WO2024124400A1 - Targeted methylation library construction system based on multiplex pcr, method, and use thereof - Google Patents

Targeted methylation library construction system based on multiplex pcr, method, and use thereof Download PDF

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WO2024124400A1
WO2024124400A1 PCT/CN2022/138685 CN2022138685W WO2024124400A1 WO 2024124400 A1 WO2024124400 A1 WO 2024124400A1 CN 2022138685 W CN2022138685 W CN 2022138685W WO 2024124400 A1 WO2024124400 A1 WO 2024124400A1
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primer
universal
dna
round
sequence
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PCT/CN2022/138685
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French (fr)
Chinese (zh)
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杨林
张艳艳
陈芳
蒋慧
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深圳华大智造科技股份有限公司
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Publication of WO2024124400A1 publication Critical patent/WO2024124400A1/en

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  • the present invention relates to the field of biotechnology, and in particular to a targeted methylation library construction system based on multiplex PCR, a method and an application thereof.
  • DNA methylation is an epigenetic regulatory modification that participates in regulating the amount of protein synthesis without changing the base sequence.
  • DNA methylation is a very wonderful chemical modification. The care of relatives, aging of the body, smoking, alcoholism and even obesity will be recorded faithfully on the genome by methylation. The genome is like a diary, and methylation is used as text to record the experience of the human body.
  • DNA methylation is an important epigenetic marker information. Obtaining methylation level data for all C sites in the whole genome is of great significance for the study of spatiotemporal specificity of epigenetics.
  • mapping of DNA methylation levels in the whole genome and the analysis of high-precision methylation modification patterns of specific species will surely have a milestone significance in epigenomic research, and lay the foundation for the study of basic mechanisms such as cell differentiation and tissue development, as well as animal and plant breeding, human health and disease research.
  • WGBS Whole Genome Bisulfite Sequencing
  • Targeted methylation sequencing technology can be divided into probe capture and multiplex PCR-based sequencing technologies.
  • probe capture the required starting amount is high, and it is difficult to capture some trace samples such as plasma free DNA.
  • design and operation process of the probe capture probe is too complicated, the detection cycle is long, and the cost is high.
  • the biggest problems encountered by the current multiplex PCR based on bisulfite are the primer dimers between primers and the specificity of primer amplification.
  • the multiplex PCR after conventional bisulfite treatment after the DNA is treated with bisulfite, the unmethylated cytosine is converted to uracil, and most (99%) cytosines on the genome are unmethylated, so the bases of most sequences are changed from the previous four compositions of A/T/C/G to A/T/G.
  • one primer is designed for the positive strand and the other is designed for the complementary strand. Therefore, one strand used for PCR is a sequence rich in ATG, and the other strand is a sequence rich in ATC.
  • This three-base complementary primer sequence is easy to form primer dimers.
  • the formation of primer dimers also increases sharply.
  • too many primers are consumed due to the generation of primer dimers, resulting in the failure of multiplex PCR. Therefore, to solve the problem of multiplex bisulfite multiplex PCR, the problem of primers easily forming primer dimers must be solved first.
  • the complexity of the genome is reduced and the base sequence on the genome becomes simple, resulting in a decrease in the specificity of primer binding on the genome, leading to the existence of non-specific products and affecting the amplification effect.
  • the purpose of the present invention is to solve at least one of the above-mentioned technical defects, especially the problems of non-specific amplification of primers and primer dimers in the multiplex PCR targeted detection of methylation sites in the prior art.
  • the inventors studied a semi-nested multiple amplification method with universal sequence introduction.
  • the enrichment of target methylation regions was integrated into the two-step semi-nested PCR, reducing primer dimers and improving primer amplification specificity, achieving amplification of tens of thousands of target methylation sites in one tube.
  • This method can be compatible with multiple target methylation sites in one tube, or multiple target methylation sites and unmethylated sites can be captured simultaneously in one tube, achieving DNA methylation detection, or DNA and DNA methylation detection at the same time, improving the accuracy and efficiency of genomic methylation site detection.
  • the present invention provides a method for multiplex PCR.
  • the method comprises: a first round of amplification reaction 1, wherein the system of the first round of amplification reaction 1 comprises: conversion DNA and one or more conversion DNA primer pairs, wherein each of the conversion DNA primer pairs comprises a forward primer and a reverse primer, and the 5' end of the forward primer or the reverse primer in each of the conversion DNA primer pairs has a universal sequence 1; a second round of amplification reaction 1, wherein the system of the second round of amplification reaction 1 comprises: the product of the first round of amplification reaction 1 and one or more primer sets, wherein each of the primer sets comprises: 1) a semi-nested primer 1 having a 3' end that binds to one or more target regions of the product of the first round of amplification reaction 1 and a 5' end that has a universal sequence 2, wherein the semi-nested primer 1 is arranged at a position close to the target region of the forward primer or the reverse
  • the inventors reduce the proportion of dimers by introducing a round of semi-nested amplification. During this round of semi-nested PCR, a semi-nested specific primer and one or two universal primers are included.
  • the above method further includes at least one of the following additional technical features:
  • the conversion DNA is DNA after cytosine methylation conversion treatment.
  • the method according to a specific embodiment of the present invention can effectively detect the methylation sites in the target region of the genome.
  • the methylation conversion treated DNA is obtained by subjecting the DNA to a bisulfite or enzyme-assisted treatment.
  • the enzyme includes at least one of the following: TET methylcytosine dioxygenase 2 (TET2).
  • the universal sequence 1 and the universal sequence 2 are the same or different.
  • the T base content in the conversion DNA primer pair is 10%-70%.
  • At least one of the universal sequence 1, universal sequence 2, universal primer 1 and universal primer 2 includes a sequencing adapter sequence, a sample tag sequence or a molecular tag sequence.
  • CG sites and SNP sites should be avoided as much as possible, and continuous polyT structures should be avoided.
  • the primer length is 15-50 bp, and the TM value is 50-65.
  • the molar ratio of the primer pair of the conversion DNA can be adjusted according to the target fragment to be detected and the primer requirements.
  • the conditions of the first round of amplification reaction 1 are: 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 10-30 cycles; 65°C-78°C, 4min-6min, 1 cycle.
  • the conditions of the second round of amplification reaction 1 are 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 10-30 cycles; 65°C-78°C, 4min-6min, 1 cycle.
  • the present invention provides a multiplex PCR method.
  • the method comprises the following steps: a first round of amplification reaction 2, wherein the system of the first round of amplification reaction 2 comprises: original DNA, conversion DNA, one or more original DNA primer pairs and one or more conversion DNA primer pairs, wherein each of the original DNA primer pairs and conversion DNA primer pairs comprises a forward primer and a reverse primer, and the 5' end of the forward primer or the reverse primer in each of the original DNA primer pairs and conversion DNA primer pairs has a universal sequence 3;
  • the second round of amplification reaction 2 includes: the product of the first round of amplification reaction 2 and the second round of amplification primer set, wherein the second round of amplification primer set includes: i) a semi-nested primer 2, having a 3' end that binds to one or more target regions in the product of the first round of amplification reaction 2 and a 5' end that has a universal sequence 4, the semi-nested primer 2 is arranged downstream of the forward primer or reverse primer that does not have the universal sequence 3 in the first round of amplification system 2, and ii) a universal primer 3 having a 3' end that is the same as the universal sequence 3 and/or a universal primer 4 having a 3' end that is the same as the universal sequence 4.
  • multiple target methylation sites and unmethylated sites can be effectively captured simultaneously in one tube, and the untreated original DNA and the methylated converted DNA can be detected simultaneously, thereby improving the accuracy and efficiency of the detection of genomic methylation sites.
  • the original DNA is untreated DNA.
  • the conversion DNA is a DNA subjected to cytosine methylation conversion treatment.
  • the methylation conversion treated DNA is obtained by subjecting the DNA to bisulfite or enzyme treatment.
  • the methylase comprises at least one of the following: TET methylcytosine dioxygenase 2 (TET2).
  • the universal sequence 3 and the universal sequence 4 are the same or different.
  • the T base content of the conversion DNA primer pair is 10%-70%.
  • At least one of the universal sequence 3, the universal sequence 4, the universal primer 3 and the universal primer 4 includes a sequencing adapter sequence, a sample tag sequence or a molecular tag sequence.
  • CG sites and SNP sites should be avoided as much as possible, continuous polyT structures should be avoided, and the TM value should be 50-65.
  • a person skilled in the art can adjust the molar ratio of the original DNA, the converted DNA, one or more original DNA primer pairs and one or more converted DNA primer pairs included in the system of the first round of amplification reaction 2 as required.
  • the molar ratio of the first round of amplification reaction 2 product and the second round of amplification primer group included in the system of the second round of amplification reaction 2 can also be adjusted.
  • the conditions of the first round of PCR amplification reaction 2 are 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 12-18 cycles; 65°C-78°C, 4min-6min, 1 cycle.
  • the conditions of the second round of PCR amplification reaction 2 are: 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 18-22 cycles; 65°C-78°C, 4min-6min, 1 cycle.
  • the present invention provides a method for preparing a sequencing library.
  • the method comprises the step of constructing a sequencing library using the method described in the first aspect or the second aspect.
  • the above method further includes at least one of the following additional technical features:
  • the method further includes purifying the product of the second round of PCR amplification system 1 or 2 in the aforementioned multiplex PCR method.
  • the purification treatment is a magnetic bead purification treatment, an ethanol precipitation treatment or a tubular kit purification treatment.
  • the present invention provides a kit.
  • the kit comprises at least one of the following: one or more first-round conversion DNA primer pairs, the first-round conversion DNA primer pairs having a forward primer and a reverse primer, wherein the 5' end of the forward primer or the reverse primer has a universal sequence 1;
  • One or more second-round conversion DNA primer sets comprising: 1) a semi-nested primer 1 having a 3' end that binds to a conversion DNA target region and a 5' end that has a universal sequence 2, and 2) a universal primer 1 having a 3' end that is identical to the universal sequence 1 and/or a universal primer 2 having a 3' end that is identical to the universal sequence 2.
  • the kit further comprises one or more first-round original DNA primer pairs, wherein the first-round original DNA primer pair comprises a forward primer and a reverse primer, wherein the 5' end of the forward primer or the reverse primer comprises a universal sequence 3; and
  • One or more second-round original DNA primer sets include: 3) semi-nested primer 2, having a 3' end binding to the original DNA target region and a 5' end having a universal sequence 4, and 4) universal primer 3 having a 3' end identical to the universal sequence 3 and/or universal primer 4 having a 3' end identical to the universal sequence 4.
  • the kit includes a set of upstream and downstream specific primers consisting of 3'-end specific sequences designed for multiple target segment DNA and/or methylated DNA sequences and different universal sequences added to their 5' ends, and a set of upstream and downstream universal primers whose 3' ends are complementary to the universal sequences and whose 5' ends are sequencing adapter sequences.
  • the kit can amplify thousands of target methylation sites in one tube, and can also capture DNA and methylated DNA in one tube simultaneously during the detection process, thereby realizing the simultaneous amplification of DNA and DNA methylation.
  • the amplified library can be used for the simultaneous detection of DNA and DNA methylation sites.
  • the sequence length of the forward primer and the reverse primer in the first round conversion DNA primer pair is 15-50 bases.
  • the first round conversion DNA primer pair is a primer pair designed for the converted DNA, and the primer design principle follows the basic design principle of primers.
  • the sequence length of the universal sequence 1 and the universal sequence 2 is 10-100 bases.
  • the universal sequence 1 and the universal sequence 2 are not particularly limited and can be any fixed sequence or functional sequence, including but not limited to sequencing adapter sequence, sample tag sequence, molecular tag sequence, etc.
  • the sequence length of the semi-nested primer 1 and the semi-nested primer 2 is 15-50 bases.
  • the semi-nested primer 1 and the semi-nested primer 2 are designed for the transformed DNA, and the primer design principle follows the basic design principle of the primer.
  • the sequences of the universal primer 1 and the universal primer 2 may be the same or different.
  • the sequence length of the universal primer 1 and the universal primer 2 is 10-100 bases.
  • the universal primer 1 and the universal primer 2 can be any fixed sequence or functional sequence, including but not limited to a sequencing adapter sequence, a sample tag sequence, a molecular tag sequence, and the like.
  • the kit may further include one or more of a DNA sample extraction reagent, a DNA methylation conversion reagent, a Taq enzyme, dNTPs, a divalent magnesium ion, and a PCR system buffer.
  • the present invention proposes the use of the above-mentioned kit in preparing a sequencing library.
  • the kit can amplify methylated DNA, or methylated and unmethylated mixed DNA, and the library obtained after amplification meets the sequencing requirements.
  • the present invention provides a sequencing library.
  • the library is obtained using the method described in the third aspect.
  • the present invention proposes a method for sequencing a target nucleic acid molecule and/or detecting methylation sites.
  • the method comprises: 1) constructing a sequencing library for the target nucleic acid molecule according to the method described in the third aspect; 2) sequencing the sequencing library to obtain sequencing results; and 3) based on the sequencing results, determining the nucleic acid sequence of the target nucleic acid molecule and/or the methylation sites of the nucleic acid molecule.
  • the method according to an embodiment of the present invention can effectively sequence the original DNA and/or detect the methylation sites of the methylated modified DNA.
  • the present invention proposes a method for detecting methylation-related diseases.
  • the method comprises the following steps: i) constructing a subject sequencing library using the method described in the third aspect for a nucleic acid molecule derived from a subject; ii) sequencing the subject sequencing library to obtain a sequencing result; iii) determining the methylation site of the nucleic acid molecule derived from the subject based on the sequencing result; and iv) judging whether the subject suffers from a methylation-related disease based on the methylation site.
  • the above detection method may further include at least one of the following additional technical features:
  • the methylation-related disease includes at least one selected from the following: cardiovascular disease, cerebrovascular disease, autoimmune disease, metabolic disease and cancer.
  • the cardiovascular disease is ischemic cardiomyopathy, atherosclerosis, hypertension or heart failure.
  • the cerebrovascular disease is cerebral hemorrhage or cerebral stroke.
  • the autoimmune disease is psoriasis, lupus erythematosus or psoriasis.
  • the metabolic disease is obesity, type 2 diabetes, non-alcoholic fatty liver disease or osteoporosis.
  • the cancer is colorectal cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer or brain glioma.
  • the methylation-specific primer pair F/R is rich in ATG and ATC and is easy to form dimers, so the inventors introduced a round of semi-nested amplification to reduce the proportion of dimers.
  • this round of semi-nested PCR 1) all semi-nested specific PCR primers are rich in ATG bases or rich in ATC bases, and all specific ATG-ATG or ATC-ATC are difficult to form dimers with each other, and 2) during the second round of amplification, the semi-nested specific primers (ATG or ATC) and the universal primer (ATCG) are also difficult to form dimers, so the proportion of dimers in amplification can also be effectively reduced in the second round of amplification.
  • two rounds of PCR are used to improve the specificity of targeted amplification, and another round of semi-nested amplification is performed on the basis of the first round of PCR amplification to improve the specificity of target amplification.
  • the sequencing library prepared by the multiplex PCR method described in the present application is compatible with the simultaneous capture of DNA and DNA methylation, and can achieve simultaneous detection of DNA and DNA methylation.
  • Fig. 1 is a schematic diagram of the design of semi-nested PCR amplification primers introduced by the universal sequence (fixed tag) T1 or T2 provided in an embodiment of the present invention, wherein three primers are designed corresponding to each target region, namely, the outer specific primer pair F and R, and the semi-nested primer F1 or R1, the 5' end of one of the primers F or R in the outer specific primer pair is connected to the universal sequence T1, the 5' end of the semi-nested primer is connected to the universal sequence T2, and the semi-nested primer is arranged downstream of the F or R primer whose 5' end is not connected to the universal sequence T1;
  • FIG2 is a schematic diagram of nested PCR amplification according to an embodiment of the present invention, wherein the fixed tag is introduced;
  • FIG3 is a schematic diagram of library preparation based on fixed tag introduction according to an embodiment of the present invention.
  • FIG4 is a fragment distribution diagram of a methylation library based on fixed tag introduction according to an embodiment of the present invention, with a fragment size of 150-180 bp;
  • FIG. 5 is a graph showing the stability (Pearson coefficient) of methylated DNA detection with different initial input amounts according to an embodiment of the present invention
  • FIG6 is a schematic diagram of preparing a mixed library of DNA and methylated DNA based on fixed tag introduction according to an embodiment of the present invention
  • FIG. 7 is a fragment distribution diagram of a mixed library of DNA and methylated DNA based on fixed tag introduction according to an embodiment of the present invention, wherein the fragment size of the methylated library is 150-180 bp, and the fragment size of the DNA library is 340-390 bp.
  • the present application proposes a method for preparing a targeted methylation sequencing library based on multiplex PCR, wherein a semi-nested multiplex amplification method for introducing universal sequences is invented.
  • a semi-nested multiplex amplification method for introducing universal sequences is invented.
  • the enrichment of the target methylation region is integrated into the two-step semi-nested PCR, reducing primer dimers and improving primer amplification specificity, thereby achieving one-tube amplification of tens of thousands of target methylation sites.
  • this method is compatible with two schemes, namely, methylated DNA is captured in one tube, or unmethylated DNA and methylated DNA are captured simultaneously in one tube, the latter of which can achieve simultaneous detection of DNA and DNA methylation.
  • the specific technical scheme is as follows:
  • This example mainly includes the following experimental contents: gDNA is chemically converted (bisulfite) or enzymatically converted (TET enzyme-assisted conversion) to convert unmethylated cytosine into uracil.
  • sample NA12878 gDNA is treated with bisulfite, and detection primers are designed. Then, the DNA and primers are used to prepare a DNA targeted methylation library, and the obtained library is placed on an MGISEQ-2000 sequencer for on-machine sequencing, with the sequencing type being PE100, and then data analysis is performed, including data utilization, alignment rate, amplicon specificity, uniformity and other performance.
  • the specific experimental operations are as follows:
  • the experiment was conducted using EZ DNA Methylation-Gold Kit TM (ZYMO), and the sample NA12878 gDNA was set to 4 mass gradients of 0.5, 1, 5, and 10 ng, with 3 replicates for each gradient.
  • the gDNA was co-treated with bisulfite, and the specific steps were as follows:
  • CT conversion reagent solution Take out CT conversion reagent (solid mixture) from the above kit, add 900 ⁇ L of water, 50 ⁇ L of M-dissolving buffer and 300 ⁇ L of M-dilution buffer to the CT conversion reagent, dissolve and shake at room temperature for 10 minutes or shake on a shaker for 10 minutes.
  • the PCR product can be immediately used for the next step or stored at 4°C (up to 20 hours) for future use.
  • step 11 Place the Zymo-Spin IC TM Column obtained in step 10) in a new 1.5 mL EP tube, add 40 ⁇ L of M-elution buffer r to the column matrix, place at room temperature for 2 minutes, and centrifuge at full speed (>10,000 x g) to elute the target DNA.
  • each pair of primers is responsible for amplifying a target region, and a fixed universal sequence T1 is added to the 5' end of one primer (forward primer F or reverse primer R) in each pair of primers (as shown in FIG. 1 );
  • step ii) designing a semi-nested specific primer F1 or R1 based on the first-round specific primer, the binding position of which is set inside the first-round specific amplification primer described in step i) (as shown in FIG1 ), wherein another universal sequence (T2, the sequences of T2 and T1 may be the same or different) is introduced into the 5′ end of the semi-nested specific primer;
  • reaction product was purified using 1.5X AMPure magnetic beads and then dissolved in 22 ⁇ L elution buffer.
  • the obtained library was subjected to high-throughput sequencing, the sequencing platform was MGISEQ-2000, and the sequencing type was PE100. After sequencing, the data were aligned and various basic parameters were statistically analyzed, including offline data, available data, alignment rate, specificity, uniformity, etc.
  • NA12878 gDNA is used for the experiment.
  • the gDNA is converted into uracil by chemical conversion (bisulfite) or enzymatic conversion (TET enzyme-assisted conversion) to convert unmethylated cytosine.
  • a methylation capture panel containing 24 methylation sites is designed; at the same time, a DNA capture panel without methylation is designed, containing 20 DNA capture regions.
  • the gDNA of the NA12878 standard is treated with bisulfite, and then mixed with genomic DNA.
  • the mixed DNA is subjected to DNA and DNA methylation mixed library preparation according to the steps of the invention.
  • the sample is repeated 3 times, and the obtained library is placed on the MGISEQ-2000 sequencer for on-machine sequencing.
  • the sequencing type is PE100, and then data analysis is performed, including data utilization, alignment rate, amplicon specificity, uniformity and other performances.
  • the specific experimental operation is as follows:
  • CT conversion reagent solution Take out CT conversion reagent (solid mixture) from the above kit, add 900 ⁇ L of water, 50 ⁇ L of M-dissolving buffer and 300 ⁇ L of M-dilution buffer to the CT conversion reagent, dissolve and shake at room temperature for 10 minutes or shake on a shaker for 10 minutes.
  • the PCR product can be immediately used for the next step or stored at 4°C (up to 20 hours) for future use.
  • step 11 Place the Zymo-Spin IC TM Column obtained in step 10) in a new 1.5 mL EP tube, add 40 ⁇ L of M-elution buffer r to the column matrix, place at room temperature for 2 minutes, and centrifuge at full speed (>10,000 x g) to elute the methylated target DNA.
  • step 12 The methylated DNA obtained in step 11) was mixed with 5 ng of genomic DNA, and the mixed DNA was used as a template for subsequent library preparation.
  • First round of specific primer design One or more pairs of specific primers (F/R) are designed for the original genomic DNA sequence or the methylated genomic DNA sequence. Each pair of primers is responsible for the amplification of a target region of the original DNA or methylated DNA genome. A fixed universal sequence T1 is added to the 5' end of one primer (F or R) in each pair of primers. All primers with T1 sequence and all primers without T1 are amplified by single or multiplex PCR in one tube to obtain the target product with T1 adapter;
  • U1 Design a universal primer U1, the 3' end sequence of U1 contains all the sequences of T1 (U1 ⁇ T1). All specific primers with T2 sequence and a universal primer (U1) are used to perform multiple amplification on the first round of PCR products to obtain the target fragment with T2 sequence and U1 universal primer; sequencing adapter sequence, sample tag sequence, molecular tag sequence, chemical modification (phosphorylation, amination, etc.), enzyme cutting site (USER enzyme, restriction endonuclease), etc. can be introduced into the T1, T2 and U1 primer sequences for subsequent different molecular experiments.
  • the obtained library was subjected to high-throughput sequencing using the sequencing platform MGISEQ-2000 and the sequencing type PE100.
  • the sequencing data was aligned and the basic parameters were statistically analyzed, including offline data, available data, alignment rate, GC content, etc.
  • the basic performance parameters of DNA sequencing obtained by the hybrid sequencing scheme are shown in Table 12.
  • the data utilization rate is 0.99 ⁇ 0.00
  • the overall alignment rate is 0.98 ⁇ 0.02
  • the specificity is 0.98 ⁇ 0.00
  • the uniformity is 1.00 ⁇ 0.00, which can achieve effective detection of DNA targeting.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

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Abstract

The present invention provides a targeted methylation library construction system based on multiplex PCR, a method, and use thereof. The method comprises: a first amplification reaction run 1, wherein the system of the first amplification reaction run 1 comprises: a template DNA and one or more primer pairs, each primer pair comprises a forward primer and a reverse primer, and the 5' end of the forward primer or the reverse primer in each primer pair is provided with a universal sequence 1; and a second amplification reaction run 1, wherein the system of the second amplification reaction run 1 comprises a product of the first amplification reaction run 1 and one or more primer sets, and each primer set comprises: 1) a semi-nested primer 1 with a 3' end linked to one or more target regions in the product of the first amplification reaction run 1 and a 5' end having a universal sequence 2, and 2) a universal primer 1 with a 3' end identical to that of the universal sequence 1 and/or a universal primer 2 with a 3' end identical to that of the universal sequence 2.

Description

一种基于多重PCR的靶向甲基化建库体系、方法及其应用A targeted methylation library construction system based on multiplex PCR, method and application thereof
优先权信息Priority information
无。none.
技术领域Technical Field
本发明涉及生物技术领域，尤其涉及一种基于多重PCR的靶向甲基化建库体系、方法及其应用。The present invention relates to the field of biotechnology, and in particular to a targeted methylation library construction system based on multiplex PCR, a method and an application thereof.
背景技术Background technique
DNA甲基化是一种表观调控修饰，它在不改变碱基序列的情况下，参与调控蛋白质合成的多少。对人类来说，DNA甲基化是一种非常奇妙的化学修饰，亲人的关怀，机体的衰老、抽烟、酗酒甚至肥胖，都会被甲基化如实地记录到基因组上。基因组就像是一个日记本，甲基化作为文字，记录下人体的经历。DNA甲基化是重要的表观遗传学标记信息，获得全基因组范围内所有C位点的甲基化水平数据，对于表观遗传学的时空特异性研究具有重要意义。以新一代高通量测序平台为基础，进行全基因组DNA甲基化水平图谱绘制，特定物种的高精确度甲基化修饰模式的分析，必将在表观基因组学研究中具有里程碑式的意义，并为细胞分化、组织发育等基础机制研究，以及动植物育种、人类健康与疾病研究奠定基础。DNA methylation is an epigenetic regulatory modification that participates in regulating the amount of protein synthesis without changing the base sequence. For humans, DNA methylation is a very wonderful chemical modification. The care of relatives, aging of the body, smoking, alcoholism and even obesity will be recorded faithfully on the genome by methylation. The genome is like a diary, and methylation is used as text to record the experience of the human body. DNA methylation is an important epigenetic marker information. Obtaining methylation level data for all C sites in the whole genome is of great significance for the study of spatiotemporal specificity of epigenetics. Based on the new generation of high-throughput sequencing platform, the mapping of DNA methylation levels in the whole genome and the analysis of high-precision methylation modification patterns of specific species will surely have a milestone significance in epigenomic research, and lay the foundation for the study of basic mechanisms such as cell differentiation and tissue development, as well as animal and plant breeding, human health and disease research.
全基因组甲基化测序WGBS(Whole Genome Bisulfite Sequencing)，即全基因组亚硫酸氢盐测序，是研究生物甲基化的最常用手段，它可以覆盖所有甲基化位点，能够获得更加全面的甲基化图谱。但其在高通量测序中遇到了很多挑战：(1)亚硫酸氢盐处理会对DNA单链化并造成严重的损伤；(2)亚硫酸氢盐处理后的未甲基化C碱基会转变成U碱基，整个基因组的GC含量发生极端变化，造成后续扩增产生极大的偏好性；(3)建库需要微克级别的起始DNA，对于微量DNA很难有很有效的建库方法。对于临床检测和某些特定的研究来讲，全基因组甲基化测序操作复杂并且成本还过于昂贵，而采用靶向甲基化测序技术可以有效解决这些问题。Whole Genome Bisulfite Sequencing (WGBS) is the most commonly used method for studying biological methylation. It can cover all methylation sites and obtain a more comprehensive methylation map. However, it encounters many challenges in high-throughput sequencing: (1) Bisulfite treatment will cause single-stranded DNA and cause serious damage; (2) Unmethylated C bases after bisulfite treatment will be converted into U bases, and the GC content of the entire genome will change drastically, resulting in great preference for subsequent amplification; (3) Library construction requires microgram-level starting DNA, and it is difficult to have an effective library construction method for trace amounts of DNA. For clinical testing and certain specific studies, whole genome methylation sequencing is complex and too expensive, and the use of targeted methylation sequencing technology can effectively solve these problems.
靶向甲基化测序技术可以分为以探针捕获和以多重PCR为基础的测序技术，对于探针捕获，其要求的起始量高，对于一些微量样本如血浆游离DNA，很难进行捕获，并且探针捕获探针的设计和操作流程也过于复杂，检测周期长，成本高。Targeted methylation sequencing technology can be divided into probe capture and multiplex PCR-based sequencing technologies. For probe capture, the required starting amount is high, and it is difficult to capture some trace samples such as plasma free DNA. In addition, the design and operation process of the probe capture probe is too complicated, the detection cycle is long, and the cost is high.
基于DNA重亚硫酸盐处理后的多重PCR起始要求量低，操作简单，灵敏度高，但技术要求高。如何有效的进行超多重靶标扩增是主要瓶颈，即使是开展上万重的基因组扩增子测序也是非常具有挑战性的工作，更别说针对Bisulfite转化后序列的多重甲基化PCR，主要是由于在PCR过程中形成了严重的引物二聚体。先前有过报道，使用微滴技术进行单分子BS-PCR，可以同时检测九千个左右的靶标，但是起始量较高，需要2μgDNA。2015年，Lu Wen等研究人员巧妙地利用CpG岛的特征序列作为引物结合位点，开发了基于PCR技术的MCTA-seq，可以同时检测大量CpG岛区域的甲基化信号，该技术极其灵敏，能够对7.5pg的gDNA进行检测，不过，MCTA-seq更像是一种固定的CGI Panel，作为靶向测序平台，灵活性稍显不足。因此开发一个起始量要求低，灵活性强的靶向甲基化技术是未来靶向甲基化的发展方向。Multiplex PCR based on DNA bisulfite treatment has low starting requirements, simple operation, high sensitivity, but high technical requirements. How to effectively perform super-multiplex target amplification is the main bottleneck. Even the sequencing of tens of thousands of genomic amplicons is a very challenging task, not to mention the multiplex methylation PCR for sequences after Bisulfite conversion, mainly due to the formation of serious primer dimers during the PCR process. It has been reported previously that single-molecule BS-PCR using droplet technology can detect about 9,000 targets at the same time, but the starting amount is high, requiring 2μg of DNA. In 2015, Lu Wen and other researchers cleverly used the characteristic sequence of CpG islands as primer binding sites to develop MCTA-seq based on PCR technology, which can simultaneously detect methylation signals in a large number of CpG island regions. The technology is extremely sensitive and can detect 7.5pg of gDNA. However, MCTA-seq is more like a fixed CGI Panel. As a targeted sequencing platform, it is slightly less flexible. Therefore, developing a targeted methylation technology with low starting requirements and strong flexibility is the future development direction of targeted methylation.
现在基于亚硫酸盐的多重PCR遇到的最大的问题是引物之间存在的引物二聚体和引物扩增的特异性。对于常规的重亚硫酸盐处理后的多重PCR，DNA经过重亚硫酸盐处理后，未甲基化的胞嘧啶转换为尿嘧啶，基因组上大部分(99％)胞嘧啶都是未甲基化的，因此大部分序列的碱基由以前A/T/C/G四种组成变为A/T/G组成。在常规的PCR中，一条引物是针对正链设计，一条是针对互补的链设计，因此用于PCR的一条链是富含ATG的序列，另一条链是富含ATC的序列，这种3碱基互补的引物序列很容易形成引物二聚体。当引物对数增加时，引物二聚体的形成也急剧增加，在多重PCR过程中，过多的引物由于引物二聚体的产生而消耗殆尽，造成多重PCR的失败，因此要解决多重亚硫酸盐多重PCR问题就先得解决引物容易形成引物二聚体的问题。其次DNA经过重亚硫酸氢盐处理后，基因组复杂度降低，基因组上碱基序列变得简单，造成引物在基因组上结合的特异性降低，导致非特异性产物的存在，影响扩增的效果。The biggest problems encountered by the current multiplex PCR based on bisulfite are the primer dimers between primers and the specificity of primer amplification. For the multiplex PCR after conventional bisulfite treatment, after the DNA is treated with bisulfite, the unmethylated cytosine is converted to uracil, and most (99%) cytosines on the genome are unmethylated, so the bases of most sequences are changed from the previous four compositions of A/T/C/G to A/T/G. In conventional PCR, one primer is designed for the positive strand and the other is designed for the complementary strand. Therefore, one strand used for PCR is a sequence rich in ATG, and the other strand is a sequence rich in ATC. This three-base complementary primer sequence is easy to form primer dimers. When the number of primer pairs increases, the formation of primer dimers also increases sharply. In the process of multiplex PCR, too many primers are consumed due to the generation of primer dimers, resulting in the failure of multiplex PCR. Therefore, to solve the problem of multiplex bisulfite multiplex PCR, the problem of primers easily forming primer dimers must be solved first. Secondly, after DNA is treated with bisulfite, the complexity of the genome is reduced and the base sequence on the genome becomes simple, resulting in a decrease in the specificity of primer binding on the genome, leading to the existence of non-specific products and affecting the amplification effect.
因此，仍需进一步开发有效检测甲基化位点的方法。Therefore, there is still a need to further develop methods to effectively detect methylation sites.
发明内容Summary of the invention
本发明的目的旨在至少能解决上述的技术缺陷之一，特别是现有技术中多重PCR靶向检测甲基化位点中存在的引物非特异性扩增以及引物二聚体问题。The purpose of the present invention is to solve at least one of the above-mentioned technical defects, especially the problems of non-specific amplification of primers and primer dimers in the multiplex PCR targeted detection of methylation sites in the prior art.
针对引物二聚体和特异性的问题，发明人研究了通用序列引入的半巢式多重扩增方法，通过通用序列的引入，将靶标甲基化区域的富集整合到两步半巢式PCR中，降低引物二聚体和提高引物扩增特异性，实现一管扩增成千上万个靶标甲基化位点。该方法能够兼容多靶标甲基化位点在一管中，或多靶标甲基化位点和未甲基化位点在一管中同时进行捕获，实现DNA甲基化的检测，或DNA和DNA甲基化同时检测，提高了基因组甲基化位点检测的准确性和效率。In response to the problems of primer dimers and specificity, the inventors studied a semi-nested multiple amplification method with universal sequence introduction. By introducing universal sequences, the enrichment of target methylation regions was integrated into the two-step semi-nested PCR, reducing primer dimers and improving primer amplification specificity, achieving amplification of tens of thousands of target methylation sites in one tube. This method can be compatible with multiple target methylation sites in one tube, or multiple target methylation sites and unmethylated sites can be captured simultaneously in one tube, achieving DNA methylation detection, or DNA and DNA methylation detection at the same time, improving the accuracy and efficiency of genomic methylation site detection.
因此，在本发明的第一方面，本发明提供了一种多重PCR的方法。根据本发明的实施例，所述方法包括：第一轮扩增反应1，所述第一轮扩增反应1的体系中包括：转化DNA和一个或多个转化DNA引物对，其中，每个所述转化DNA引物对中包含一条正向引物和一条反向引物，每个所述转化DNA引物对中所述正向引物或反向引物的5’端具有通用序列1；第二轮扩增反应1，所述第二轮扩增反应1的体系中包括：所述第一轮扩增反应1产物和一个或多个引物组，其中，每个所述引物组包括：1)半巢式引物1，具有与所述第一轮扩增反应1产物的一个或多个目标区域结合的3’端和具有通用序列2的5’端，所述半巢式引物1设置在所述第一轮扩增反应1的体系中不具有通用序列1的所述正向引物或反向引物的靠近目标区域位置，和2)具有3’端和所述通用序列1相同的通用引物1和/或3’端和所述通用序列2相同的通用引物2。根据本发明实施例的方法，在所述扩增反应过程中由于正反向引物3’端富含ATG和ATC容易形成二聚体，因此发明人通过引入一轮半巢式扩增来降低二聚体的比例，在该轮半巢式PCR的过程中，包含一种半巢式特异性引物和一种或两种通用引物，引入半巢式扩增反应后，1)由于所有的半巢式特异性PCR引物都富含ATG碱基或都富含ATC碱基，所有特异性ATG-ATG或ATC-ATC互相之间难以形成二聚体，2)半巢式特异性引物(ATG或ATC)和通用引物(ATCG)也很难形成二聚体，因此经过二轮扩增后，可以有效降低二聚体在扩增中的比例。Therefore, in the first aspect of the present invention, the present invention provides a method for multiplex PCR. According to an embodiment of the present invention, the method comprises: a first round of amplification reaction 1, wherein the system of the first round of amplification reaction 1 comprises: conversion DNA and one or more conversion DNA primer pairs, wherein each of the conversion DNA primer pairs comprises a forward primer and a reverse primer, and the 5' end of the forward primer or the reverse primer in each of the conversion DNA primer pairs has a universal sequence 1; a second round of amplification reaction 1, wherein the system of the second round of amplification reaction 1 comprises: the product of the first round of amplification reaction 1 and one or more primer sets, wherein each of the primer sets comprises: 1) a semi-nested primer 1 having a 3' end that binds to one or more target regions of the product of the first round of amplification reaction 1 and a 5' end that has a universal sequence 2, wherein the semi-nested primer 1 is arranged at a position close to the target region of the forward primer or the reverse primer that does not have the universal sequence 1 in the system of the first round of amplification reaction 1, and 2) a universal primer 1 having a 3' end identical to the universal sequence 1 and/or a universal primer 2 having a 3' end identical to the universal sequence 2. According to the method of the embodiment of the present invention, during the amplification reaction, since the 3' ends of the forward and reverse primers are rich in ATG and ATC and are easy to form dimers, the inventors reduce the proportion of dimers by introducing a round of semi-nested amplification. During this round of semi-nested PCR, a semi-nested specific primer and one or two universal primers are included. After the introduction of the semi-nested amplification reaction, 1) since all the semi-nested specific PCR primers are rich in ATG bases or rich in ATC bases, all specific ATG-ATG or ATC-ATC are difficult to form dimers with each other, and 2) the semi-nested specific primers (ATG or ATC) and the universal primer (ATCG) are also difficult to form dimers. Therefore, after two rounds of amplification, the proportion of dimers in the amplification can be effectively reduced.
根据本发明的实施例，上述方法进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above method further includes at least one of the following additional technical features:
根据本发明的实施例，所述转化DNA为胞嘧啶甲基化转化处理后的DNA。根据本发明的具体实施例的方法能够有效检测基因组中目标区域的甲基化位点。According to an embodiment of the present invention, the conversion DNA is DNA after cytosine methylation conversion treatment. The method according to a specific embodiment of the present invention can effectively detect the methylation sites in the target region of the genome.
根据本发明的实施例，所述甲基化转化处理DNA是将所述DNA进行重亚硫酸盐或者酶法辅助的处理获得的。According to an embodiment of the present invention, the methylation conversion treated DNA is obtained by subjecting the DNA to a bisulfite or enzyme-assisted treatment.
根据本发明的实施例，所述酶包括下列中的至少之一：TET甲基胞嘧啶双加氧酶2(TET2)。According to an embodiment of the present invention, the enzyme includes at least one of the following: TET methylcytosine dioxygenase 2 (TET2).
根据本发明的实施例，所述通用序列1和通用序列2相同或不同。According to an embodiment of the present invention, the universal sequence 1 and the universal sequence 2 are the same or different.
根据本发明的实施例，所述转化DNA引物对中T碱基含量为10％-70％。According to an embodiment of the present invention, the T base content in the conversion DNA primer pair is 10%-70%.
根据本发明的实施例，所述通用序列1、通用序列2、通用引物1和通用引物2中的至少之一包括测序接头序列、样本标签序列或分子标签序列。According to an embodiment of the present invention, at least one of the universal sequence 1, universal sequence 2, universal primer 1 and universal primer 2 includes a sequencing adapter sequence, a sample tag sequence or a molecular tag sequence.
优选地，设计所述转化DNA引物对时，应尽量避免存在CG位点和SNP位点，避免连续的polyT结构，引物长度15-50bp，TM值为50-65。Preferably, when designing the conversion DNA primer pair, CG sites and SNP sites should be avoided as much as possible, and continuous polyT structures should be avoided. The primer length is 15-50 bp, and the TM value is 50-65.
本领域技术人员可以理解，所述第一轮扩增反应1的体系中，所述转化DNA的引物对摩尔比可以根据检测的目标片段以及引物需求进行调整。Those skilled in the art will appreciate that in the system of the first round of amplification reaction 1, the molar ratio of the primer pair of the conversion DNA can be adjusted according to the target fragment to be detected and the primer requirements.
此外，本领域技术人员可以根据不同待测的转化DNA及使用的相应引物调整所述第一轮PCR扩增反应1以及所述第二轮PCR扩增反应1的条件。In addition, those skilled in the art can adjust the conditions of the first round of PCR amplification reaction 1 and the second round of PCR amplification reaction 1 according to different transformed DNAs to be tested and the corresponding primers used.
根据本发明的一些具体实施例，所述第一轮扩增反应1的条件依次为：90℃-97℃、0.5-5min，1个循环；90℃-97℃、25-35s，55-65℃、1.5-2.5min，65℃-78℃、25-35s，10-30个循环；65℃-78℃、4min-6min，1个循环。According to some specific embodiments of the present invention, the conditions of the first round of amplification reaction 1 are: 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 10-30 cycles; 65°C-78°C, 4min-6min, 1 cycle.
根据本发明的一些具体的实施例，所述第二轮扩增反应1的条件依次为90℃-97℃、0.5-5min，1个循环；90℃-97℃、25-35s，55-65℃、1.5-2.5min，65℃-78℃、25-35s，10-30个循环；65℃-78℃、4min-6min，1个循环。According to some specific embodiments of the present invention, the conditions of the second round of amplification reaction 1 are 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 10-30 cycles; 65°C-78°C, 4min-6min, 1 cycle.
在本发明的第二方面，本发明提出了一种多重PCR方法。根据本发明的实施例，包括以下步骤： 第一轮扩增反应2，所述第一轮扩增反应2的体系中包括：原始DNA、转化DNA、一个或多个原始DNA引物对和一个或多个转化DNA引物对，其中，每个所述原始DNA引物对和转化DNA引物对中包含一条正向引物和一条反向引物，所述每个原始DNA引物对和转化DNA引物对中的正向引物或反向引物的5’端具有通用序列3；In the second aspect of the present invention, the present invention provides a multiplex PCR method. According to an embodiment of the present invention, the method comprises the following steps: a first round of amplification reaction 2, wherein the system of the first round of amplification reaction 2 comprises: original DNA, conversion DNA, one or more original DNA primer pairs and one or more conversion DNA primer pairs, wherein each of the original DNA primer pairs and conversion DNA primer pairs comprises a forward primer and a reverse primer, and the 5' end of the forward primer or the reverse primer in each of the original DNA primer pairs and conversion DNA primer pairs has a universal sequence 3;
第二轮扩增反应2，所述第二轮扩增反应2的体系中包括：所述第一轮扩增反应2产物和第二轮扩增引物组，其中，所述第二轮扩增引物组包括：i)半巢式引物2，具有与所述第一轮扩增反应2产物中的一个或多个目标区域结合的3’端和具有通用序列4的5’端，所述半巢式引物2设置在所述第一轮扩增体系2中不具有通用序列3的正向引物或反向引物的下游，和ii)具有3’端和通用序列3相同的通用引物3和/或具有3’端和通用序列4相同的通用引物4。根据本发明实施例的多重PCR方法，可以有效的对多靶标甲基化位点和未甲基化位点在一管中同时进行捕获，实现未处理的原始DNA和经过甲基化处理的转化DNA同时检测，提高了基因组甲基化位点检测的准确性和效率。The second round of amplification reaction 2, the system of the second round of amplification reaction 2 includes: the product of the first round of amplification reaction 2 and the second round of amplification primer set, wherein the second round of amplification primer set includes: i) a semi-nested primer 2, having a 3' end that binds to one or more target regions in the product of the first round of amplification reaction 2 and a 5' end that has a universal sequence 4, the semi-nested primer 2 is arranged downstream of the forward primer or reverse primer that does not have the universal sequence 3 in the first round of amplification system 2, and ii) a universal primer 3 having a 3' end that is the same as the universal sequence 3 and/or a universal primer 4 having a 3' end that is the same as the universal sequence 4. According to the multiplex PCR method of the embodiment of the present invention, multiple target methylation sites and unmethylated sites can be effectively captured simultaneously in one tube, and the untreated original DNA and the methylated converted DNA can be detected simultaneously, thereby improving the accuracy and efficiency of the detection of genomic methylation sites.
根据本发明的实施例，所述原始DNA为不经过处理的DNA。According to an embodiment of the present invention, the original DNA is untreated DNA.
根据本发明的实施例，所述转化DNA为胞嘧啶甲基化转化处理的DNA。According to an embodiment of the present invention, the conversion DNA is a DNA subjected to cytosine methylation conversion treatment.
根据本发明的实施例，所述甲基化转化处理DNA是将所述DNA进行重亚硫酸盐或者酶处理获得的。According to an embodiment of the present invention, the methylation conversion treated DNA is obtained by subjecting the DNA to bisulfite or enzyme treatment.
根据本发明的实施例，所述甲基化酶包括下列中的至少之一：TET甲基胞嘧啶双加氧酶2(TET2)。According to an embodiment of the present invention, the methylase comprises at least one of the following: TET methylcytosine dioxygenase 2 (TET2).
根据本发明的实施例，所述通用序列3和通用序列4相同或不同。According to an embodiment of the present invention, the universal sequence 3 and the universal sequence 4 are the same or different.
根据本发明的实施例，所述转化DNA引物对的T碱基含量为10％-70％。According to an embodiment of the present invention, the T base content of the conversion DNA primer pair is 10%-70%.
根据本发明的实施例，所述通用序列3、通用序列4、通用引物3和通用引物4中的至少之一包括测序接头序列、样本标签序列或分子标签序列。According to an embodiment of the present invention, at least one of the universal sequence 3, the universal sequence 4, the universal primer 3 and the universal primer 4 includes a sequencing adapter sequence, a sample tag sequence or a molecular tag sequence.
优选地，设计所述转化DNA引物对和/或原始DNA引物对时，应尽量避免存在CG位点和SNP位点，避免连续的polyT结构，TM值为50-65。Preferably, when designing the conversion DNA primer pair and/or the original DNA primer pair, CG sites and SNP sites should be avoided as much as possible, continuous polyT structures should be avoided, and the TM value should be 50-65.
根据本发明的实施例，本领域技术人员可以根据需求对所述第一轮扩增反应2的体系中包括的原始DNA、转化DNA、一个或多个原始DNA引物对和一个或多个转化DNA引物对的摩尔比进行调整，同理，也可以对所述第二轮扩增反应2的体系中包括的所述第一轮扩增反应2产物和第二轮扩增引物组的摩尔比进行调整。According to an embodiment of the present invention, a person skilled in the art can adjust the molar ratio of the original DNA, the converted DNA, one or more original DNA primer pairs and one or more converted DNA primer pairs included in the system of the first round of amplification reaction 2 as required. Similarly, the molar ratio of the first round of amplification reaction 2 product and the second round of amplification primer group included in the system of the second round of amplification reaction 2 can also be adjusted.
此外，本领域技术人员可以根据不同待测的原始DNA和转化DNA及使用的相应引物调整所述第一轮PCR扩增反应2以及所述第二轮PCR扩增反应2的条件。In addition, those skilled in the art can adjust the conditions of the first round PCR amplification reaction 2 and the second round PCR amplification reaction 2 according to the different original DNA and converted DNA to be tested and the corresponding primers used.
根据本发明的一些具体实施例，所述第一轮PCR扩增反应2的条件依次为90℃-97℃、0.5-5min，1个循环；90℃-97℃、25-35s，55-65℃、1.5-2.5min，65℃-78℃、25-35s，12-18个循环；65℃-78℃、4min-6min，1个循环。According to some specific embodiments of the present invention, the conditions of the first round of PCR amplification reaction 2 are 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 12-18 cycles; 65°C-78°C, 4min-6min, 1 cycle.
根据本发明的一些具体实施例，所述第二轮PCR扩增反应2的条件依次为：90℃-97℃、0.5-5min，1个循环；90℃-97℃、25-35s，55-65℃、1.5-2.5min，65℃-78℃、25-35s，18-22个循环；65℃-78℃、4min-6min，1个循环。According to some specific embodiments of the present invention, the conditions of the second round of PCR amplification reaction 2 are: 90°C-97°C, 0.5-5min, 1 cycle; 90°C-97°C, 25-35s, 55-65°C, 1.5-2.5min, 65°C-78°C, 25-35s, 18-22 cycles; 65°C-78°C, 4min-6min, 1 cycle.
在本发明的第三方面，本发明提出了一种制备测序文库的方法。根据本发明的实施例，所述方法包括利用第一方面或第二方面所述的方法进行测序文库构建的步骤。In the third aspect of the present invention, the present invention provides a method for preparing a sequencing library. According to an embodiment of the present invention, the method comprises the step of constructing a sequencing library using the method described in the first aspect or the second aspect.
根据本发明的实施例，上述方法进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above method further includes at least one of the following additional technical features:
根据本发明的实施例，还包括对前面所述多重PCR方法中的所述第二轮PCR扩增体系1或2的产物进行纯化处理。According to an embodiment of the present invention, the method further includes purifying the product of the second round of PCR amplification system 1 or 2 in the aforementioned multiplex PCR method.
根据本发明的实施例，所述纯化处理为磁珠纯化处理、乙醇沉淀处理或管式试剂盒纯化处理。According to an embodiment of the present invention, the purification treatment is a magnetic bead purification treatment, an ethanol precipitation treatment or a tubular kit purification treatment.
在本发明的第四方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括下列中的至少之一：一个或多个第一轮转化DNA引物对，所述第一轮转化DNA引物对具有一条正向引物和一条反向引物，其中，所述正向引物或反向引物的5’端具有通用序列1；In a fourth aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises at least one of the following: one or more first-round conversion DNA primer pairs, the first-round conversion DNA primer pairs having a forward primer and a reverse primer, wherein the 5' end of the forward primer or the reverse primer has a universal sequence 1;
一个或多个第二轮转化DNA引物组，所述第二轮转化DNA引物组包括：1)半巢式引物1，具有与转化DNA目标区域结合的3’端和具有通用序列2的5’端，和2)具有3’端和所述通用序列1相同的通用引物1和/或3’端和所述通用序列2相同的通用引物2。One or more second-round conversion DNA primer sets, the second-round conversion DNA primer sets comprising: 1) a semi-nested primer 1 having a 3' end that binds to a conversion DNA target region and a 5' end that has a universal sequence 2, and 2) a universal primer 1 having a 3' end that is identical to the universal sequence 1 and/or a universal primer 2 having a 3' end that is identical to the universal sequence 2.
根据本发明的实施例，所述试剂盒中还进一步包括一个或多个第一轮原始DNA引物对，所述第一轮原始DNA引物对具有一条正向引物和一条反向引物，其中，所述正向引物或反向引物的5’端具有通用序列3；和According to an embodiment of the present invention, the kit further comprises one or more first-round original DNA primer pairs, wherein the first-round original DNA primer pair comprises a forward primer and a reverse primer, wherein the 5' end of the forward primer or the reverse primer comprises a universal sequence 3; and
一个或多个第二轮原始DNA引物组，所述第二轮原始DNA引物组包括：3)半巢式引物2，具有与原始DNA目标区域结合的3’端和具有通用序列4的5’端，和4)具有3’端和所述通用序列3相同的通用引物3和/或3’端和所述通用序列4相同的通用引物4。One or more second-round original DNA primer sets, the second-round original DNA primer sets include: 3) semi-nested primer 2, having a 3' end binding to the original DNA target region and a 5' end having a universal sequence 4, and 4) universal primer 3 having a 3' end identical to the universal sequence 3 and/or universal primer 4 having a 3' end identical to the universal sequence 4.
根据本发明的实施例，所述试剂盒中包括一套针对多个目标区段DNA和/或甲基化DNA序列设计的3’端特异序列及其5’端分别添加不同的通用序列所构成的上下游特异引物，和一套3’端与通用序列互补且5’端为测序接头序列的上下游接头通用引物，所述试剂盒能够实现一管扩增成千上万个靶标甲基化位点，同时检测过程中还能够兼容DNA和甲基化DNA在一管中同时进行捕获，实现DNA和DNA甲基化的同步扩增，扩增后的文库可用于DNA和DNA甲基化位点的同时检测。According to an embodiment of the present invention, the kit includes a set of upstream and downstream specific primers consisting of 3'-end specific sequences designed for multiple target segment DNA and/or methylated DNA sequences and different universal sequences added to their 5' ends, and a set of upstream and downstream universal primers whose 3' ends are complementary to the universal sequences and whose 5' ends are sequencing adapter sequences. The kit can amplify thousands of target methylation sites in one tube, and can also capture DNA and methylated DNA in one tube simultaneously during the detection process, thereby realizing the simultaneous amplification of DNA and DNA methylation. The amplified library can be used for the simultaneous detection of DNA and DNA methylation sites.
根据本发明的实施例，所述第一轮转化DNA引物对中正向引物和反向引物的序列长度为15-50个碱基。所述第一轮转化DNA引物对是针对转化后的DNA设计的引物对，引物设计原理遵循引物的基本设计原则。According to an embodiment of the present invention, the sequence length of the forward primer and the reverse primer in the first round conversion DNA primer pair is 15-50 bases. The first round conversion DNA primer pair is a primer pair designed for the converted DNA, and the primer design principle follows the basic design principle of primers.
根据本发明的实施例，所述通用序列1和通用序列2的序列长度为10-100个碱基。所述通用序列1和通用序列2不受特别限制，可以为任意固定序列或功能序列，包括但不限于测序接头序列、样本标签序列、分子标签序列等。According to an embodiment of the present invention, the sequence length of the universal sequence 1 and the universal sequence 2 is 10-100 bases. The universal sequence 1 and the universal sequence 2 are not particularly limited and can be any fixed sequence or functional sequence, including but not limited to sequencing adapter sequence, sample tag sequence, molecular tag sequence, etc.
根据本发明的实施例，所述半巢式引物1和半巢式引物2的序列长度为15-50个碱基。所述半巢式引物1和半巢式引物2针对转化后的DNA设计引物，引物设计原理遵循引物的基本设计原则。According to an embodiment of the present invention, the sequence length of the semi-nested primer 1 and the semi-nested primer 2 is 15-50 bases. The semi-nested primer 1 and the semi-nested primer 2 are designed for the transformed DNA, and the primer design principle follows the basic design principle of the primer.
根据本发明的实施例，所述通用引物1和通用引物2序列可以相同也可以不同。According to an embodiment of the present invention, the sequences of the universal primer 1 and the universal primer 2 may be the same or different.
根据本发明的实施例，所述通用引物1和通用引物2的序列长度为10-100个碱基。本领域技术人员可以理解，所述通用引物1和通用引物2可以为任意固定序列或功能序列，包括但不限于测序接头序列、样本标签序列、分子标签序列等。According to an embodiment of the present invention, the sequence length of the universal primer 1 and the universal primer 2 is 10-100 bases. Those skilled in the art will appreciate that the universal primer 1 and the universal primer 2 can be any fixed sequence or functional sequence, including but not limited to a sequencing adapter sequence, a sample tag sequence, a molecular tag sequence, and the like.
根据本发明的实施例，所述试剂盒中还可以包括DNA样本提取试剂、DNA甲基化转化试剂、Taq酶、dNTPs、二价镁离子和PCR体系缓冲液中的一种或几种。According to an embodiment of the present invention, the kit may further include one or more of a DNA sample extraction reagent, a DNA methylation conversion reagent, a Taq enzyme, dNTPs, a divalent magnesium ion, and a PCR system buffer.
在本发明的第五方面，本发明提出了前面所述的试剂盒在制备测序文库中的用途。如前所述，所述试剂盒能够对实现甲基化DNA，或甲基化与未甲基化混合DNA进行扩增，扩增后获得的文库满足测序要求。In a fifth aspect of the present invention, the present invention proposes the use of the above-mentioned kit in preparing a sequencing library. As mentioned above, the kit can amplify methylated DNA, or methylated and unmethylated mixed DNA, and the library obtained after amplification meets the sequencing requirements.
在本发明的第六方面，本发明提出了一种测序文库。根据本发明的实施例，所述文库是利用第三方面所述的方法获得的。In a sixth aspect of the present invention, the present invention provides a sequencing library. According to an embodiment of the present invention, the library is obtained using the method described in the third aspect.
在本发明的第七方面，本发明提出了一种对目标核酸分子进行测序和/或甲基化位点检测的方法。根据本发明的实施例，所述方法包括：1)针对所述目标核酸分子，根据第三方面所述的方法构建测序文库；2)针对所述测序文库进行测序，以便获得测序结果；和3)基于所述测序结果，确定所述目标核酸分子的核酸序列和/或所述核酸分子的甲基化位点。根据本发明实施例的方法能够有效的对原始DNA进行测序和/或对甲基化修饰DNA进行甲基化位点的检测。In the seventh aspect of the present invention, the present invention proposes a method for sequencing a target nucleic acid molecule and/or detecting methylation sites. According to an embodiment of the present invention, the method comprises: 1) constructing a sequencing library for the target nucleic acid molecule according to the method described in the third aspect; 2) sequencing the sequencing library to obtain sequencing results; and 3) based on the sequencing results, determining the nucleic acid sequence of the target nucleic acid molecule and/or the methylation sites of the nucleic acid molecule. The method according to an embodiment of the present invention can effectively sequence the original DNA and/or detect the methylation sites of the methylated modified DNA.
在本发明的第八方面，本发明提出了一种甲基化相关疾病的检测方法。根据本发明的实施例，包括以下步骤：i)针对受试者来源的核酸分子，利用第三方面所述的方法，构建受试者测序文库；ii)针对所述受试者测序文库进行测序，以便获得测序结果；iii)基于所述测序结果，确定所述受试者来源的核酸分子的甲基化位点；和iv)基于所述甲基化位点判断所述受试者是否患有和甲基化相关疾病。In the eighth aspect of the present invention, the present invention proposes a method for detecting methylation-related diseases. According to an embodiment of the present invention, the method comprises the following steps: i) constructing a subject sequencing library using the method described in the third aspect for a nucleic acid molecule derived from a subject; ii) sequencing the subject sequencing library to obtain a sequencing result; iii) determining the methylation site of the nucleic acid molecule derived from the subject based on the sequencing result; and iv) judging whether the subject suffers from a methylation-related disease based on the methylation site.
根据本发明的实施例，上述检测方法还可以进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above detection method may further include at least one of the following additional technical features:
根据本发明的实施例，所述甲基化相关疾病包括选自下列中的至少之一：心血管疾病、脑血管疾病、自身免疫性疾病、代谢性疾病和癌症。According to an embodiment of the present invention, the methylation-related disease includes at least one selected from the following: cardiovascular disease, cerebrovascular disease, autoimmune disease, metabolic disease and cancer.
根据本发明的实施例，所述心血管疾病为缺血性心肌病、动脉粥样硬化、高血压或心力衰竭。According to an embodiment of the present invention, the cardiovascular disease is ischemic cardiomyopathy, atherosclerosis, hypertension or heart failure.
根据本发明的实施例，所述脑血管疾病为脑出血或脑卒中。According to an embodiment of the present invention, the cerebrovascular disease is cerebral hemorrhage or cerebral stroke.
根据本发明的实施例，所述自身免疫性疾病为牛皮癣、红斑狼疮或银屑病。According to an embodiment of the present invention, the autoimmune disease is psoriasis, lupus erythematosus or psoriasis.
根据本发明的实施例，所述代谢性疾病为肥胖、2型糖尿病、非酒精性脂肪肝或骨质疏松症。According to an embodiment of the present invention, the metabolic disease is obesity, type 2 diabetes, non-alcoholic fatty liver disease or osteoporosis.
根据本发明的实施例，所述癌症为直肠癌、肺癌、肝癌、胃癌、胰腺癌或脑胶质瘤。According to an embodiment of the present invention, the cancer is colorectal cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer or brain glioma.
从以上技术方案可以看出，本发明提供的多重PCR检测方法至少具有以下优势：It can be seen from the above technical solutions that the multiplex PCR detection method provided by the present invention has at least the following advantages:
1、通过通用引物引入的半巢式PCR扩增实现引物二聚体的降低1. Reduction of primer dimers by semi-nested PCR amplification using universal primers
在第一轮PCR扩增过程中甲基化特异性引物对F/R由于富含ATG和ATC容易形成二聚体，因此发明人通过引入一轮半巢式扩增来降低二聚体的比例。在该轮半巢式PCR的过程中，1)所有的半巢式特异性PCR引物都富含ATG碱基或都富含ATC碱基，所有特异性ATG-ATG或ATC-ATC互相之间难以形成二聚体，2)在第二轮扩增过程中，所述半巢式特异性引物(ATG或ATC)和通用引物(ATCG)也很难形成二聚体，因此在第二轮扩增中也可以有效降低二聚体在扩增中的比例。During the first round of PCR amplification, the methylation-specific primer pair F/R is rich in ATG and ATC and is easy to form dimers, so the inventors introduced a round of semi-nested amplification to reduce the proportion of dimers. During this round of semi-nested PCR, 1) all semi-nested specific PCR primers are rich in ATG bases or rich in ATC bases, and all specific ATG-ATG or ATC-ATC are difficult to form dimers with each other, and 2) during the second round of amplification, the semi-nested specific primers (ATG or ATC) and the universal primer (ATCG) are also difficult to form dimers, so the proportion of dimers in amplification can also be effectively reduced in the second round of amplification.
2、通过通用引物引入的半巢式PCR扩增实现特异性的提高2. Improved specificity through semi-nested PCR amplification using universal primers
本发明中，通过两轮PCR提高靶向扩增的特异性，在第一轮PCR扩增的基础上再进行一轮半巢式扩增，以提高目标扩增的特异性。In the present invention, two rounds of PCR are used to improve the specificity of targeted amplification, and another round of semi-nested amplification is performed on the basis of the first round of PCR amplification to improve the specificity of target amplification.
3、利用本申请所述多重PCR方法制备的测序文库，能够兼容DNA和DNA甲基化进行同时捕获，可以实现DNA和DNA甲基化的同时检测。3. The sequencing library prepared by the multiplex PCR method described in the present application is compatible with the simultaneous capture of DNA and DNA methylation, and can achieve simultaneous detection of DNA and DNA methylation.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.
图1是本发明实施例提供的所述通用序列(固定标签)T1或T2引入的半巢式PCR扩增引物设计示意图，其中，每个目标区域对应设计3条引物，即外侧的特异性引物对F和R，和半巢式引物F1或R1，所述外侧特异性引物对中的其中一条引物F或R的5’端连接通用序列T1，半巢式引物5’端连接通用序列T2，且半巢式引物设置在5’端未连接通用序列T1的F或R引物的下游；Fig. 1 is a schematic diagram of the design of semi-nested PCR amplification primers introduced by the universal sequence (fixed tag) T1 or T2 provided in an embodiment of the present invention, wherein three primers are designed corresponding to each target region, namely, the outer specific primer pair F and R, and the semi-nested primer F1 or R1, the 5' end of one of the primers F or R in the outer specific primer pair is connected to the universal sequence T1, the 5' end of the semi-nested primer is connected to the universal sequence T2, and the semi-nested primer is arranged downstream of the F or R primer whose 5' end is not connected to the universal sequence T1;
图2是根据本发明实施例提供的固定标签引入的巢式PCR扩增示意图；FIG2 is a schematic diagram of nested PCR amplification according to an embodiment of the present invention, wherein the fixed tag is introduced;
图3是根据本发明实施例提供的基于固定标签引入的文库制备示意图；FIG3 is a schematic diagram of library preparation based on fixed tag introduction according to an embodiment of the present invention;
图4是根据本发明实施例的基于固定标签引入的甲基化文库片段分布图，片段大小150-180bp；FIG4 is a fragment distribution diagram of a methylation library based on fixed tag introduction according to an embodiment of the present invention, with a fragment size of 150-180 bp;
图5是根据本发明实施例的不同起始投入量的甲基化DNA检测稳定性(皮尔森系数)结果图；5 is a graph showing the stability (Pearson coefficient) of methylated DNA detection with different initial input amounts according to an embodiment of the present invention;
图6是根据本发明实施例的基于固定标签引入的DNA和甲基化DNA混合文库制备示意图；FIG6 is a schematic diagram of preparing a mixed library of DNA and methylated DNA based on fixed tag introduction according to an embodiment of the present invention;
图7是根据本发明实施例的基于固定标签引入的DNA和甲基化DNA混合文库片段分布图，甲基化文库片段大小150-180bp，DNA文库片段大小340-390bp。7 is a fragment distribution diagram of a mixed library of DNA and methylated DNA based on fixed tag introduction according to an embodiment of the present invention, wherein the fragment size of the methylated library is 150-180 bp, and the fragment size of the DNA library is 340-390 bp.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
本申请提出了以多重PCR为基础的靶向甲基化测序文库的制备方法，其中，发明了通用序列引入的半巢式多重扩增方法，通过通用引物的引入，将靶标甲基化区域的富集整合到两步半巢式PCR中，降低引物二聚体和提高引物扩增特异性，实现一管扩增成千上万个靶标甲基化位点。同时该方法能够兼容2种方案，即甲基化DNA在一管中进行捕获，或未甲基化DNA和甲基化DNA在一管中同时进行捕获，后者可以实现DNA和DNA甲基化同时检测。具体的技术方案如下：The present application proposes a method for preparing a targeted methylation sequencing library based on multiplex PCR, wherein a semi-nested multiplex amplification method for introducing universal sequences is invented. By introducing universal primers, the enrichment of the target methylation region is integrated into the two-step semi-nested PCR, reducing primer dimers and improving primer amplification specificity, thereby achieving one-tube amplification of tens of thousands of target methylation sites. At the same time, this method is compatible with two schemes, namely, methylated DNA is captured in one tube, or unmethylated DNA and methylated DNA are captured simultaneously in one tube, the latter of which can achieve simultaneous detection of DNA and DNA methylation. The specific technical scheme is as follows:
实施例1甲基化多重PCR建库测序Example 1 Methylation multiplex PCR library construction and sequencing
本实施例主要包括以下实验内容：gDNA经过化学转化(亚硫酸氢盐)或酶转化(TET酶辅助的转化)，将未甲基化的胞嘧啶转化为尿嘧啶，本实施例中取样品NA12878gDNA进行重亚硫酸盐处理，并设计检测引物，然后利用所述DNA和引物进行DNA靶向甲基化文库制备，将获得的文库置于MGISEQ-2000测序仪上进行上机测序，测序类型PE100，然后进行数据分析，包括数据利用率、比对率、扩增子特异性、均一性等性能。其中，具体的实验操作如下：This example mainly includes the following experimental contents: gDNA is chemically converted (bisulfite) or enzymatically converted (TET enzyme-assisted conversion) to convert unmethylated cytosine into uracil. In this example, sample NA12878 gDNA is treated with bisulfite, and detection primers are designed. Then, the DNA and primers are used to prepare a DNA targeted methylation library, and the obtained library is placed on an MGISEQ-2000 sequencer for on-machine sequencing, with the sequencing type being PE100, and then data analysis is performed, including data utilization, alignment rate, amplicon specificity, uniformity and other performance. Among them, the specific experimental operations are as follows:
1.1重亚硫酸盐处理法获得甲基化目的DNA1.1 Bisulfite treatment to obtain methylated target DNA
采用EZ DNA Methylation-Gold Kit TM(ZYMO)进行实验，将样品NA12878gDNA设置为4个质量梯度0.5，1，5，10ng，每个梯度设置3个重复，将所述gDNA进行重亚硫酸盐共处理，具体步骤如下： The experiment was conducted using EZ DNA Methylation-Gold Kit TM (ZYMO), and the sample NA12878 gDNA was set to 4 mass gradients of 0.5, 1, 5, and 10 ng, with 3 replicates for each gradient. The gDNA was co-treated with bisulfite, and the specific steps were as follows:
1)制备CT转换试剂(CT Conversion Reagent)溶液：从上述试剂盒中取出CT转换试剂(固体混合物)，向所述CT转换试剂中分别加入900μL的水、50μL的M-溶解缓冲液(M-Dissolving Buffer)和300μL的M-稀释缓冲液(M-Dilution Buffer)，室温下溶解并且震荡10分钟或在摇床上摇动10分钟。1) Prepare CT conversion reagent solution: Take out CT conversion reagent (solid mixture) from the above kit, add 900 μL of water, 50 μL of M-dissolving buffer and 300 μL of M-dilution buffer to the CT conversion reagent, dissolve and shake at room temperature for 10 minutes or shake on a shaker for 10 minutes.
2)M-洗涤缓冲液的制备：向M-洗涤缓冲液中添加24mL 100％的乙醇，备用。2) Preparation of M-wash buffer: Add 24 mL of 100% ethanol to M-wash buffer and set aside.
3)在PCR管中加入130μL的CT转换试剂溶液和上述DNA，轻弹或移液器吹悬混合样品。3) Add 130 μL of CT conversion reagent solution and the above DNA into a PCR tube, and flick or pipette to suspend and mix the sample.
4)将上述处理后的PCR管放到PCR仪上按以下步骤进行操作：98℃下持续5分钟，64℃下持续2.5小时。4) Place the treated PCR tube on a PCR instrument and follow the steps below: 98°C for 5 minutes and 64°C for 2.5 hours.
5)完成上述操作后，PCR产物立刻进行下一步操作或者在4℃下存储(最多20小时)备用。5) After completing the above operations, the PCR product can be immediately used for the next step or stored at 4°C (up to 20 hours) for future use.
6)将Zymo-Spin IC TMColumn(离心柱)放入收集管(Collection Tube)中，并加入600μL的M-结合缓冲液(M-Binding Buffer)。 6) Place the Zymo-Spin IC ™ Column into a collection tube and add 600 μL of M-Binding Buffer.
7)将重亚硫酸盐处理的样品加入到含M-结合缓冲液的Zymo-Spin IC TMColumn中，盖上盖子颠倒混匀。 7) Add the bisulfite-treated sample to the Zymo-Spin IC TM Column containing M-binding buffer, cover the column and invert to mix.
8)将步骤7)获得的样品进行全速(>10,000x g)离心30秒，弃收集管中的收集液。继续向离心柱中加入100μL的M-洗涤缓冲液，全速(>10,000x g)离心30秒，弃收集管中的液体。8) Centrifuge the sample obtained in step 7) at full speed (>10,000x g) for 30 seconds and discard the collected liquid in the collection tube. Continue to add 100μL of M-wash buffer to the centrifuge column, centrifuge at full speed (>10,000x g) for 30 seconds and discard the liquid in the collection tube.
9)向步骤8)获得的离心柱中添加200μL的M-Desulphonation Buffer，室温放置15min，全速(>10,000x g)离心30s，弃收集管中的液体。9) Add 200 μL of M-Desulphonation Buffer to the centrifuge column obtained in step 8), incubate at room temperature for 15 min, centrifuge at full speed (>10,000 x g) for 30 s, and discard the liquid in the collection tube.
10)继续向离心柱中添加200μL的M-洗涤缓冲液，全速(>10,000x g)离心30s，弃收集管中的液体，并再重复此步骤1次。10) Add 200 μL of M-wash buffer to the centrifuge column, centrifuge at full speed (>10,000 x g) for 30 seconds, discard the liquid in the collection tube, and repeat this step once more.
11)将步骤10)获得的Zymo-Spin IC TMColumn置于新的1.5mL EP管中，加入40μL的M-洗脱缓冲液r到柱基质中，室温放置2min，全速(>10,000x g)离心洗脱目的DNA。 11) Place the Zymo-Spin IC TM Column obtained in step 10) in a new 1.5 mL EP tube, add 40 μL of M-elution buffer r to the column matrix, place at room temperature for 2 minutes, and centrifuge at full speed (>10,000 x g) to elute the target DNA.
1.2 DNA甲基化检测引物的设计1.2 Design of primers for DNA methylation detection
依据实验1.1获得的甲基化DNA进行引物设计，其设计方法如图1-3所示，具体实验步骤如下：Primers were designed based on the methylated DNA obtained in Experiment 1.1. The design method is shown in Figure 1-3. The specific experimental steps are as follows:
i)针对甲基化的基因组序列设计一个或多个特异性引物对(F/R)，每对引物负责一个目标区域的扩增，每对引物中的一条引物(正向引物F或反向引物R)的5’端加上一个固定的通用序列T1(如图1所示)；i) designing one or more specific primer pairs (F/R) for the methylated genomic sequence, each pair of primers is responsible for amplifying a target region, and a fixed universal sequence T1 is added to the 5' end of one primer (forward primer F or reverse primer R) in each pair of primers (as shown in FIG. 1 );
ii)在第一轮特异性引物的基础上设计一条半巢式特异性引物F1或R1，该引物的结合位置设置在步骤i)中所述的第一轮特异性扩增引物的内侧(如图1所示)，其中，该条半巢式特异性引物的5’端引入另一个通用序列(T2，T2和T1的序列可以相同也可以不同)；ii) designing a semi-nested specific primer F1 or R1 based on the first-round specific primer, the binding position of which is set inside the first-round specific amplification primer described in step i) (as shown in FIG1 ), wherein another universal sequence (T2, the sequences of T2 and T1 may be the same or different) is introduced into the 5′ end of the semi-nested specific primer;
iii)设计一条通用引物U1，U1的3’端序列包含T1的所有序列(U1≥T1)。所有带T2序列的特异性引物和一条通用引物(U1)对第一轮的PCR产物进行第二轮多重扩增，获得带有T2序列和U1通用引物序列的目标片段(如图2所示)；或者iii) Design a universal primer U1, whose 3' end sequence contains all the sequences of T1 (U1 ≥ T1). All specific primers with T2 sequence and a universal primer (U1) are used to perform a second round of multiplex amplification on the PCR products of the first round to obtain the target fragment with T2 sequence and U1 universal primer sequence (as shown in Figure 2); or
iv)设计一条通用引物U1，U1的3’端序列包含上述T1的所有序列(U1≥T1)。再设计一条通用引物U2，U2的3’端序列包含上述T2的所有序列(U2≥T2)。所有5’端带T2序列的特异性引物、通用引物U1和通用引物U2对第一轮的PCR产物进行第二轮多重扩增，获得带有U2序列和U1通用引物序列的目标片段(如图3所示)；其中，所述T1、T2、U1和U2引物序列中可以引入样本标签序列、分子标签序列、化学修饰(磷酸化、氨基化等)、酶切位点(USER酶、限制性内切酶)等，用于后续不同的实验流程。本实施例中最终获得的引物序列如表1和表2所示。iv) Design a universal primer U1, the 3' end sequence of U1 contains all the sequences of T1 above (U1 ≥ T1). Design a universal primer U2, the 3' end sequence of U2 contains all the sequences of T2 above (U2 ≥ T2). All specific primers with T2 sequences at the 5' end, universal primer U1 and universal primer U2 perform a second round of multiple amplification on the PCR products of the first round to obtain a target fragment with U2 sequence and U1 universal primer sequence (as shown in Figure 3); wherein, sample tag sequences, molecular tag sequences, chemical modifications (phosphorylation, amination, etc.), enzyme cutting sites (USER enzymes, restriction endonucleases), etc. can be introduced into the T1, T2, U1 and U2 primer sequences for subsequent different experimental processes. The primer sequences finally obtained in this embodiment are shown in Tables 1 and 2.
表1：甲基化特异性引物Table 1: Methylation-specific primers
Figure PCTCN2022138685-appb-000001
Figure PCTCN2022138685-appb-000001
Figure PCTCN2022138685-appb-000002
Figure PCTCN2022138685-appb-000002
Figure PCTCN2022138685-appb-000003
Figure PCTCN2022138685-appb-000003
注：所有引物以10uM的初始浓度进行混合，得到甲基化特异性引物池。Note: All primers were mixed at an initial concentration of 10uM to obtain a methylation-specific primer pool.
表2：甲基化半巢式特异性引物Table 2: Methylation semi-nested specific primers
Figure PCTCN2022138685-appb-000004
Figure PCTCN2022138685-appb-000004
Figure PCTCN2022138685-appb-000005
Figure PCTCN2022138685-appb-000005
注：所有引物以10μM的初始浓度进行混合，得到半巢式甲基化特异性引物池。Note: All primers were mixed at an initial concentration of 10 μM to obtain a semi-nested methylation-specific primer pool.
1.3第一轮PCR1.3 First round of PCR
1)在PCR管中按照表3所示的反应体系配置。1) Prepare the reaction system in a PCR tube according to Table 3.
表3：table 3:
组份Component 体积(μL)Volume (μL)
1.1部分获得的甲基化DNA1.1 Methylated DNA obtained 2020
2×KAPA2G Fast ReadyMix2×KAPA2G Fast ReadyMix 2525
DNA甲基化特异性引物池(10μM)DNA methylation specific primer pool (10 μM) 5.05.0
总体积 total capacity 5050
2)第一轮PCR反应条件如下：2) The first round of PCR reaction conditions are as follows:
Figure PCTCN2022138685-appb-000006
Figure PCTCN2022138685-appb-000006
3)反应完后的产物用1.5X AMPure磁珠进行纯化，最后将纯化产物溶于22μL洗脱缓冲液中。3) The reaction product was purified using 1.5X AMPure magnetic beads and then dissolved in 22 μL elution buffer.
1.4第二轮PCR1.4 Second round of PCR
1)在PCR管中按照表4所示的反应体系配置，其中，通用引物的具体序列如表5所示。1) The reaction system shown in Table 4 was configured in a PCR tube, wherein the specific sequences of the universal primers are shown in Table 5.
表4：Table 4:
组分Components 体积(μL)Volume (μL)
1.3部分获得的纯化DNAPurified DNA obtained in section 1.3 17.517.5
2×KAPA2G Fast ReadyMix2×KAPA2G Fast ReadyMix 2525
DNA甲基化半巢式引物池(10μM)DNA methylation semi-nested primer pool (10 μM) 2.52.5
第一通用引物U1(10μM)First universal primer U1 (10 μM) 2.52.5
第二通用引物U2(10μM)Second universal primer U2 (10 μM) 2.52.5
总体积 total capacity 5050
表5：通用引物和标签引物Table 5: Universal primers and index primers
ID编号ID Number SEQ(5’-3’)SEQ(5'-3')
U1U1 TGTGAGCCAAGGAGTTGBBBBBBBBBB*TTGTCTTCCTAAGACCGCTTGGCCTCCGACTTTGTGAGCCAAGGAGTTGBBBBBBBBBB*TTGTCTTCCTAAGACCGCTTGGCCTCCGACTT
 The (SEQ ID NO:136)(SEQ ID NO:136)
U2U2 PHOS#GAACGACATGGCTACGATCCGACTT(SEQ ID NO:137)PHOS#GAACGACATGGCTACGATCCGACTT (SEQ ID NO:137)
2)第二轮PCR反应条件如下：2) The second round of PCR reaction conditions are as follows:
Figure PCTCN2022138685-appb-000007
Figure PCTCN2022138685-appb-000007
3)第二轮PCR反应完后获得的产物用1.0X AMPure磁珠进行纯化，最后将纯化产物溶于22μL洗脱缓冲液。3) The product obtained after the second round of PCR reaction was purified using 1.0X AMPure magnetic beads, and finally the purified product was dissolved in 22 μL elution buffer.
1.5文库检测：1.5 Library detection:
将实验1.3和1.4部分PCR扩增反应后获得的产物通过Bioanalyzer分析系统(Agilent,Santa Clara,USA)检测文库插入片段的大小及含量，检测方法为本领域常规方法，具体结果如附图4所示。获得的文库满足上机要求。The products obtained after the PCR amplification reaction in Experiment 1.3 and 1.4 were tested for the size and content of the library insert fragments by the Bioanalyzer analysis system (Agilent, Santa Clara, USA). The detection method was conventional in the art, and the specific results are shown in Figure 4. The obtained library met the requirements for the machine.
1.6上机测序1.6 Sequencing
将得到的文库进行高通量测序，测序平台为MGISEQ-2000，测序类型PE100，测序后数据经过比对后统计各项基本参数，包括下机数据、可用数据、比对率、特异性、均一性等。The obtained library was subjected to high-throughput sequencing, the sequencing platform was MGISEQ-2000, and the sequencing type was PE100. After sequencing, the data were aligned and various basic parameters were statistically analyzed, including offline data, available data, alignment rate, specificity, uniformity, etc.
1.6.1结果分析：1.6.1 Result analysis:
1)扩增方法得到的基本性能参数如表6所示，其中，数据利用率为0.97～0.99，唯一比对率为0.91～0.93，目标区域比例为0.95～0.97，0.1X平均深度均一性为0.92～0.96；1) The basic performance parameters obtained by the amplification method are shown in Table 6, where the data utilization rate is 0.97-0.99, the unique alignment rate is 0.91-0.93, the target area ratio is 0.95-0.97, and the 0.1X average depth uniformity is 0.92-0.96;
2)根据图5显示的结果，在不同投入量的情况下，甲基化检测准确性都有非常好的一致性(皮尔森系数>0.87)，一定条件下投入量越高，稳定性越好。2) According to the results shown in Figure 5, under different input amounts, the accuracy of methylation detection has very good consistency (Pearson coefficient>0.87). Under certain conditions, the higher the input amount, the better the stability.
表6：甲基化多重PCR测序数据统计Table 6: Methylation multiplex PCR sequencing data statistics
Figure PCTCN2022138685-appb-000008
Figure PCTCN2022138685-appb-000008
注： @表示统计方式为Mean±SD。 Note: @ indicates the statistical method is Mean±SD.
实施例2 DNA和甲基化DNA多重PCR混合建库测序Example 2 DNA and methylated DNA multiplex PCR mixed library construction and sequencing
本实施例主要包括以下实验内容：使用NA12878gDNA进行实验，gDNA经过化学转化(亚硫酸氢盐)或酶转化(TET酶辅助的转化)，将未甲基化的胞嘧啶转化为尿嘧啶，设计一个包含24个甲基化位点的甲基化捕获panel；同时设计一个没有进行甲基化的DNA捕获panel，包含20个DNA捕获区域。将NA12878标准品的gDNA进行重亚硫酸盐处理，然后混合基因组DNA，按照发明的步骤对混合DNA进行DNA和DNA甲基化混合文库制备，样本重复3次，将获得的文库置于MGISEQ-2000测序仪上进行上机测序，测序类型PE100，然后进行数据分析，包括数据利用率、比对率、扩增子特异性、均一性等性能，具体的实验操作如下：This embodiment mainly includes the following experimental contents: NA12878 gDNA is used for the experiment. The gDNA is converted into uracil by chemical conversion (bisulfite) or enzymatic conversion (TET enzyme-assisted conversion) to convert unmethylated cytosine. A methylation capture panel containing 24 methylation sites is designed; at the same time, a DNA capture panel without methylation is designed, containing 20 DNA capture regions. The gDNA of the NA12878 standard is treated with bisulfite, and then mixed with genomic DNA. The mixed DNA is subjected to DNA and DNA methylation mixed library preparation according to the steps of the invention. The sample is repeated 3 times, and the obtained library is placed on the MGISEQ-2000 sequencer for on-machine sequencing. The sequencing type is PE100, and then data analysis is performed, including data utilization, alignment rate, amplicon specificity, uniformity and other performances. The specific experimental operation is as follows:
2.1重亚硫酸盐处理法获得甲基化目的DNA2.1 Bisulfite treatment to obtain methylated target DNA
采用EZ DNA Methylation-Gold Kit TM(ZYMO)进行实验，将样品NA12878 gDNA设置3个重复进行 重亚硫酸盐共处理，具体步骤如下： The experiment was conducted using the EZ DNA Methylation-Gold Kit TM (ZYMO). Sample NA12878 gDNA was set up for three replicates for bisulfite co-treatment. The specific steps are as follows:
1)制备CT转换试剂(CT Conversion Reagent)溶液：从上述试剂盒中取出CT转换试剂(固体混合物)，向所述CT转换试剂中分别加入900μL的水、50μL的M-溶解缓冲液(M-Dissolving Buffer)和300μL的M-稀释缓冲液(M-Dilution Buffer)，室温下溶解并且震荡10分钟或在摇床上摇动10分钟。1) Prepare CT conversion reagent solution: Take out CT conversion reagent (solid mixture) from the above kit, add 900 μL of water, 50 μL of M-dissolving buffer and 300 μL of M-dilution buffer to the CT conversion reagent, dissolve and shake at room temperature for 10 minutes or shake on a shaker for 10 minutes.
2)M-洗涤缓冲液的制备：向M-洗涤缓冲液中添加24mL 100％的乙醇，备用。2) Preparation of M-wash buffer: Add 24 mL of 100% ethanol to M-wash buffer and set aside.
3)在PCR管中加入130μL的CT转换试剂溶液和10ng上述DNA，轻弹或移液器吹悬混合样品。3) Add 130 μL of CT conversion reagent solution and 10 ng of the above DNA into a PCR tube, and flick or pipette to suspend and mix the sample.
4)将上述处理后的PCR管放到PCR仪上按以下步骤进行操作：98℃下持续5分钟，64℃下持续2.5小时。4) Place the treated PCR tube on a PCR instrument and perform the following steps: 98°C for 5 minutes and 64°C for 2.5 hours.
5)完成上述操作后，PCR产物立刻进行下一步操作或者在4℃下存储(最多20小时)备用。5) After completing the above operations, the PCR product can be immediately used for the next step or stored at 4°C (up to 20 hours) for future use.
6)将Zymo-Spin IC TMColumn(离心柱)放入收集管(Collection Tube)中，并加入600μL的M-结合缓冲液(M-Binding Buffer)。 6) Place the Zymo-Spin IC ™ Column into a collection tube and add 600 μL of M-Binding Buffer.
7)将重亚硫酸盐处理的样品加入到含M-结合缓冲液的Zymo-Spin IC TMColumn中，盖上盖子颠倒混匀。 7) Add the bisulfite-treated sample to the Zymo-Spin IC TM Column containing M-binding buffer, cover the column and invert to mix.
8)将步骤7)获得的样品进行全速(>10,000x g)离心30秒，弃收集管中的收集液。继续向离心柱中加入100μL的M-洗涤缓冲液，全速(>10,000x g)离心30秒，弃收集管中的液体。8) Centrifuge the sample obtained in step 7) at full speed (>10,000x g) for 30 seconds and discard the collected liquid in the collection tube. Continue to add 100μL of M-wash buffer to the centrifuge column, centrifuge at full speed (>10,000x g) for 30 seconds and discard the liquid in the collection tube.
9)向步骤8)获得的离心柱中添加200μL的M-Desulphonation Buffer，室温放置15min，全速(>10,000x g)离心30s，弃收集管中的液体。9) Add 200 μL of M-Desulphonation Buffer to the centrifuge column obtained in step 8), incubate at room temperature for 15 min, centrifuge at full speed (>10,000 x g) for 30 s, and discard the liquid in the collection tube.
10)继续向离心柱中添加200μL的M-洗涤缓冲液，全速(>10,000x g)离心30s，弃收集管中的液体，并再重复此步骤1次。10) Add 200 μL of M-wash buffer to the centrifuge column, centrifuge at full speed (>10,000 x g) for 30 seconds, discard the liquid in the collection tube, and repeat this step once more.
11)将步骤10)获得的Zymo-Spin IC TMColumn置于新的1.5mL EP管中，加入40μL的M-洗脱缓冲液r到柱基质中，室温放置2min，全速(>10,000x g)离心洗脱甲基化的目的DNA。 11) Place the Zymo-Spin IC TM Column obtained in step 10) in a new 1.5 mL EP tube, add 40 μL of M-elution buffer r to the column matrix, place at room temperature for 2 minutes, and centrifuge at full speed (>10,000 x g) to elute the methylated target DNA.
12)将步骤11)获得的甲基化DNA和5ng的基因组DNA进行混合，以该混合DNA作为模板进行后续的文库制备。12) The methylated DNA obtained in step 11) was mixed with 5 ng of genomic DNA, and the mixed DNA was used as a template for subsequent library preparation.
2.2 DNA和甲基化DNA检测引物的设计2.2 Design of primers for DNA and methylated DNA detection
依据实验2.1获得的未甲基化的DNA和甲基化DNA进行引物设计，具体实验步骤如下：Primers were designed based on the unmethylated DNA and methylated DNA obtained in Experiment 2.1. The specific experimental steps are as follows:
i)第一轮特异性引物设计：针对原始基因组DNA序列或甲基化的基因组DNA序列设计一对或多对特异性引物(F/R)，每对引物负责一个原始DNA或甲基化DNA基因组目标区域的扩增，每对引物中的一条引物(F或R)的5’端加上一个固定的通用序列T1。所有带T1序列的引物和所有不带T1的引物在一管中进行单重或多重PCR扩增，获得带有T1接头的目标产物；i) First round of specific primer design: One or more pairs of specific primers (F/R) are designed for the original genomic DNA sequence or the methylated genomic DNA sequence. Each pair of primers is responsible for the amplification of a target region of the original DNA or methylated DNA genome. A fixed universal sequence T1 is added to the 5' end of one primer (F or R) in each pair of primers. All primers with T1 sequence and all primers without T1 are amplified by single or multiplex PCR in one tube to obtain the target product with T1 adapter;
ii)在第一轮特异性引物的基础上设计一条半巢式特异性引物F1或R1，该引物的位置在第一轮特异性扩增引物的内侧，在该条半巢式特异性引物的5’端引入另一个通用序列T2，(T2和T1的序列可以相同也可以不同)；ii) designing a semi-nested specific primer F1 or R1 based on the first-round specific primers, the position of the primer is inside the first-round specific amplification primer, and introducing another universal sequence T2 at the 5' end of the semi-nested specific primer (the sequences of T2 and T1 may be the same or different);
iii)设计一条通用引物U1，U1的3’端序列包含T1的所有序列(U1≥T1)。所有带T2序列的特异性引物和一条通用引物(U1)对第一轮的PCR产物进行多重扩增，获得带有T2序列和U1通用引物的目标片段；T1、T2和U1引物序列中可以引入测序接头序列、样本标签序列、分子标签序列、化学修饰(磷酸化、氨基化等)、酶切位点(USER酶、限制性内切酶)等，用于后续不同的分子学实验。iii) Design a universal primer U1, the 3' end sequence of U1 contains all the sequences of T1 (U1 ≥ T1). All specific primers with T2 sequence and a universal primer (U1) are used to perform multiple amplification on the first round of PCR products to obtain the target fragment with T2 sequence and U1 universal primer; sequencing adapter sequence, sample tag sequence, molecular tag sequence, chemical modification (phosphorylation, amination, etc.), enzyme cutting site (USER enzyme, restriction endonuclease), etc. can be introduced into the T1, T2 and U1 primer sequences for subsequent different molecular experiments.
iv)再设计一条通用引物U2，U2的3’端序列包含T2的所有序列(U2≥T2)。所有5’端带T2序列的特异性引物、通用引物U1和通用引物U2对本实施例中第一轮PCR扩增反应产物进行多重扩增，获得带有U2序列和U1通用引物序列的目标片段，具体的引物设计以及使用过程如表7所示；T1、T2、U1和U2引物序列中可以引入测序接头序列、样本标签序列、分子标签序列、化学修饰(磷酸化、氨基化等)、酶切位点(USER酶、限制性内切酶)等，用于后续不同的分子学实验。最终获得的甲基化DAN特异性引物序列F/R和甲基化DNA半巢式引物R1如表1和表2所示，获得的DNA特异性引物序列F/R和DNA半巢式引物R1分别如表7和表8所示。iv) Design another universal primer U2, the 3' end sequence of U2 contains all the sequences of T2 (U2 ≥ T2). All specific primers with T2 sequences at the 5' end, universal primer U1 and universal primer U2 perform multiple amplification on the products of the first round of PCR amplification reaction in this embodiment to obtain target fragments with U2 sequences and U1 universal primer sequences. The specific primer design and use process are shown in Table 7; sequencing adapter sequences, sample tag sequences, molecular tag sequences, chemical modifications (phosphorylation, amination, etc.), enzyme cutting sites (USER enzymes, restriction endonucleases), etc. can be introduced into the T1, T2, U1 and U2 primer sequences for subsequent different molecular experiments. The methylated DNA specific primer sequence F/R and the methylated DNA semi-nested primer R1 finally obtained are shown in Tables 1 and 2, and the obtained DNA specific primer sequence F/R and DNA semi-nested primer R1 are shown in Tables 7 and 8, respectively.
表7：DNA特异性引物Table 7: DNA specific primers
Figure PCTCN2022138685-appb-000009
Figure PCTCN2022138685-appb-000009
Figure PCTCN2022138685-appb-000010
Figure PCTCN2022138685-appb-000010
Figure PCTCN2022138685-appb-000011
Figure PCTCN2022138685-appb-000011
注：所有引物以10μM的初始浓度进行混合，得到特异性引物池。Note: All primers were mixed at an initial concentration of 10 μM to obtain a specific primer pool.
表8：DNA半巢式特异性引物Table 8: DNA semi-nested specific primers
Figure PCTCN2022138685-appb-000012
Figure PCTCN2022138685-appb-000012
Figure PCTCN2022138685-appb-000013
Figure PCTCN2022138685-appb-000013
注：所有引物10μM混合，得到半巢式特异性引物池。Note: All primers were mixed at 10 μM to obtain a semi-nested specific primer pool.
2.3第一轮PCR2.3 First round of PCR
1)于PCR管中配置表7所示的反应体系1) Prepare the reaction system shown in Table 7 in a PCR tube
表9：Table 9:
组分Components 体积(μL)Volume (μL)
上一步处理后的DNA+不处理的基因组DNADNA treated in the previous step + untreated genomic DNA 2020
2 X KAPA2G Fast ReadyMix2 X KAPA2G Fast ReadyMix 2525
DNA甲基化特异性引物池(10μM)DNA methylation specific primer pool (10 μM) 2.52.5
DNA特异性引物池(10μM)DNA specific primer pool (10 μM) 2.52.5
总体积 total capacity 5050
2)第一轮PCR反应条件如下：2) The first round of PCR reaction conditions are as follows:
Figure PCTCN2022138685-appb-000014
Figure PCTCN2022138685-appb-000014
3)第一轮PCR反应完后获得的产物用1.5X AMPure磁珠进行纯化，最后将纯化产物溶于22μL洗脱缓冲液。3) The product obtained after the first round of PCR reaction was purified using 1.5X AMPure magnetic beads, and finally the purified product was dissolved in 22 μL elution buffer.
2.4第二轮PCR2.4 Second round of PCR
1)在PCR管中按照表10配置反应体系1) Prepare the reaction system in the PCR tube according to Table 10
表10：Table 10:
组分Components 体积(μL)Volume (μL)
2.2部分获得的纯化DNAPurified DNA obtained in section 2.2 17.517.5
2 X KAPA2G Fast ReadyMix2 X KAPA2G Fast ReadyMix 2525
DNA甲基化半巢式引物池(10μM)DNA methylation semi-nested primer pool (10 μM) 1.251.25
DNA半巢式引物池(10μM)DNA semi-nested primer pool (10 μM) 1.251.25
第一通用引物U1(10μM)First universal primer U1 (10 μM) 2.52.5
第二通用引物U2(10μM)Second universal primer U2 (10 μM) 2.52.5
总体积 total capacity 5050
2)PCR反应条件2) PCR reaction conditions
Figure PCTCN2022138685-appb-000015
Figure PCTCN2022138685-appb-000015
3)反应完后用1.0X AMPure磁珠进行纯化，最后将纯化产物溶于22μl洗脱缓冲液。3) After the reaction, purify with 1.0X AMPure magnetic beads and finally dissolve the purified product in 22μl elution buffer.
2.5文库检测：2.5 Library detection:
将实验2.3和2.4部分PCR后获得的产物通过Bioanalyzer分析系统(Agilent,Santa Clara,USA)检测文库插入片段的大小及含量，检测方法为本领域常规方法，具体结果如附图7所示。获得恶的文库满足上机要求。The products obtained after PCR in Experiment 2.3 and 2.4 were tested for the size and content of the library insert fragments using a Bioanalyzer analysis system (Agilent, Santa Clara, USA). The detection method was a conventional method in the art, and the specific results are shown in Figure 7. The obtained library met the requirements for the machine.
2.6上机测序2.6 Sequencing
将得到的文库进行高通量测序，测序平台MGISEQ-2000，测序类型PE100,测序后数据经过比对后统计各项基本参数，包括下机数据、可用数据、比对率、GC含量等。The obtained library was subjected to high-throughput sequencing using the sequencing platform MGISEQ-2000 and the sequencing type PE100. The sequencing data was aligned and the basic parameters were statistically analyzed, including offline data, available data, alignment rate, GC content, etc.
2.6.1结果：2.6.1 Results:
1)混合测序方案得到的甲基化测序基本性能参数如表11所示，数据利用率为0.99±0.00，整体比对率0.98±0.02，特异性0.96±0.00，均一性0.96±0.00，和DNA甲基化得到的甲基化参数无差异，DNA扩增不影响DNA甲基化的扩增；1) The basic performance parameters of methylation sequencing obtained by the hybrid sequencing scheme are shown in Table 11, with data utilization rate of 0.99±0.00, overall alignment rate of 0.98±0.02, specificity of 0.96±0.00, and uniformity of 0.96±0.00. There is no difference in methylation parameters obtained by DNA methylation, and DNA amplification does not affect the amplification of DNA methylation;
2)混合测序方案得到的DNA测序基本性能参数如表12所示，数据利用率为0.99±0.00，整体比对率为0.98±0.02，特异性为0.98±0.00，均一性为1.00±0.00，可以实现DNA靶向的有效检测。2) The basic performance parameters of DNA sequencing obtained by the hybrid sequencing scheme are shown in Table 12. The data utilization rate is 0.99±0.00, the overall alignment rate is 0.98±0.02, the specificity is 0.98±0.00, and the uniformity is 1.00±0.00, which can achieve effective detection of DNA targeting.
表11：DNA甲基化数据统计Table 11: DNA methylation data statistics
Figure PCTCN2022138685-appb-000016
Figure PCTCN2022138685-appb-000016
表12：DNA数据统计Table 12: DNA data statistics
Figure PCTCN2022138685-appb-000017
Figure PCTCN2022138685-appb-000017
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (15)

  1. 一种多重PCR的方法，其特征在于，包括：A multiplex PCR method, comprising:
    第一轮扩增反应1，所述第一轮扩增反应1的体系中包括：转化DNA和一个或多个转化DNA引物对，其中，每个所述转化DNA引物对中包含一条正向引物和一条反向引物，每个所述转化DNA引物对中所述正向引物或反向引物的5’端具有通用序列1；A first round of amplification reaction 1, wherein the system of the first round of amplification reaction 1 comprises: a conversion DNA and one or more conversion DNA primer pairs, wherein each of the conversion DNA primer pairs comprises a forward primer and a reverse primer, and the 5' end of the forward primer or the reverse primer in each of the conversion DNA primer pairs has a universal sequence 1;
    第二轮扩增反应1，所述第二轮扩增反应1的体系中包括：所述第一轮扩增反应1产物和一个或多个引物组，其中，每个所述引物组包括：1)半巢式引物1，具有与所述第一轮扩增反应1产物的一个或多个目标区域结合的3’端和具有通用序列2的5’端，所述半巢式引物1设置在所述第一轮扩增反应1的体系中不具有通用序列1的所述正向引物或反向引物的靠近目标区域位置，和2)具有3’端和所述通用序列1相同的通用引物1和/或3’端和所述通用序列2相同的通用引物2。A second round of amplification reaction 1, wherein the system of the second round of amplification reaction 1 includes: the product of the first round of amplification reaction 1 and one or more primer sets, wherein each of the primer sets includes: 1) a semi-nested primer 1 having a 3' end that binds to one or more target regions of the product of the first round of amplification reaction 1 and a 5' end that has a universal sequence 2, wherein the semi-nested primer 1 is arranged at a position close to the target region of the forward primer or reverse primer that does not have the universal sequence 1 in the system of the first round of amplification reaction 1, and 2) a universal primer 1 having a 3' end that is identical to the universal sequence 1 and/or a universal primer 2 having a 3' end that is identical to the universal sequence 2.
  2. 根据权利要求1所述的方法，其特征在于，所述转化DNA为胞嘧啶甲基化转化处理后的DNA；The method according to claim 1, characterized in that the conversion DNA is DNA that has been subjected to cytosine methylation conversion treatment;
    任选地，所述甲基化酶包括下列中的至少之一：TET甲基胞嘧啶双加氧酶2。Optionally, the methylase comprises at least one of the following: TET methylcytosine dioxygenase 2.
  3. 根据权利要求1或2所述的方法，其特征在于，所述通用序列1和通用序列2相同或不同。The method according to claim 1 or 2, characterized in that the universal sequence 1 and the universal sequence 2 are the same or different.
    任选地，所述转化DNA引物对中T碱基含量为10-70％；Optionally, the T base content in the conversion DNA primer pair is 10-70%;
    任选地，所述通用序列1、通用序列2、通用引物1和通用引物2中的至少之一包括测序接头序列、样本标签序列或分子标签序列。Optionally, at least one of the universal sequence 1, universal sequence 2, universal primer 1 and universal primer 2 comprises a sequencing adapter sequence, a sample tag sequence or a molecular tag sequence.
    任选地，所述甲基化转化处理DNA是将所述DNA进行重亚硫酸盐或者酶法辅助处理获得的；Optionally, the methylation conversion treated DNA is obtained by subjecting the DNA to bisulfite or enzyme-assisted treatment;
  4. 一种多重PCR的方法，其特征在于，包括以下步骤：A multiplex PCR method, characterized in that it comprises the following steps:
    第一轮扩增反应2，所述第一轮扩增反应2的体系中包括：原始DNA、转化DNA、一个或多个原始DNA引物对和一个或多个转化DNA引物对，其中，每个所述原始DNA引物对和转化DNA引物对中包含一条正向引物和一条反向引物，所述每个原始DNA引物对和转化DNA引物对中的正向引物或反向引物的5’端具有通用序列3；A first round of amplification reaction 2, wherein the system of the first round of amplification reaction 2 includes: original DNA, conversion DNA, one or more original DNA primer pairs and one or more conversion DNA primer pairs, wherein each of the original DNA primer pairs and conversion DNA primer pairs includes a forward primer and a reverse primer, and the 5' end of the forward primer or the reverse primer in each of the original DNA primer pairs and conversion DNA primer pairs has a universal sequence 3;
    第二轮扩增反应2，所述第二轮扩增反应2的体系中包括：所述第一轮扩增反应2产物和第二轮扩增引物组，其中，所述第二轮扩增引物组包括：i)半巢式引物2，具有与所述第一轮扩增反应2产物中的一个或多个目标区域结合的3’端和具有通用序列4的5’端，所述半巢式引物2设置在所述第一轮扩增体系2中不具有通用序列3的正向引物或反向引物的靠近目标区域位置，和ii)具有3’端和通用序列3相同的通用引物3和/或具有3’端和通用序列4相同的通用引物4。A second round of amplification reaction 2, wherein the system of the second round of amplification reaction 2 includes: the product of the first round of amplification reaction 2 and a second round of amplification primer set, wherein the second round of amplification primer set includes: i) a semi-nested primer 2 having a 3' end that binds to one or more target regions in the product of the first round of amplification reaction 2 and a 5' end that has a universal sequence 4, wherein the semi-nested primer 2 is arranged at a position close to the target region of a forward primer or a reverse primer that does not have a universal sequence 3 in the first round of amplification system 2, and ii) a universal primer 3 having a 3' end that is identical to the universal sequence 3 and/or a universal primer 4 having a 3' end that is identical to the universal sequence 4.
  5. 根据权利要求4所述的方法，其特征在于，所述转化DNA为胞嘧啶甲基化转化处理后的DNA；The method according to claim 4, characterized in that the conversion DNA is DNA that has been subjected to cytosine methylation conversion treatment;
    任选地，所述甲基化转化处理DNA是将所述DNA进行重亚硫酸盐或者酶法辅助处理获得的；Optionally, the methylation conversion treated DNA is obtained by subjecting the DNA to bisulfite or enzyme-assisted treatment;
    任选地，所述甲基化酶包括下列中的至少之一：TET甲基胞嘧啶双加氧酶2(TET2)。Optionally, the methylase comprises at least one of the following: TET methylcytosine dioxygenase 2 (TET2).
  6. 根据权利要求4或5所述的方法，其特征在于，所述通用序列3和通用序列4相同或不同；The method according to claim 4 or 5, characterized in that the universal sequence 3 and the universal sequence 4 are the same or different;
    任选地，所述转化DNA引物对的T碱基含量为10-70％；Optionally, the T base content of the conversion DNA primer pair is 10-70%;
    任选地，所述通用序列3、通用序列4、通用引物3和通用引物4中的至少之一包括测序接头序列、样本标签序列或分子标签序列。Optionally, at least one of the universal sequence 3, universal sequence 4, universal primer 3 and universal primer 4 comprises a sequencing adapter sequence, a sample tag sequence or a molecular tag sequence.
  7. 一种测序文库的构建方法，其特征在于，包括利用权利要求1-6任一项所述的多重PCR方法进行测序文库构建的步骤。A method for constructing a sequencing library, characterized in that it comprises the step of constructing a sequencing library using the multiplex PCR method described in any one of claims 1 to 6.
  8. 根据权利要求7所述的方法，其特征在于，还包括对所述多重PCR方法中的所述第一轮扩增反应1或2的产物进行纯化处理；The method according to claim 7, characterized in that it also includes purifying the product of the first round of amplification reaction 1 or 2 in the multiplex PCR method;
    任选地，所述纯化处理为磁珠纯化处理、乙醇沉淀纯化或管式试剂盒纯化。Optionally, the purification treatment is magnetic bead purification treatment, ethanol precipitation purification or tubular kit purification.
  9. 一种试剂盒，其特征在于，包括下列中的至少之一：A kit, characterized in that it comprises at least one of the following:
    一个或多个第一轮转化DNA引物对，所述第一轮转化DNA引物对具有一条正向引物和一条反向引物，其中，所述正向引物或反向引物的5’端具有通用序列1；One or more first-round conversion DNA primer pairs, wherein the first-round conversion DNA primer pair has a forward primer and a reverse primer, wherein the 5' end of the forward primer or the reverse primer has universal sequence 1;
    一个或多个第二轮转化DNA引物组，所述第二轮转化DNA引物组包括：1)半巢式引物1，具有与转化DNA目标区域结合的3’端和具有通用序列2的5’端，和2)具有3’端和所述通用序列1相同的通用引物1和/或3’端和所述通用序列2相同的通用引物2。One or more second-round conversion DNA primer sets, the second-round conversion DNA primer sets comprising: 1) semi-nested primer 1, having a 3' end that binds to the conversion DNA target region and a 5' end having a universal sequence 2, and 2) universal primer 1 having a 3' end identical to the universal sequence 1 and/or universal primer 2 having a 3' end identical to the universal sequence 2.
  10. 根据权利要求9所述的试剂盒，其特征在于，所述试剂盒进一步包括：The kit according to claim 9, characterized in that the kit further comprises:
    一个或多个第一轮原始DNA引物对，所述第一轮原始DNA引物对具有一条正向引物和一条反向引物，其中，所述正向引物或反向引物的5’端具有通用序列3；和One or more first-round original DNA primer pairs, wherein the first-round original DNA primer pairs have a forward primer and a reverse primer, wherein the 5' end of the forward primer or the reverse primer has a universal sequence 3; and
    一个或多个第二轮原始DNA引物组，所述第二轮原始DNA引物组包括：3)半巢式引物2，具有与原始DNA目标区域结合的3’端和具有通用序列4的5’端，和4)具有3’端和所述通用序列3相同的通用引物3和/或3’端和所述通用序列4相同的通用引物4。One or more second-round original DNA primer sets, the second-round original DNA primer sets include: 3) semi-nested primer 2, having a 3' end binding to the original DNA target region and a 5' end having a universal sequence 4, and 4) universal primer 3 having a 3' end identical to the universal sequence 3 and/or universal primer 4 having a 3' end identical to the universal sequence 4.
  11. 根据权利要求9所述的试剂盒，其特征在于，所述通用引物1和通用引物2的序列可以相同也可以不同；The kit according to claim 9, characterized in that the sequences of the universal primer 1 and the universal primer 2 may be the same or different;
    任选地，所述通用序列1、通用序列2、通用引物1和通用引物2中的至少之一包括但不限于测序接头序列、样本标签序列或分子标签序列。Optionally, at least one of the universal sequence 1, universal sequence 2, universal primer 1 and universal primer 2 includes but is not limited to a sequencing adapter sequence, a sample tag sequence or a molecular tag sequence.
  12. 权利要求9-11任一项所述的试剂盒在制备测序文库中的用途。Use of the kit according to any one of claims 9 to 11 in preparing a sequencing library.
  13. 一种对目标核酸分子进行测序和/或甲基化位点检测的方法，其特征在于，包括：A method for sequencing a target nucleic acid molecule and/or detecting methylation sites, comprising:
    1)针对所述目标核酸分子，根据权利要求7或8所述的方法构建测序文库；1) constructing a sequencing library for the target nucleic acid molecule according to the method of claim 7 or 8;
    2)针对所述测序文库进行测序，以便获得测序结果；和2) performing sequencing on the sequencing library to obtain sequencing results; and
    3)基于所述测序结果，确定所述目标核酸分子的核酸序列和/或所述核酸分子的甲基化位点。3) Based on the sequencing results, determining the nucleic acid sequence of the target nucleic acid molecule and/or the methylation site of the nucleic acid molecule.
  14. 一种甲基化相关疾病的检测方法，其特征在于，包括以下步骤：A method for detecting a methylation-related disease, characterized in that it comprises the following steps:
    i)针对受试者来源的核酸分子，利用权利要求7或8所述的方法，构建受试者测序文库；i) constructing a subject sequencing library for nucleic acid molecules derived from the subject using the method described in claim 7 or 8;
    ii)针对所述受试者测序文库进行测序，以便获得测序结果；ii) performing sequencing on the subject sequencing library to obtain sequencing results;
    iii)基于所述测序结果，确定所述受试者来源的核酸分子的甲基化位点；和iii) determining the methylation sites of the nucleic acid molecule derived from the subject based on the sequencing results; and
    iv)基于所述甲基化位点判断所述受试者是否患有甲基化相关疾病。iv) judging whether the subject suffers from a methylation-related disease based on the methylation site.
  15. 根据权利要求14所述的方法，其特征在于，所述甲基化相关疾病包括选自下列中的至少之一：心血管疾病、脑血管疾病、自身免疫性疾病、代谢性疾病和癌症；The method according to claim 14, characterized in that the methylation-related disease comprises at least one selected from the group consisting of: cardiovascular disease, cerebrovascular disease, autoimmune disease, metabolic disease and cancer;
    任选地，所述脑血管疾病为脑出血或脑卒中；Optionally, the cerebrovascular disease is cerebral hemorrhage or cerebral stroke;
    任选地，所述自身免疫性疾病为牛皮癣、红斑狼疮或银屑病；Optionally, the autoimmune disease is psoriasis, lupus erythematosus or psoriasis;
    任选地，所述代谢性疾病为肥胖、2型糖尿病、非酒精性脂肪肝或骨质疏松症；Optionally, the metabolic disease is obesity, type 2 diabetes, non-alcoholic fatty liver disease or osteoporosis;
    任选地，所述癌症为直肠癌、肺癌、肝癌、胃癌、胰腺癌或脑胶质瘤。Optionally, the cancer is colorectal cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer or brain glioma.
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