CN114717662A - Micro free DNA methylation library building method, kit and sequencing method - Google Patents
Micro free DNA methylation library building method, kit and sequencing method Download PDFInfo
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Abstract
The invention relates to the technical field of gene detection, in particular to a method, a kit and a sequencing method for establishing a micro free DNA methylation library. The database building method comprises the following steps: extracting and separating free DNA samples; performing double-end enzyme digestion trimming on the free DNA sample; connecting a linker sequence containing methylation and biotin modification to the 5' end of the free DNA fragment; performing magnetic bead capture and purification on free DNA fragments connected with the adaptor sequences; carrying out sulfite conversion by using a sulfite conversion solution; and (3) carrying out double-strand extension on the free DNA fragment after sulfite conversion and carrying out PCR amplification to obtain a DNA methylation library. The method can obviously increase the number of CG detection sites of free DNA, and has obvious covering effect on the CG sites. Compared with whole genome cfDNA methylation sequencing, the sequencing depth is obviously increased, the sequencing data volume is lower, and the cost is lower.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a method, a kit and a sequencing method for methylation library construction of trace free DNA.
Background
DNA methylation of 5mC is a methyltransferase mediated transfer of the methyl group in the methyl donor S-adenosylmethionine (SAM) molecule to DNA cytosine. In eukaryotes, DNA methylation occurs predominantly at the 5 carbon position of the CpG dinucleotide cytosine, forming 5-methylcytosine (5 mC). The DNA methylation modification plays an important role in the expression regulation of genes and is an important analysis marker for the occurrence and development of cancer and the treatment prognosis.
cfDNA is extracellular DNA (cell-free DNA), fragment lengths of usually around 150bp, as discovered by Mandel and M etais early in 1984. The isolated free DNA of a tumor patient is also called ctDNA because it is mostly derived from tumor cells, carrying information of all genetic variations of each diseased organ or tissue. Detection of cfDNA methylation, particularly marker screening and identification of ctDNA methylation applied to early tumor screening and patient prognosis risk assessment, has become one of the technical problems solved by scientists and enterprises in various countries around the world competing for time and time. The most important of them are the very low content of free DNA, short fragment length and large individual difference. Healthy individuals contain 0-100ng per ml of blood, with an average of 30ng/ml, and even patients with advanced tumors have an average of only 180ng/ml due to apoptosis and necrosis of a large number of tumor cells. In the industry, gene detection companies at home and abroad often fund hundreds of millions of dollars for development in order to quickly establish detection technologies with independent intellectual property rights, for example, GRAIL corporation in the United states finances more than 10 hundred million dollars for the development of layout free DNA in early screening of breast cancer. There are also several hundred million units in China that have completed financing and are beginning to develop relevant detection techniques.
The detection technology applied to the free DNA omics screening is mainly whole genome methylation sequencing, for example, the extracellular free DNA methylation characteristics published in 2020 and invested by Grail company are used for the research of multi-tumor detection and localization, and the key methylation markers are mainly identified through the whole genome methylation sequencing. However, since the fragment characteristic of free DNA is only about 140bp, even the 90G data is sequenced, the average depth is only 15X, the sequencing depth of most detection sites is less than 5X, the detection cost of a single sample is nearly ten thousand yuan, the cost is extremely high, and the large sample sequencing is difficult to perform by a general unit. In addition, cfMeDIP technology recently developed by the Margare Burgunner cancer center, Canada, can perform methylation sequencing on free DNA as low as 1ng, and the basic principle is to capture DNA fragments containing methylation modified cytosine bases through methylation specific antibodies, and determine the methylation relative content of different fragments of a genome by sequencing the captured fragments.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method, a kit and a sequencing method for methylation library construction of trace free DNA.
The technical scheme for solving the technical problems is as follows:
the invention provides a method for building a library by methylation of trace free DNA, which comprises the following steps:
1) extracting and separating free DNA samples;
2) performing double-end enzyme digestion and trimming on the free DNA sample to obtain a free DNA fragment;
3) connecting a linker sequence containing methylation and biotin modification to the 5' end of the free DNA fragment;
4) capturing and purifying the free DNA fragment connected with the linker sequence by using magnetic beads to obtain a product;
5) carrying out sulfite conversion on the product obtained in the step 4) by using a sulfite conversion solution;
6) carrying out two-chain extension on the product after the sulfite conversion;
7) and carrying out PCR amplification on the product of the two-strand extension to obtain a DNA methylation library.
Further, in the step 2), the enzyme used for double-ended enzyme cutting and trimming is one of MspI, AluI, BstNI, HpyCH4V, HaeIII, HpyCH4III, ApekI, BanII, SphI, BssSI, BglII, BamHI and KpnI.
Further, the linker sequence in step 3) is Ad1, and the step 3) further comprises the step of ligating Ad2 to the 3' end of the free DNA fragment; in the step 5), after sulfite conversion, Ad2 is bound on magnetic beads and separated from the free DNA fragments, and the free DNA fragments connected with Ad1 are transferred into the sulfite conversion solution.
Further, the linker sequence is designed based on the Illumina platform;
wherein, the nucleotide sequence of Ad1 is shown as SEQ ID NO 1, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 2; or the nucleotide sequence of Ad1 is shown as SEQ ID NO 3, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 4.
Further, the adaptor sequence is designed based on an MGI platform;
wherein, the nucleotide sequence of Ad1 is shown as SEQ ID NO 5, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 6; or, the nucleotide sequence of Ad1 is shown in SEQ ID NO 7, and the nucleotide sequence of Ad2 is shown in SEQ ID NO 8.
Further, the amount of the free DNA sample extracted and separated in the step 1) is 0.3-2 ng.
Further, in the step 6), the reagent for performing the double strand extension includes DNA Polymerase, which is T4 DNA Polymerase, Bst DNA Polymerase, Klenow Fragment (3 '-5' exo-), PCR Polymerase, Hemo KlenotaQ (NEB), Amplitaq DNA Polymerase, Stoffel Fragment (Life), and NEB(exo-) DNA polymerase,One kind of DNA polymerase.
Further, in the step 7), after PCR amplification is carried out on the product of the double-strand extension, the method also comprises a step of quality detection; the step of quality detection detects the length of the amplified product fragment, and when the length of the product fragment is 150bp-300bp, the obtained product is a DNA methylation library.
The invention provides a micro free DNA methylation library building kit, which is used for the micro free DNA methylation library building method;
comprises a free DNA sample extraction and separation reagent, an enzyme digestion reagent, a linker sequence containing methylation and biotin modification, a linker sequence connecting reagent, a sulfite conversion reagent, a double-strand extension reagent and a PCR amplification reagent.
The invention provides a free DNA sequencing method, which comprises the steps of firstly building a library by adopting the micro free DNA methylation library building method and then sequencing.
The invention has the beneficial effects that:
1) according to the method for building the library by methylation of the trace free DNA, almost all CCGG loci in the fragmented free DNA can be detected by sequencing by performing the joint on the 5' end of the free DNA fragment subjected to enzyme digestion, and CG methylation information on two sides of the loci is added.
2) According to the method for building the database by methylation of the trace free DNA, after the free DNA fragments are jointed, sulfite conversion is carried out, and conversion products are extended randomly, so that the input amount of the DNA is reduced, each sequencing sequence is ensured to contain methylation information of at least one CG locus, the loss of purification is reduced, the initial amount of the DNA is detected as much as possible, the aim of low initial amount is fulfilled, and the trace detection is realized while the accurate detection is realized.
3) According to the method for building the library by methylation of the trace free DNA, the free DNA sample size can be as low as less than 1ng, and compared with the prior art, the requirement on the sample size is greatly reduced.
4) The methylation library construction method of the trace free DNA can obviously increase the number of CG detection sites of the free DNA and has obvious covering effect on the CG sites.
5) Compared with whole genome cfDNA methylation sequencing, the free DNA sequencing method provided by the invention has the advantages that the sequencing depth is obviously increased, the sequencing data amount is lower, and the cost is lower.
Drawings
FIG. 1 is a chromatogram for detecting the extraction effect of cfDNA in step 1 of example 1 in the methylation database construction method for trace free DNA of the present invention;
FIG. 2 is a graph showing the number and ratio of 1X and 5X CG site coverage in the sequencing results of example 1, example 2 and comparative example 1 in the free DNA sequencing method of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
Because the length of the DNA fragments is obviously reduced due to degradation, the conventional simplified genome methylation sequencing is difficult to enrich enough CG sites and carry out methylation sequencing. Particularly, samples such as cfDNA and the like have the fragment length of about 150bp and are completely contained in the fragment range (40bp-300bp) detected by the simplified genome methylation technology. And the fragments containing CCGG sites are processed by a simplified genome methylation technology, effective fragments are further cut to be shorter and lost, and the fragments not containing the CCGG sites in the fragments are reserved and sequenced, so that effective data is wasted, and the detection number of CG sites is reduced.
The free DNA methylation library construction method provided by the invention can be used for constructing a library aiming at trace free DNA, and is based on the principle that the 5' end of a free DNA enzyme digestion product is connected with a joint, almost all CCGG sites subjected to enzyme digestion can be detected theoretically, and CG methylation information on two sides of the sites is increased. Compared with the whole genome free DNA methylation sequencing, the invention adopts a fragmentation treatment mode of firstly enzyme cutting and then jointing, so that the sequencing depth of free DNA methylation is obviously increased, the sequencing data volume is lower, and the cost is lower.
The invention relates to a method for building a library by methylation of trace free DNA, which comprises the following steps:
1) extracting and separating free DNA samples;
the library construction method mainly aims at free DNA in blood, namely cfDNA, or free DNA of circulating tumor cells in blood of a tumor patient, namely ctDNA; it can also be directed against normal tissue or cell genomic DNA, i.e., gDNA.
The free DNA sample can be from peripheral blood, cerebrospinal fluid, saliva, bronchial lavage fluid, pleural effusion, gastric juice, bile, feces, urine and the like.
Preferably, the cfDNA sample of the invention is derived from plasma.
The sample size of the invention can be lower than 1ng and can be as low as a single cell level, and the sample size of the invention is preferably between 0.3ng and 2 ng.
Wherein, the input amount of the DNA can be lower than 1ng or the single cell level. The extraction of the sample DNA can be carried out by selecting the existing commercial extraction kit or the laboratory self-built technology according to different sample properties.
Preferably, the elution of the finally extracted free DNA sample can only use ultrapure water or EB buffer solution, TE or a kit with unknown components cannot be used for elution, the elution volume of the sample does not exceed 10ul, and the buffer solution is prevented from interfering the free DNA sample.
Preferably, for cfDNA methylation detection, in order to avoid methylation signal interference of intracellular DNA, capillary electrophoresis may be performed after sample extraction to detect distribution characteristics of cfDNA fragments, fragments of cfDNA products with good extraction effects should be mainly distributed around 140bp, a small amount of DNA may exist around 280bp, and genomic DNA fragments should not exist above 1000 bp.
2) And (3) carrying out double-end trimming on the free DNA sample by using MspI enzyme to obtain a free DNA fragment.
The DNA fragments are subjected to double-end pruning under the action of MspI enzyme, preferably, the reaction system is 10-50ul, a fragment structure with CG base protruding from the 5' end is constructed, and the substrate is reacted for 20min-16h at 37 ℃.
The enzyme digestion system is specifically as follows:
3) the 5' end of the free DNA fragment is connected with a linker sequence containing methylation modification.
Adding a linker and DNA ligase which are connected with Buffer A, methylated and biotin jointly modified into the free DNA fragment obtained in the step 2), and reacting for 20min-6h at 16-30 ℃.
The linker sequence contains biotin modifications, which enable the magnetic beads in the subsequent steps to capture free DNA fragments better.
The reaction system is specifically as follows:
the Buffer A comprises
Wherein the linker sequences comprise Ad1 and Ad2, both of which are linked to the free DNA fragment;
the linker sequence is designed based on an Illumina platform or MGI platform; in the sequence designed based on the Illumina platform, the nucleotide sequence of Ad1 is shown as SEQ ID NO 1, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 2.
The nucleotide sequence of Ad1 in the sequence designed based on the MGI platform is shown as SEQ ID NO 5, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 6.
Preferably, the sequences designed based on the Illumina platform can also have the following alternatives: the nucleotide sequence of Ad1 is shown as SEQ ID NO. 3, and the nucleotide sequence of Ad2 is shown as SEQ ID NO. 4;
the sequences designed based on the MGI platform may also have the following alternatives: the nucleotide sequence of Ad1 is shown as SEQ ID NO 7, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 8.
The sequence designed based on the Illumina platform has two expression modes of SEQ ID NO 1, wherein the first expression mode is 5' -GGAGTTCAGACGTGTGCTCTTCCGATCT-3', wherein the base marked with a horizontal line is a methylated base; in a second way of presentation, the display is,
5 '-GGAGTTmCAGAmCGTGmCTTmCmCGATmCT-3', m is methyl, and indicates the subsequent base methylation.
SEQ ID NO 2.:5’-CGAGATCGGAAGAGCACACGTCTGAACTCC-Bio-3’。
In the linker sequence which may be substituted with an index, there are two representations of SEQ ID NO 3, the first representation being 5' -GGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNN-3', wherein the base marked with a horizontal line is a methylated base, and N is an arbitrary base; in the second expression, 5 '-GGAGTTmCAGAmCGTGTGmCTmCMCGATmCTNNNNNNNNNNN-3', m is a methyl group and indicates the methylation of the base located therebehind, and N is an arbitrary base.
SEQ ID NO 4.:5’-CGNNNNNNNNAGATCGGAAGAGCACACGTCTGAACTCC-Bio-3’。
In the sequence designed based on the MGI sequencing joint, SEQ ID NO 5 has two expression modes, the first expression mode is 5' -TTGTCTTCCTAAGGAACGACATGGCTACGATCCGACTT-3', wherein the underlined bases are methylated bases; in the second expression, 5 '-TTGTCTTCCTAAGGAAmCGAmCATGGmCTAmCGATmCCGAmCAMCTT-3', m is methyl, indicating the methylation of the base located therebehind.
SEQ ID NO 6.:
5’-CGAAGTCGGATCGTAGCCATGTCGTTCCTTAGGAAGACAA-Bio-3’。
In alternative linker sequences, SEQ ID NO 7 has two representations, the first being 5' -GAACGACATGGCTACGATCCGACTT-3', wherein the underlined bases are methylated bases; in the second expression, 5 '-GAAmCGAmCATGGmCTAmCGATmCMCGAmCTT-3' is methyl, which indicates the methylation of the base located after it.
SEQ ID NO 8.:5’-CGAAGTCGGATCGTAGCCATGTCGTTC-Bio-3’。
4) And capturing and purifying the free DNA fragments connected with the joint by using magnetic beads to obtain a magnetic bead-free DNA capturing mixed solution.
In the step, streptomycin-labeled magnetic beads are adopted to capture free DNA connection products connected with a joint modified by methylation and biotin together, and the capture products are resuspended in 20ul of ultrapure water. The specific operation steps are as follows:
first, 20-40ul of streptavidin-conjugated magnetic beads were taken and rinsed three times with 50ul of rinse solution, and the beads were resuspended in 15ul of EB buffer.
Secondly, adding the free DNA joint connection product into 15ul of magnetic beads, incubating for 10-30min at room temperature, and capturing the free DNA connection fragment by a magnetic frame;
finally, the beads were rinsed 2 times with 50ml of EB, and after the supernatant was removed by a magnetic stand, the beads were resuspended in 20ul of ultrapure water or 0.1M sodium hydroxide.
If the elution is carried out with ultrapure water, a magnetic bead-free DNA mixture is finally obtained in this step. If the magnetic beads are dissolved by replacing ultrapure water with strong alkali such as sodium hydroxide, the eluted supernatant is recovered and the beads are demagnetized to obtain a supernatant containing no magnetic beads, and the supernatant is directly subjected to the next sulfite conversion.
5) Carrying out sulfite conversion on the product obtained in the step 4).
Free DNA was sulphite transformed using a commercial kit.
If ultra pure water is used for elution in step 4), before the experiment according to the kit instructions, firstly, 20ul of the sulfite reaction solution with the recommended volume of the kit is added into the magnetic bead capture solution, denaturation is carried out for 1-10min at 98 ℃, free DNA is denatured from the magnetic beads, centrifugation is carried out at 16000rpm for 5-10min or a magnetic frame is kept still, and the supernatant is transferred to a new PCR tube.
If 0.1M sodium hydroxide is used for elution, the eluent is put on a magnetic frame, 20ul of supernatant is absorbed into a PCR tube, and sulfite reaction liquid with the volume recommended by the kit is added, and then sulfite conversion is directly carried out according to the kit specification. After the reaction, the free DNA after C-T conversion was eluted into 26ul of ultrapure water or EB.
After sulfite conversion by this step, the linker sequence Ad2 was bound to the magnetic beads and the cfDNA fragment ligated to the linker sequence Ad1 was denatured into sulfite conversion solution.
6) The sulfite-converted free DNA fragments were subjected to two-strand extension.
The following reagents were added to the sulfite-converted DNA PCR tubes, respectively, to extend the two strands of the sulfite-converted product.
Primer features were designed using Primer1 sequences, and primers containing the following features were used for duplex extension.
A primer sequence designed based on an Illumina sequencing platform is shown in SEQ ID NO 9, wherein n is a random base:
Oligo 1:5’-tacacgacgctcttccgatctnnnnnn-3’(SEQ ID NO 9.)。
a primer sequence designed based on an MGI sequencing platform is shown as SEQ ID NO 10, s is an index sequence, and n is a random base:
Oligo 1:5’-tgtgagccaaggagttgssssssssssttgtcttcctaagaccgcttggcctccgacttnnnnnn-3’(SEQ ID NO 10.)。
the specific operation steps are as follows:
mixing the reaction solution uniformly, placing the mixture in a PCR instrument, heating the mixture at 95 ℃ for 45s, quickly transferring the mixture to ice after reaction, standing the mixture for 5min, adding DNA polymerase Klenow (3 '→ 5' exo-)100U, mixing the mixture uniformly, placing the mixture on the PCR instrument, incubating the mixture for 5min at 4 ℃, raising the temperature to 37 ℃ according to the temperature rise rate of 0.1-1 ℃/s after incubation, then incubating the mixture for 1.5h at 37 ℃, purifying the mixture by using 1 time of magnetic beads after reaction, and eluting the purified mixture into 20ul of EB buffer solution.
The reactive DNA Polymerase includes, but is not limited to, T4 DNA Polymerase, Bst DNA Polymerase, Klenow Fragment (3 '-5' exo-), PCR Polymerase, Hemo KlenoTaq (NEB), Amplitaq DNA Polymerase, Stoffel Fragment (Life), and those of NEB(exo-) DNA polymerase,DNA polymerase, and the like.
In the library building method, after the enzyme digestion in the step 2), the single-end connection is carried out through the joint in the step 3), and after the sulfite conversion is completed in the steps 4) and 5), the random extension of the sulfite conversion product in the step is carried out.
7) PCR amplification is performed on the product of the two-strand extension.
The primers adopted in the step are primers with the following characteristics:
for the Illumina sequencing platform, the sequence characteristics of the upstream Primer (Primer1) and the downstream Primer (Primer2) of the PCR amplification are shown in SEQ ID NO 11 and SEQ ID NO 12, respectively, wherein n is a random base:
Primer 1:5′-aatgatacggcgaccaccgagatctacacnnnnnnnnacactctttccctacacgacgctcttccgatct-3’(SEQ ID NO 11.);
Primer 2:5′-caagcagaagacggcatacgagatnnnnnnnngtgactggagttcagacgtgtgc-3’(SEQ ID NO 12.)。
for the MGI sequencing platform, the sequence characteristics of the upstream Primer (Primer1) and the downstream Primer (Primer2) amplified by PCR are respectively shown in SEQ ID NO 13 and SEQ ID NO 14, wherein n is a random base, and the 5 end of the Primer2 is subjected to phosphorylation modification:
Primer 1:5’-tgtgagccaaggagttg-3’(SEQ ID NO 13.);
Primer 2:5Phos-gaacgacatggctacga-3’(SEQ ID NO 14.)。
the PCR amplification system of the step is as follows:
the PCR amplification conditions were as follows:
at 98 ℃ for 1 min; at 98 deg.C, 30s, 50-68 deg.C, 30s, 72 deg.C, 30s, 10-18 cycles; 72 ℃ and 5 mm.
After PCR amplification and purification, the mixture was eluted into 20ul of buffer.
After PCR amplification, the method also comprises a quality detection step; the step of quality detection detects the length of the amplified product fragment, and when the length of the product fragment is 150bp-300bp, the obtained product is a free DNA methylation library. If the length of the product fragment is not in the range, the library building fails, and the library building needs to be carried out again.
The invention also provides a DNA sequencing method, which is used for sequencing the obtained library after the DNA methylation library is established by adopting the steps.
The specific steps are that the DNA methylation library obtained by library construction by the library construction method is subjected to QPCR (quantitative polymerase chain reaction) to quantify the Mohr concentration of the sample DNA, capillary electrophoresis or 2100 bioanalyzer is used for detecting the size of the library fragment, the qualified fragment is 150bp-270bp, the corresponding molar library is measured according to the sequencing data, and the sequencing is carried out on a sequencer.
After sequencing, performing joint removal, low-quality value reads filtration and statistics, methylation conversion rate statistics, comparison and comparison rate statistics, genome coverage statistics and repetition rate statistics on off-line data respectively, and calculating the methylation level of each locus to obtain the CG locus coverage number.
The process of the invention is illustrated below by means of specific examples:
example 1
This example provides a specific library construction and sequencing procedure using the method of the invention. The specific process is as follows:
1) sample collection and cfDNA extraction: collecting 2-5ml of whole blood in an EDTA-containing blood collection tube or cfDNA blood collection tube, reversing and mixing uniformly, centrifuging 1600g, immediately centrifuging the upper layer of plasma part at 16000g and 4 ℃ for 10min, removing residual cells and debris, separating supernatant plasma into a new centrifuge tube, and storing at-80 ℃.
Plasma collection was extracted using a commercial cfDNA extraction kit. The kit is a DSP Blood Mini kit (Qiagen), the concentration of DNA is quantitatively extracted by the Qubit HS, the extraction effect of cfDNA is detected by Agilent 2100, and whether genome pollution exists is judged. When there is no genomic contamination, the next step is carried out.
Fig. 1 is a chromatogram for detecting the extraction effect of cfDNA, and it can be seen from fig. 1 that there is no genomic contamination in the cfDNA extracted in this example.
2) MspI enzyme digestion of cfDNA fragments: taking 1-2ng of free DNA without genome pollution, and carrying out CCGG site digestion according to the following system: MspI enzyme 50U, 10X buffer 1ul, water supplementing to total volume 10ul, and reacting at 37 ℃ for 2 hours.
3) cfDNA end linker sequence: 1uM of adaptor sequence Ad1 and Ad2 annealing product 1ul, 3.5ul of buffer A and 200U of DNA ligase are sequentially added into the cfDNA enzyme digestion system, mixed uniformly and incubated for 2 hours at 23 ℃.
The linker sequence used in this example is the nucleotide sequence of Ad1, i.e., the sequence shown in SEQ ID NO 1 and SEQ ID NO 2, in the sequence designed based on the Illumina platform.
4) Capturing a joint connection product: taking 40ul of streptomycin coupled magnetic beads, rinsing the streptomycin coupled magnetic beads for three times by using 50ul of rinsing liquid, and resuspending the magnetic beads in 15ul of EB buffer solution; secondly, adding the cfDNA adaptor ligation product into 15ul of magnetic beads, incubating for 30min at room temperature, and capturing the cfDNA ligation fragments by a magnetic frame; the beads were rinsed 2 times with 100ml EB, the supernatant was removed by a magnetic stand, and the beads were resuspended in 20ul ultrapure water
5) Linker Ad2 removal and sulfite treatment: EZ DNA Methylation-Gold from ZYMO Research was usedTMThe Kit sulfite conversion Kit was used for sulfite treatment.
The processing flow is as follows: adding 20ul of the mixed solution of the capture of the magnetic bead-cfDNA into 130ul of sulfite reaction solution, denaturing at 98 ℃ for 8min, centrifuging at 16000rpm for 10min, still binding the adapter Ad2 on the magnetic bead, denaturing the cfDNA into sulfite conversion solution, transferring the supernatant into a new PCR tube, and continuing the reaction on a PCR instrument according to the following procedures: at 98 ℃ for 2 min; after reaction at 64 ℃ for 2.5 hours, purification was carried out according to the kit instructions and eluted into 26ul of ultrapure water.
6) Performing double-strand extension on the cfDNA fragment after sulfite conversion: adding 5ul of 10 Xreaction buffer solution, 2ul of 10mM dNTP and 5ul of 20mM Oligo 1 into purified 26ul of converted DNA respectively, supplementing water to the total volume of 50ul, mixing the reaction solution uniformly, placing the mixture in a PCR instrument, heating at 95 ℃ for 45s, quickly transferring the mixture to ice after reaction, standing for 5min, adding DNA polymerase Klenow (3 '→ 5' exo-)100U, mixing uniformly, placing the mixture on the PCR instrument, incubating at 4 ℃ for 5min, raising the temperature to 37 ℃ according to the temperature raising rate of 1 ℃/s after incubation, then incubating at 37 ℃ for 1.5h, purifying by using 1-time DNA purification magnetic beads after reaction, and eluting to 20ul of EB buffer solution.
7) PCR amplification
In the PCR tube, a PCR reaction solution was prepared in the following system
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; at 95 ℃ for 30 s; at 58 ℃ for 30 s; performing 12 cycles of amplification at 72 deg.C for 45s, extending at 72 deg.C for 5min, and storing at 16 deg.C; after amplification of the PCR product, it was purified with 1-fold volume of Ampure XP Beads and eluted into 20ul of EB eluent.
After the library is constructed, fragment size detection and QPCR concentration quantitative detection are respectively carried out by using Agilent 2100, and Hiseq is used for sequencing after the library is qualified.
The library of this example was each subjected to two-sided sequencing at 1X and 5X sequencing depth, respectively.
Example 2
gDNA was pooled and sequenced using the same procedures and reagents as in example 1. The libraries of this example were each subjected to two-sided sequencing at 1X and 5X depth, respectively.
Comparative example 1
gDNA was pooled and sequenced using conventional simplified methylation methods. The libraries of this comparative example were each subjected to two-sided sequencing at 1X and 5X depth, respectively.
The number and ratio of CG site coverage at 1X and 5X sequencing depth in the sequencing results of examples 1 and 2 and comparative example 1, respectively, are examined, and the results are shown in FIG. 2.
As can be seen from the results of fig. 2, the 1xcg sites of examples 1 and 2 cover 16.2M and 13.3M, respectively, accounting for 23% and 24% of the total CG sites of the genome; whereas the conventional simplified genome methylation 1XCG site covers only about 10M and accounts for about 16% of the CG sites of the genome. The 5X CG site coverage was 10.97M and 8.83M, respectively; whereas the conventional simplified genomic methylation 1XCG site coverage is only around 5.56M.
Therefore, the library construction method and the sequencing method are suitable for detecting trace cfDNA or gDNA, and have obvious coverage effect on CG sites.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Shenzhen Shuyi Gene technology Limited
<120> micro free DNA methylation library building method, kit and sequencing method
<141> 2022-04-20
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Claims (10)
1. A method for building a library by methylation of trace free DNA is characterized by comprising the following steps:
1) extracting and separating free DNA samples;
2) performing double-end enzyme digestion and trimming on the free DNA sample to obtain a free DNA fragment;
3) connecting a linker sequence containing methylation and biotin modification to the 5' end of the free DNA fragment;
4) capturing and purifying the free DNA fragment connected with the linker sequence by using magnetic beads to obtain a product;
5) carrying out sulfite conversion on the product obtained in the step 4) by using a sulfite conversion solution;
6) carrying out two-chain extension on the product after the sulfite conversion;
7) and carrying out PCR amplification on the product of the two-strand extension to obtain a DNA methylation library.
2. The method for building a library through methylation of trace free DNA according to claim 1, wherein in the step 2), the enzyme used for double-ended enzyme cutting and trimming is one of MspI, AluI, BstNI, HpyCH4V, HaeIII, HpyCH4III, ApekI, BanII, SphI, BssSI, BglII, BamHI and KpnI.
3. The method for creating a library of trace free DNA methylation according to claim 1, wherein the linker sequence in the step 3) is Ad1, and the step 3) further comprises the step of ligating Ad2 to the 3' end of the free DNA fragment;
in the step 5), after sulfite conversion, Ad2 is bound on magnetic beads and separated from the free DNA fragments, and the free DNA fragments connected with Ad1 are transferred into the sulfite conversion solution.
4. The method for building a library of trace free DNA methylation according to claim 3, wherein the linker sequence is a sequence designed based on the Illumina platform;
wherein, the nucleotide sequence of Ad1 is shown as SEQ ID NO 1, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 2;
or the nucleotide sequence of Ad1 is shown as SEQ ID NO 3, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 4.
5. The method for building a library of trace free DNA methylation according to claim 3, wherein the linker sequence is a sequence designed based on MGI platform;
wherein, the nucleotide sequence of Ad1 is shown as SEQ ID NO 5, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 6;
or, the nucleotide sequence of Ad1 is shown in SEQ ID NO 7, and the nucleotide sequence of Ad2 is shown in SEQ ID NO 8.
6. The method for creating a library by methylation of trace free DNA according to any one of claims 1 to 5, wherein the amount of the free DNA sample extracted and separated in the step 1) is 0.3ng to 2 ng.
7. The method for creating a library by methylation of trace free DNA according to any one of claims 1 to 5, wherein in the step 6), the reagent for performing double-strand extension comprises DNA Polymerase, and the DNA Polymerase is T4 DNA Polymerase, Bst DNA Polymerase, Klenow Fragment (3 '-5' exo-), PCR Polymerase, Hemo KlenotaQ (NEB), Amplitaq DNA Polymerase, Stoffel Fragment (Life) and NEB(exo-) DNA polymerase,One kind of DNA polymerase.
8. The method for methylation library construction of trace free DNA according to any one of claims 1 to 5, wherein the step 7) further comprises a quality detection step after PCR amplification of the product of the double-strand extension: detecting the length of the amplified product fragment, and when the length of the product fragment is 150bp-300bp, obtaining a product which is a DNA methylation library.
9. A micro free DNA methylation library building kit, which is used for the micro free DNA methylation library building method according to any one of claims 1-8;
comprises a free DNA sample extraction and separation reagent, an enzyme digestion reagent, a linker sequence containing methylation and biotin modification, a linker sequence connecting reagent, a sulfite conversion reagent, a double-strand extension reagent and a PCR amplification reagent.
10. A free DNA sequencing method, characterized in that, the methylation database building method of trace free DNA according to any one of claims 1 to 8 is adopted for database building, and then sequencing is carried out.
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