific): A260/280 = 1.8-2.0, indicating that the DNA purity was high and there were fewer impurities such as protein and RNA in the DNA sample; A260/230 = 2.0-2.2, indicating that there were fewer impurities such as organic solvents and salts in the DNA sample;

DNA concentration was determined using Qubit4.0 quantitative analysis (Thermo Scientific): C(DNA) ≥180 ng/μL;

The integrity of DNA was determined using TapeStation 2200 (Agilent).

(2) Preparation of RNP complex

Targeting probe crRNA pool (SEQ ID No.1-No.4), tracRNA and Cas9 enzyme, dilute crRNA and tracRNA to a final concentration of 100μM with buffer (pH7.5). Add crRNApool (1μL), tracRNA (1μL) and Buffer (8μL) to a 0.2mL PCR tube and mix and centrifuge. Incubate at 95℃ for 5min and place at room temperature. Pair under natural cooling to form crRNA·tracRNA complex (10μM). Then mix and centrifuge the prepared crRNA·tracRNA complex (10μM), Cas9 enzyme and buffer in a 1.5mL EP tube and place at room temperature for 30min to form an RNP complex.

(3) Cas9 targeted cleavage of genomic DNA

The crRNA guides the RNP complex to target and cut the dephosphorylated genomic DNA. The DNA end at the Cas9 cutting site generates a 5′ phosphate group. When the target DNA end is added with an A tail, it is connected to the adapter sequence. In a 1.5mL EP tube, 30μL of the prepared dephosphorylated genomic DNA, 10μL of RNP complex, 1μL of dATP and 1μL of Taq polymerase were mixed and centrifuged, and incubated at 37℃ for 30min and 72℃ for 5min in a thermal cycler. After incubation, place on ice for later use.

(4) Preparation of sequencing library

The library was prepared using the Nanopore Ligation Kit (SQK-CS9109) from ONT (Oxford Nanopore Technologies). In a 1.5 mL EP tube, the prepared targeted genomic DNA was added with Ligation Buffer, T4 DNA Ligase and other components according to the instructions of the ligation kit, mixed and centrifuged, and incubated at room temperature for 15 to 20 minutes. Subsequently, the library needs to be purified using AMPure XP magnetic beads to remove excess adapter sequences and DNA fragments without adapters to reduce sequencing background. The library was purified using 0.3X AMPure XP, and the library was eluted with 12 μL of elution buffer to obtain a purified DNA library as a sequencing library.

(5) Sequencing chip pretreatment

The DNA sequencing equipment can be MinION, GridION or PromethION from Oxford Nanopore Technologies. In this embodiment, the handheld sequencer MinION Mk1B is used. Thaw a tube of Flush Tether (FLT) and Flush Buffer (FB) at room temperature. Open the lid of MinION Mk1B and install the chip. Rotate the lid of the priming port clockwise to check if there are small bubbles under the lid. Draw back a small amount of buffer in the chip to remove any bubbles. The specific operations are as follows: (a) Set the P1000 pipette to 200 μL; (b) Insert the tip into the priming port; (c) Turn the turntable until a small amount of buffer is seen entering the pipette tip.

Add 30 μL of Flush Tether (FLT) to a tube of Flush Buffer (FB) as the priming mix, and then vortex mix at room temperature. Inject 800 μL of priming mix into the sequencing chip through the priming port, avoiding the introduction of bubbles during this process; leave at room temperature for 5 minutes.

(6) Sample loading

Thaw the sequencing buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes at room temperature, vortex and centrifuge. In a new 1.5 mL EP tube, prepare the loading library as follows:

Sequencing Buffer(SQB) 37.5μL Loading Beads (LB), mix immediately before use 25.5μL Step (4) Sequencing library 12μL total 75μL

Mix the prepared library by gently blowing (2-3 times). Open the cover of the sample port and inject 75μL of the prepared library dropwise into the sequencing chip through the sample port; finally, close the covers of the sample port and the priming port; connect the sequencing device MinION Mk1B to the computer and open the sequencing software MinKNOW to conduct the sequencing experiment.

(7) Results Analysis

Nanopore sequencing equipment MinION Mk1B and software MinKNOW are used to obtain the fast5 file of the original electrical signal; the fast5 file is base-identified and converted into a FASTQ file containing the base sequence and quality value, and the FASTQ file is compared with the reference genome to generate a Bam file; the sequencing depth, coverage, and on-target and off-target rates of the targeted probe crRNA in the target region are calculated based on the Bam file; the methylation sites are identified based on the original electrical signal and the reference genome, and the methylation level and sequencing depth, methylation and non-methylation depth, and methylation rate of the methylation sites are calculated. The sequencing equipment can also be GridION or PromethION.

Table 1: Analysis and processing results of methylation results of the targeted region of the SHOX2 gene promoter

Table 1 selects reference sequences containing CpG island sites in the reference target region according to the sequencing results, compares them with the reference gene sequence to identify methylation sites, and calculates the methylation rate of the methylation site (called_sites methylation number/called_sites number). This shows that this analysis method can evaluate the methylation level at the overall level and can also obtain the methylation status of a single methylation site.

Figure 1 shows the scope of the SHOX2 gene promoter targeting region, and the SHOX2 gene promoter region (including the core CpG island) is selected to design four crRNAs for targeted shearing. The crRNA for cutting upstream of the target region is designed for the positive strand (+) of DNA, and the crRNA for cutting downstream of the target region is designed for the negative strand (-) of DNA. The crRNA cutting points upstream of the target region: chr3:158098496(+), chr3:158101300(+), the crRNA cutting points downstream of the target region: chr3:158104878(-), chr3:158106968(-).

Figure 2 shows the quality control result of the methylation data of the targeted region of the SHOX2 gene promoter. The accuracy of DNA base reading in the sequencing experiment can be measured by the read length quality value Q value (Quality scores), and the Q value is logarithmically related to the error probability of the base (P): Q = -10 × log ₁₀ P, that is, the larger the Q value, the lower the base error rate, and the higher the accuracy of the sequencing reads. According to the analysis of the data quality control result chart, the quality values of the sequencing reads are concentrated between Q13-Q15, among which the median quality value (Medianread quality) is Q13.0, that is, the base accuracy (Read accuracy) reaches 95.0%.

Figure 3 shows the methylation status diagram of the targeted region of the SHOX2 gene promoter. Normal tissue and colorectal cancer tissue of the same patient were selected for sequencing analysis according to the method. The results showed that the methylation site frequencies of different CG sites in normal tissue and colorectal cancer tissue were different. Based on the data quality control results in Figure 2, the feasibility of the method of the present invention is illustrated. The methylation level can be evaluated from the overall level, and the methylation status of a single methylation site can also be obtained.

The method for detecting gene promoter methylation can detect methylated DNA and is applicable to various types of cancer. The detection method itself is not specific to a certain type of cancer. The present invention can be used to detect gene methylation changes in various cancers such as colorectal cancer, breast cancer, lung cancer, liver cancer, and gastric cancer.

Bisulfite treatment of samples is a common step in genomic methylation sequencing experiments. However, bisulfite treatment may make it difficult to detect the methylation status of some CpG sites, especially for highly methylated regions, because these regions may be degraded during bisulfite treatment. During the treatment process, the concentration and treatment time of bisulfite need to be strictly controlled to avoid excessive damage to DNA and nonspecific adsorption. After the treatment, the samples need to be properly washed and neutralized to remove residual bisulfite and sulfite. If the treatment is not complete, it is easy to cause false positive results. Common methylation detection methods based on bisulfite treatment include: methylation-specific PCR (MSP), methylation-specific quantitative PCR method (qMSP), bisulfite sequencing PCR (BSP) and methylation-sensitive high-resolution melting curve analysis (MS-HRM). Among them, the methylation-specific PCR (MSP) method designs two different primers for methylation and non-methylation for the DNA sequence after sulfite conversion, and performs polymerase chain reaction (PCR) amplification respectively. The quality of the detection results depends heavily on the primer design, and quantitative detection cannot be achieved, and there is a high risk of false positives; the methylation-specific quantitative PCR method (qMSP) is a technology developed based on MSP, mainly by adding TaqMan probes during the detection process to ensure high sensitivity and accuracy, but if more methylation sites are detected, only overall analysis can be achieved, so this method is not suitable for the detection of more CG sites and large-scale samples. The bisulfite sequencing PCR (BSP) method mainly detects the methylation status by combining PCR with Sanger sequencing technology. The BSP method designs primers that do not contain CG sites for sulfite-converted DNA sequences for PCR, then connects the products to the vector, transforms them into Escherichia coli for culture, selects single clones from the positive clones for sequencing, and counts the methylation level of the promoter CG site based on the sequencing results. However, since this method requires a large number of single clones to be selected for sequencing, the operation is cumbersome, the detection cycle is long, it is not suitable for large-scale detection, and incomplete sulfite treatment is prone to false positive results. Methylation-sensitive high-resolution melting curve analysis (MS-HRM) mainly determines whether methylation exists by converting the difference in base sequence into the difference in melting curve based on the difference in sequence length and GC content. However, this method has high requirements for instruments and fluorescent dyes, requiring a fluorescent quantitative PCR instrument with an HRM module, and this method can only analyze the overall methylation status of the fragment, but cannot clarify the methylation status of each CpG site.

Compared with the above-mentioned methylation detection technology, the method of the present invention has three major advantages in detecting DNA methylation: first, there is no need to interrupt DNA, and long read sequencing can be achieved, thereby obtaining methylation information of longer DNA fragments; second, the library construction step does not require PCR amplification and has no GC preference; third, DNA does not need to be treated with chemicals or enzymes, and can be directly sequenced to obtain the original methylation modification information of the DNA double strand. The method of the present invention does not require the sample to be treated with bisulfite to detect methylation sites, and can quickly and efficiently obtain the most original methylation state of the sample DNA, which can evaluate the methylation level from the overall level and obtain the methylation state of a single methylation site. Provide a basis for early screening, risk assessment, diagnosis, prognosis and treatment monitoring of tumors.

The above embodiments are only preferred embodiments of the present invention and cannot be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims (7)

1. A group of crRNA spacer sequences targeting the SHOX2 gene promoter, characterized in that the nucleotide sequences are shown in the sequence table SEQ ID No. 1 to No. 4, specifically as follows: SEQ ID No.1:UGGGUCUCCGAAACUCGCUU; SEQ ID No.2:UCUGCUAUUUAGUACAACAG; SEQ ID No.3:AGCGUUCUAUACCAAUUCAA; SEQ ID No. 4: AUGCGAAGCUGAGCAUUGCG. 2. A methylation detection kit containing the crRNA spacer sequence targeting the SHOX2 gene promoter as claimed in claim 1. 3. The methylation detection kit as described in claim 2, characterized in that it comprises: RNPs formed by incubating crRNA·tracRNA mix with Cas9 enzyme at room temperature, dATP, NEB Taq polymerase, Adapter Mix, Ligation Buffer, NEBNextQuick T4 DNA Ligase, Flush Tether, Flush Buffer, Sequencing Buffer, and Loading Beads. 4. The methylation detection kit according to claim 3, characterized in that the RNPs formed by incubating the crRNA·tracRNA mix with the Cas9 enzyme at room temperature are prepared by the following method: 1) Mixing equal volumes of crRNA spacer sequences targeting the SHOX2 gene promoter as shown in SEQ ID No. 1 to No. 4 in the sequence listing to form a crRNA pool; 2) Mix equal amounts of crRNApool and tracRNA, add buffer, heat in a thermal cycler, and then cool to room temperature to anneal to form crRNA·tracRNAmix; 3) Incubate crRNA·tracRNAmix with Cas9 enzyme at room temperature to form RNPs. 5. The methylation detection kit according to claim 4, characterized in that in step 2), the heating temperature in the thermal cycler is 95°C and the heating time is 5 minutes. 6. The methylation detection kit according to claim 4, characterized in that in step 3), the incubation time at room temperature is 30 min. 7. Use of the crRNA spacer sequence targeting the SHOX2 gene promoter as claimed in claim 1 or the methylation detection kit as claimed in claim 2 in detecting methylation of the SHOX2 gene promoter.