A method for library construction of cell-free DNA methylation, a kit and a sequencing method

Abstract

The application relates to the technical field of gene detection, in particular to a micro free DNA methylation library construction method, a kit and a sequencing method. The library construction method comprises the following steps: extracting and separating a free DNA sample; performing double-end enzyme cutting 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 the free DNA fragment connected with the linker sequence; performing sulfite conversion on the free DNA fragment connected with the linker sequence by adopting a sulfite conversion solution; performing double-strand extension on the free DNA fragment after sulfite conversion and performing PCR amplification to obtain a DNA methylation library. The method can significantly increase the number of CG detection sites of free DNA and has a significant covering effect on CG sites. Compared with whole genome cfDNA methylation sequencing, the sequencing depth is significantly increased, the sequencing data volume is lower, and the cost is lower.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, in particular to a micro-free DNA methylation library construction method, a kit and a sequencing method. BACKGROUND

[0002] DNA methylation 5mC is a process in which the methyl group in the methyl donor S-adenosyl methionine (SAM) molecule is transferred to the DNA cytosine by methyltransferase. In eukaryotes, DNA methylation mainly occurs at the 5-carbon position of the CpG dinucleotide cytosine to form 5-methylcytosine (5mC). DNA methylation modification plays an important role in the expression regulation of genes and is an important analysis marker for cancer occurrence, development and treatment prognosis.

[0003] cfDNA is DNA that is free in the extracellular space (cell-free DNA, cfDNA), and the fragment length is usually about 150bp. It was discovered as early as 1984 by Mandel and Métais. The isolated free DNA of tumor patients is derived from tumor cells and carries all genetic variation information of the diseased organs or tissues, so it is also called ctDNA. The detection of cfDNA methylation, especially the application of ctDNA methylation as a marker for screening and identification of early tumor screening and patient prognosis risk assessment, has become one of the technical problems that scientists and enterprises around the world are competing for. The most core is that the content of free DNA is extremely low, the fragment length is short, and the individual difference is large. Healthy individuals contain 0-100ng of DNA per milliliter of blood, with an average of 30ng / ml. Even in patients with advanced tumors, due to the apoptosis and necrosis of a large number of tumor cells, the average content is only 180ng / ml. In the industry, various gene detection companies at home and abroad often invest hundreds of millions of dollars in research and development in order to quickly establish detection technologies with independent intellectual property rights. For example, the American GRAIL company has invested more than 1 billion US dollars to develop plasma free DNA for early breast cancer screening. There are also many domestic units that have completed hundreds of millions of financing to develop related detection technologies.

[0004] The detection technology currently applied to free DNA omics screening is mainly whole genome methylation sequencing, such as the research published in 2020 based on the methylation characteristics of extracellular free DNA for multi-tumor detection and positioning, which is mainly to identify key methylation markers through whole genome methylation sequencing. However, due to the fragment characteristics of free DNA being only about 140bp, even if 90G of data is sequenced, the average depth is only 15X, and the sequencing depth of most detection sites is less than 5X, the cost of single sample detection is nearly ten thousand yuan, and the cost is extremely high, so it is difficult for general units to conduct large sample sequencing. In addition, the cfMeDIP technology developed by the Princess Margaret Cancer Center in Canada in recent years can perform methylation sequencing on as little as 1ng of free DNA. The basic principle is to capture DNA fragments containing methylated modified cytosine bases through methylation-specific antibodies, and determine the relative methylation content of different fragments of the genome by sequencing the captured fragments. This technology cannot detect the precise methylation modification level of the base, and has capture bias. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a trace free DNA methylation library construction method, kit and sequencing method.

[0006] The technical solution of the present application to solve the above technical problem is as follows:

[0007] The present application provides a trace free DNA methylation library construction method, comprising the following steps:

[0008] 1) Extract and separate the free DNA sample;

[0009] 2) Perform double-end enzyme cutting trimming on the free DNA sample to obtain free DNA fragments;

[0010] 3) Connect a linker sequence containing methylation and biotin modification to the 5' end of the free DNA fragments;

[0011] 4) Capture and purify the free DNA fragments connected with the linker sequence using magnetic beads, and obtain the product;

[0012] 5) Perform bisulfite conversion on the product obtained in step 4) using a bisulfite conversion solution;

[0013] 6) Perform double-strand extension on the product after bisulfite conversion;

[0014] 7) Perform PCR amplification on the double-strand extended product to obtain a DNA methylation library.

[0015] Further, in the step 2), the enzyme used for the double-end enzyme digestion and trimming is one of MspI, AluI, BstNI, HpyCH4V, HaeIII, HpyCH4III, ApekI, BanII, SphI, BssSI, BglII, BamHI, KpnI.

[0016] Further, the adaptor sequence in the step 3) is Ad1, and the step 3) further comprises a step of connecting Ad2 at the 3’ end of the free DNA fragments; in the step 5), after the bisulfite conversion, Ad2 is combined with the magnetic beads and separated from the free DNA fragments, and the free DNA fragments with Ad1 are transferred to the bisulfite conversion solution.

[0017] Further, the adaptor sequence is a sequence designed based on the Illumina platform.

[0018] 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.

[0019] Further, the adaptor sequence is a sequence designed based on the MGI platform.

[0020] 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 as SEQ ID NO 7, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 8.

[0021] Further, in the step 1), the amount of the free DNA sample extracted and separated is 0.3-2 ng.

[0022] Further, in the step 6), the reagent for the double-strand extension comprises a DNA polymerase, which is one of T4 DNA Polymerase, Bst DNA polymerase, Klenow Frgment (3’-5’exo-), PCR polymerase, Hemo KlenTaq (NEB), Amplitaq DNA polymerase, Stoffel Fragment (Life) and NEB’s exo- ) DNA polymerase, DNA polymerase.

[0023] Further, the step 7) further comprises a quality detection step after the PCR amplification of the double-stranded extension product; the quality detection step detects the length of the amplified product fragment, and when the length of the product fragment is 150-300 bp, the obtained product is a DNA methylation library.

[0024] The present application provides a micro-free DNA methylation library construction kit for the micro-free DNA methylation library construction method as described above.

[0025] The kit comprises extraction and separation reagents for free DNA samples, enzyme digestion reagents, adapter sequences containing methylation and biotin modification, adapter sequence ligation reagents, bisulfite conversion reagents, double-stranded extension reagents, and PCR amplification reagents.

[0026] The present application provides a free DNA sequencing method, which is constructed by the micro-free DNA methylation library construction method as described above, and then sequenced.

[0027] The present application has the following advantages:

[0028] 1) The micro-free DNA methylation library construction method of the present application enables almost all CCGG sites in fragmented free DNA to be detected by sequencing by adding adapters to the 5' end of the enzyme-digested free DNA fragments, and increases the CG methylation information on both sides of the site.

[0029] 2) The micro-free DNA methylation library construction method of the present application reduces the amount of DNA input by performing bisulfite conversion after adding adapters to the free DNA fragments and randomly extending the conversion product, ensures that each sequencing sequence contains at least one CG site methylation information, reduces the loss of purification, and detects as much starting DNA as possible, achieving the purpose of low starting amount, so as to realize accurate detection while performing micro-detection.

[0030] 3) The micro-free DNA methylation library construction method of the present application can reduce the sample amount to less than 1 ng, which is significantly lower than the prior art.

[0031] 4) The micro-free DNA methylation library construction method of the present application can significantly increase the number of CG detection sites of free DNA, and has a significant effect on CG site coverage.

[0032] 5) The free DNA sequencing method of the present application significantly increases the sequencing depth and reduces the sequencing data volume and cost compared to whole genome cfDNA methylation sequencing. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1The chromatogram for detecting the cfDNA extraction effect in step 1 of Example 1 in the library construction method for trace free DNA methylation of the application;

[0034] Figure 2 The number and proportion 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 application. DETAILED DESCRIPTION

[0035] The principles and characteristics of the application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the application and not to limit the scope of the application.

[0036] Due to degradation, the fragment length of DNA is significantly reduced, and it is difficult to enrich enough CG sites and perform methylation sequencing by conventional reduced genome methylation sequencing. Especially for samples such as cfDNA, the fragment length thereof is usually about 150 bp, which is completely included in the fragment range (40-300 bp) detected by reduced genome methylation technology. The fragments containing CCGG sites are processed by reduced genome methylation technology, and the effective fragments are further cleaved to be shorter and lost, and instead the fragments not containing CCGG sites are retained and sequenced, which wastes effective data and reduces the number of CG site detection.

[0037] The principle of the free DNA methylation library construction method of the application for constructing a library for trace free DNA is that, in the application, a linker is connected to the 5' end of the free DNA cleavage product, and theoretically almost all CCGG sites cleaved can be detected, and the CG methylation information on both sides of the site is increased. Compared with whole genome free DNA methylation sequencing, the fragmentation processing mode of cleaving first and then connecting is adopted in the application, which can significantly increase the sequencing depth of free DNA methylation, and the sequencing data amount is lower and the cost is lower.

[0038] The trace free DNA methylation library construction method of the application comprises the following steps:

[0039] 1) Extracting and separating free DNA samples;

[0040] The library construction method of the application is mainly for free DNA in blood, i.e. cfDNA, or circulating tumor cell free DNA in blood of tumor patients, i.e. ctDNA; or for normal tissue or cell genomic DNA, i.e. gDNA.

[0041] The free DNA sample of the application can be derived from peripheral blood, cerebrospinal fluid, saliva, bronchial lavage fluid, pleural effusion, gastric juice, bile, feces, urine, etc.

[0042] Preferably, the cfDNA sample of the application is derived from plasma.

[0043] The sample amount of the present application can be less than 1 ng and as low as a single cell level, preferably, the sample amount of the present application is between 0.3 ng and 2 ng.

[0044] The input amount of DNA can be less than 1 ng or a single cell level. The extraction of sample DNA can be performed according to different sample properties by using existing commercial extraction kits or laboratory self-built technology.

[0045] Preferably, the elution of the finally extracted free DNA sample can only use ultrapure water or EB buffer, and cannot use TE or elution buffer provided by the kit with unknown components, and the sample elution volume is not more than 10 ul, to prevent the interference of the buffer on the free DNA sample.

[0046] Preferably, for cfDNA methylation detection, in order to avoid the methylation signal interference of intracellular DNA, the sample can be subjected to capillary electrophoresis detection of cfDNA fragment distribution characteristics after extraction, and the cfDNA product fragments with good extraction effect should be mainly distributed at about 140 bp, and a small amount of DNA can exist near 280 bp, and no genomic DNA fragments above 1000 bp should exist.

[0047] 2) The free DNA sample is subjected to double-end trimming by using MspI enzyme to obtain free DNA fragments.

[0048] The DNA fragments are subjected to double-end trimming under the action of MspI enzyme, and preferably, the total reaction system is 10-50 ul, the fragment structure with protruding CG base at the 5' end is constructed, and the substrate is reacted at 37℃ for 20 min-16 h.

[0049] The enzyme digestion system is specifically as follows:

[0050]

[0051] 3) The 5' end of the free DNA fragments is connected with a linker sequence containing methylation modification.

[0052] In the free DNA fragments obtained in step 2), the linker sequence containing methylation and biotin co-modification and DNA ligase are added into the free DNA fragments, and the reaction is carried out at 16-30℃ for 20 min-6 h.

[0053] The linker sequence contains biotin modification, which can make the magnetic beads in the subsequent step better capture the free DNA fragments.

[0054] The reaction system is specifically as follows:

[0055]

[0056] The components of Buffer A are specifically as follows:

[0057]

[0058] Wherein, the adapter sequence comprises Ad1 and Ad2, and both of the adapter sequences are connected with the free DNA fragment;

[0059] The adapter sequence is a sequence designed based on an Illumina platform or a sequence designed based on an MGI platform; wherein, in the sequence designed based on the Illumina platform, the nucleotide sequence of Ad1 is as shown in SEQ ID NO 1, and the nucleotide sequence of Ad2 is as shown in SEQ ID NO 2.

[0060] In the sequence designed based on the MGI platform, the nucleotide sequence of Ad1 is as shown in SEQ ID NO 5, and the nucleotide sequence of Ad2 is as shown in SEQ ID NO 6.

[0061] Preferably, the sequence designed based on the Illumina platform can also have the following alternatives: the nucleotide sequence of Ad1 is as shown in SEQ ID NO 3, and the nucleotide sequence of Ad2 is as shown in SEQ ID NO 4.

[0062] The sequence designed based on the MGI platform can also have the following alternatives: the nucleotide sequence of Ad1 is as shown in SEQ ID NO 7, and the nucleotide sequence of Ad2 is as shown in SEQ ID NO 8.

[0063] The sequence designed based on the Illumina platform, wherein, SEQ ID NO 1 has two representation methods, the first representation method is 5'-GGAGTT C AGA C GTGTG C T C TT CC GAT C T-3', wherein, the base with a horizontal line is a methylated base; the second representation method is,

[0064] 5'-GGAGTTmCAGAmCGTGTGmCTmCTTmCmCGATmCT-3', m is a methyl group, indicating that the base after it is methylated.

[0065] SEQ ID NO 2: 5'-CGAGATCGGAAGAGCACACGTCTGAACTCC-Bio-3'.

[0066] The sequence designed based on the Illumina platform can also have the following alternatives: the nucleotide sequence of Ad1 is as shown in SEQ ID NO 3, and the nucleotide sequence of Ad2 is as shown in SEQ ID NO 4. C AGAC GTGTG C T C TT CC GAT C TNNNNNNNN-3', wherein the bases with a line through them are methylated bases, and N is any base; the second representation is 5'-GGAGTTmCAGAmCGTGTGmCTmCTTmCmCGATmCTNNNNNNNN-3', m is a methyl group, indicating that the base following it is methylated, and N is any base.

[0067] SEQ ID NO 4. 5'-CGNNNNNNNNAGATCGGAAGAGCACACGTCTGAACTCC-Bio-3'.

[0068] The sequence based on the MGI sequencing adaptor design has two representations, the first representation is 5'-TTGTCTTCCTAAGGAA C GA C ATGG C TA C GAT CC GA C TT-3', wherein the bases with a line through them are methylated bases; the second representation is 5'-TTGTCTTCCTAAGGAAmCGAmCATGGmCTAmCGATmCmCGAmCTT-3', m is a methyl group, indicating that the base following it is methylated.

[0069] SEQ ID NO 6.

[0070] 5'-CGAAGTCGGATCGTAGCCATGTCGTTCCTTAGGAAGACAA-Bio-3'.

[0071] The sequence based on the MGI sequencing adaptor design has two representations, the first representation is 5'-GAA C GA C ATGG C TA C GAT CC GA C TT-3', wherein the bases with a line through them are methylated bases; the second representation is 5'-GAAmCGAmCATGGmCTAmCGATmCmCGAmCTT-3', m is a methyl group, indicating that the base following it is methylated.

[0072] SEQ ID NO 8. 5'-CGAAGTCGGATCGTAGCCATGTCGTTC-Bio-3'.

[0073] 4) Capture and purification of the free DNA fragments connected to the adaptor by magnetic beads, and obtain a magnetic bead-free DNA capture mixture.

[0074] In this step, streptomycin-labeled magnetic beads are used to capture the free DNA ligation product connected to the adaptor modified by methylation and biotin, and the captured product is resuspended in 20ul ultrapure water. The specific operation steps are as follows:

[0075] First, take 20-40ul streptomycin-coupled magnetic beads, rinse with 50ul rinsing solution for three times, and resuspend the magnetic beads in 15ul EB buffer.

[0076] Second, add the free DNA adaptor ligation product to 15ul magnetic beads, incubate at room temperature for 10-30min, and capture the free DNA ligation fragment by magnetic stand;

[0077] Finally, rinse the magnetic beads with 50ml EB for two times, remove the supernatant by magnetic stand, and resuspend the magnetic beads in 20ul ultrapure water or 0.1M sodium hydroxide.

[0078] It should be noted that if ultrapure water is used for elution, the final product of this step is a magnetic bead-free DNA mixture. If sodium hydroxide or other strong alkali is used to replace ultrapure water to dissolve the magnetic beads, the elution supernatant is recovered, the magnetic beads are removed, and the supernatant without magnetic beads is obtained. The supernatant is directly subjected to the next step of bisulfite conversion.

[0079] 5) Bisulfite conversion of the product obtained in step 4).

[0080] Commercial kits are used for bisulfite conversion of free DNA.

[0081] If ultrapure water is used for elution in step 4), first add the recommended volume of bisulfite reaction solution in the kit to the 20ul magnetic bead capture solution before following the kit instructions, denature at 98℃ for 1-10min, and denature the free DNA from the magnetic beads. Centrifuge at 16000rpm for 5-10min or stand by magnetic stand, and transfer the supernatant to a new PCR tube.

[0082] If 0.1M sodium hydroxide is used for elution, the eluate is placed on the magnetic stand, 20ul supernatant is taken to a PCR tube, the recommended volume of bisulfite reaction solution in the kit is added, and then the bisulfite conversion is directly performed according to the kit instructions. After the reaction, the free DNA after C-T conversion is eluted into 26ul ultrapure water or EB.

[0083] After the step of sulfite conversion, the linker sequence Ad2 is bound to the magnetic beads, and the cfDNA fragments connected with the linker sequence Ad1 are denatured into the sulfite conversion solution.

[0084] 6) Perform double-strand extension on the free DNA fragments after sulfite conversion.

[0085] In the PCR tube of the DNA after sulfite conversion, the following reagents are added respectively, and the double-strand extension of the sulfite conversion product is performed.

[0086]

[0087] The primer characteristics are designed using Primer1 sequence, and the primer containing the following characteristics is used for double-strand extension.

[0088] The primer sequence designed based on the Illumina sequencing platform is shown in SEQ ID NO 9, wherein n is a random base:

[0089] Oligo 1: 5'-tacacgacgctcttccgatctnnnnnn-3' (SEQ ID NO 9.).

[0090] The primer sequence designed based on the MGI sequencing platform is shown in SEQ ID NO 10, s is an index sequence, and n is a random base:

[0091] Oligo 1: 5'-tgtgagccaaggagttgssssssssssttgtcttcctaagaccgcttggcctccgacttnnnnnn-3' (SEQ ID NO 10.).

[0092] The specific operation steps are as follows:

[0093] After mixing the above reaction solution, it is placed in a PCR instrument, heated at 95°C for 45s, and then quickly transferred to ice for 5min. DNA polymerase Klenow (3'→5'exo-) 100U is added, mixed, and then placed in a PCR instrument for 4°C incubation for 5min. After incubation, the temperature is raised to 37°C at a rate of 0.1-1°C / s, and then incubated at 37°C for 1.5h. After reaction, 1-fold magnetic beads are used for purification, and eluted into 20ul EB buffer.

[0094] The reaction DNA polymerase includes but is not limited to T4 DNA Polymerase, Bst DNA polymerase, Klenow Frgment (3'-5'exo-), PCR polymerase, Hemo KlenTaq (NEB), Amplitaq DNA polymerase, Stoffel Fragment (Life) and NEB's (exo–)DNA polymerase, DNA polymerase, etc.

[0095] In the library construction method of the present application, after the enzyme digestion of step 2), the single-end ligation of the linker of step 3), and the completion of the bisulfite conversion of steps 4) and 5), the random extension of the bisulfite conversion product is performed in this step. Such a method reduces the input amount of DNA, ensures that each sequencing sequence contains at least one methylation information of CG site, reduces the loss of purification, and makes the initial amount of DNA as much as possible to be detected, achieving the purpose of low initial amount.

[0096] 7) PCR amplification of the double-stranded extended product.

[0097] The primer used in this step is a primer containing the following characteristics:

[0098] For the Illumina sequencing platform, the sequence characteristics of the upstream primer (Primer 1) and the downstream primer (Primer 2) of PCR amplification are shown in SEQ ID NO 11. and SEQ ID NO 12. respectively, wherein n is a random base:

[0099] Primer 1: 5'-aatgatacggcgaccaccgagatctacacnnnnnnnnacactctttccctacacgacgctcttccgatct-3' (SEQ ID NO 11.);

[0100] Primer 2: 5'-caagcagaagacggcatacgagatnnnnnnnngtgactggagttcagacgtgtgc-3' (SEQ ID NO 12.).

[0101] For the MGI sequencing platform, the sequence characteristics of the upstream primer (Primer 1) and the downstream primer (Primer 2) of PCR amplification are shown in SEQ ID NO 13. and SEQ ID NO 14. respectively, wherein n is a random base, and the 5' end of Primer 2 is phosphorylated:

[0102] Primer 1: 5'-tgtgagccaaggagttg-3' (SEQ ID NO 13.);

[0103] Primer 2: 5Phos-gaacgacatggctacga-3' (SEQ ID NO 14.).

[0104] The PCR amplification system of this step is as follows:

[0105]

[0106]

[0107] The PCR amplification conditions are as follows:

[0108] 98℃, 1min; 98℃, 30s, 50-68℃, 30s, 72℃, 30s, 10-18 cycles; 72℃, 5min.

[0109] After PCR amplification and purification, elute into 20ul buffer.

[0110] After PCR amplification, a quality detection step is further included; the quality detection step 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 within the above range, the library construction fails, and the library construction needs to be performed again.

[0111] The present application also provides a DNA sequencing method, after the DNA methylation library is established by the above steps, the obtained library is sequenced.

[0112] The specific steps are as follows: after the DNA methylation library obtained by the library construction method of the present application is constructed, the molar concentration of the sample DNA is quantified by QPCR, the library fragment size is detected by capillary electrophoresis or 2100 bioanalyzer, the qualified fragment is between 150bp and 270bp, the corresponding molar library is taken according to the sequencing data volume, and sequencing is performed on a sequencer.

[0113] After sequencing, the data are respectively subjected to the following steps: de-adapter, low-quality read filtering and statistics, methylation conversion rate statistics, alignment and alignment rate statistics, genome coverage statistics, repeat rate statistics, and calculation of the methylation level of each site, to obtain the CG site coverage number.

[0114] The method of the present application is described below through specific examples:

[0115] Example 1

[0116] The embodiment provides a specific step of library construction and sequencing by using the method of the application. The specific process is as follows:

[0117] 1) Sample collection and cfDNA extraction: collect 2-5 ml of whole blood in an EDTA-containing blood collection tube or a cfDNA blood collection tube, mix well by inverting, centrifuge at 1600g, immediately remove the upper plasma part, further centrifuge at 16000g at 4 DEG C for 10 min, remove residual cells and debris, separate the supernatant plasma into a new centrifuge tube, and store at -80 DEG C.

[0118] Collect the plasma and extract by using a commercial cfDNA extraction kit. The kit used is DSP Blood Mini kit (Qiagen). The concentration of the extracted DNA is quantified by Qubit HS, and the cfDNA extraction effect is detected by Agilent 2100 to determine whether there is genomic contamination. When there is no genomic contamination, the next step is performed.

[0119] Figure 1 The chromatogram for detecting the cfDNA extraction effect is obtained by Figure 1 It can be seen that the cfDNA extracted in the embodiment does not have genomic contamination.

[0120] 2) MspI enzyme digestion of cfDNA fragments: take 1-2 ng of free DNA without genomic contamination, and perform CCGG site enzyme digestion according to the following system: MspI enzyme 50 U, 10X buffer 1 ul, and water to a total volume of 10 ul, and react at 37 DEG C for 2 hours.

[0121] 3) cfDNA end connection linker sequence: in the above cfDNA enzyme digestion system, sequentially add 1 ul of 1 uM linker sequence Ad1 and Ad2 annealing product, 3.5 ul of buffer A and 200 U of DNA ligase, mix well, and incubate at 23 DEG C for 2 hours.

[0122] The linker sequence used in the embodiment is a sequence based on the Illumina platform, and the nucleotide sequence of Ad1 is shown in SEQ ID NO 1 and SEQ ID NO 2.

[0123] 4) Linker connection product capture: take 40 ul of streptomycin-coupled magnetic beads, rinse with 50 ul of rinsing solution for three times, resuspend the magnetic beads in 15 ul of EB buffer; secondly, add the cfDNA linker connection product into 15 ul of magnetic beads, incubate at room temperature for 30 min, capture the cfDNA connection fragments by using a magnetic stand; rinse the magnetic beads with 100 ml of EB for two times, remove the supernatant by using a magnetic stand, and resuspend the magnetic beads in 20 ul of ultrapure water

[0124] 5) Adaptor Ad2 removal and bisulfite treatment: Bisulfite treatment was performed using the EZ DNA Methylation-Gold Kit from ZYMO Research. TM The bisulfite treatment was performed using the EZ DNA Methylation-Gold Kit from ZYMO Research.

[0125] The procedure was as follows: 20ul of the magnetic bead-cfDNA capture mixture was added to 130ul of the bisulfite reaction solution, denatured at 98℃ for 8min, centrifuged at 16000rpm for 10min, the adaptor Ad2 was still bound to the magnetic beads, and the cfDNA was denatured into the bisulfite conversion solution. The supernatant was transferred to a new PCR tube, and the following program was continued on the PCR instrument: 98℃, 2min; 64℃, 2.5h. After the reaction, purification was performed according to the kit instructions, and eluted into 26ul of ultrapure water.

[0126] 6) Double-strand extension of the bisulfite-converted cfDNA fragments: 5ul of 10X reaction buffer, 2ul of 10mM dNTP, and 5ul of 20mM Oligo 1 were added to the purified 26ul of the converted DNA, and the total volume was made up to 50ul with water. After mixing the above reaction solution, it was placed in the PCR instrument, heated at 95℃ for 45s, and after the reaction, it was quickly transferred to ice and incubated for 5min. Then, 100U of DNA polymerase Klenow (3'→5'exo-) was added, mixed, and then placed in the PCR instrument for 4℃ incubation for 5min. After incubation, the temperature was raised to 37℃ at a rate of 1℃ / s, and then incubated at 37℃ for 1.5h. After the reaction, 1-fold DNA purification magnetic beads were used for purification, and eluted into 20ul of EB buffer.

[0127] 7) PCR amplification

[0128] The PCR reaction solution was prepared in a PCR tube according to the following system

[0129]

[0130] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 1min; 95℃, 30s; 58℃, 30s; 72℃, 45s for 12 cycles of amplification, 72℃ extension for 5min, and 16℃ storage. After PCR product amplification, 1-fold volume of Ampure XP Beads was used for purification, and eluted into 20ul of EB eluent.

[0131] After library construction, Agilent 2100 was used for fragment size detection and QPCR concentration quantitative detection, respectively. After passing the qualification, Hiseq was used for sequencing.

[0132] The library of this example was sequenced on both sides, with a sequencing depth of 1X and 5X, respectively.

[0133] Example 2

[0134] The gDNA library was constructed and sequenced using the same steps and reagents as in Example 1. The library from this example was sequenced bilaterally at depths of 1X and 5X.

[0135] Comparative Example 1

[0136] The gDNA library was constructed and sequenced using a standard simplified methylation method. The comparative library was sequenced bilaterally at depths of 1X and 5X.

[0137] The number and proportion of CG sites covered at 1X and 5X sequencing depths were examined in the sequencing results of Examples 1 and 2 and Comparative Example 1, respectively. The results are as follows: Figure 2 As shown.

[0138] pass Figure 2 The results show that the 1X CG site coverage in Examples 1 and 2 was 16.2M and 13.3M, respectively, accounting for 23% and 24% of the total CG sites in the genome; while the coverage of 1X CG sites in conventional simplified genomic methylation was only about 10M, accounting for about 16% of the genomic CG sites. The 5X CG site coverage was 10.97M and 8.83M, respectively; while the coverage of 1X CG sites in conventional simplified genomic methylation was only about 5.56M.

[0139] Therefore, the library construction method and sequencing method of the present invention are suitable for the detection of trace amounts of cfDNA or gDNA, and have a significant coverage effect on CG sites.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. sequence list <110> Shenzhen Egene Technology Co., Ltd. <120> A method for library construction, kit, and sequencing of trace free DNA methylation <141> 2022-04-20 <160> 14 <170> SIPOSequenceListing 1.0 <210> 1 <211> 28 <212> DNA / RNA <213> Artificial Sequence <400> 1 ggagttcaga cgtgtgctct tccgatct 28 <210> 2 <211> 30 <212> DNA / RNA <213> Artificial Sequence <400> 2 cgagatcgga agagcacacg tctgaactcc 30 <210> 3 <211> 36 <212> DNA / RNA <213> Artificial Sequence <220> <221> misc_feature <222> (29)..(36) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (29)..(29) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (30)..(30) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (31)..(31) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (32)..(32) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (33)..(33) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (34)..(34) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (35)..(35) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (36)..(36) <223> n is a, c, g, t or u <400> 3 ggagttcaga cgtgtgctct tccgatctnn nnnnnn 36 <210> 4 <211> 38 <212> DNA / RNA <213> Artificial Sequence <220> <221> misc_feature <222> (3)..(10) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (3)..(3) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (4)..(4) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (5)..(5) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (6)..(6) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (7)..(7) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (8)..(8) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (9)..(9) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (10)..(10) <223> n is a, c, g, t or u <400> 4 cgnnnnnnnn agatcggaag agcacacgtc tgaactcc 38 <210> 5 <211> 38 <212> DNA / RNA <213> Artificial Sequence <400> 5 ttgtcttcct aaggaacgac atggctacga tccgactt 38 <210> 6 <211> 40 <212> DNA / RNA <213> Artificial Sequence <400> 6 cgaagtcgga tcgtagccat gtcgttcctt aggaagacaa 40 <210> 7 <211> 25 <212> DNA / RNA <213> Artificial Sequence <400> 7 gaacgacatg gctacgatcc gactt 25 <210> 8 <211> 27 <212> DNA / RNA <213> Artificial Sequence <400> 8<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <222> (25)..(25) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (26)..(26) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (27)..(27) <223> n is a, c, g, t or u <400> 9 tacacgacgc tcttccgatc tnnnnnn 27 <210> 10 <211> 65 <212> DNA / RNA <213> Artificial Sequence <220> <221> misc_feature <222> (18)..(27) <223> s is a, t, c or g <220> <221> misc_feature <222> (60)..(65) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (60)..(60) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (61)..(61) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (62)..(62) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (63)..(63) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (64)..(64) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (65)..(65) <223> n is a, c, g, t or u <400> 10 tgtgagccaa ggagttgsss sssssssttg tcttcctaag accgcttggc ctccgacttn 60 nnnnn 65 <210> 11 <211> 70 <212> DNA / RNA <213> Artificial Sequence <220> <221> misc_feature <222> (30)..(37) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (30)..(30) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (31)..(31) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (32)..(32) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (33)..(33) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (34)..(34) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (35)..(35) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (36)..(36) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (37)..(37) <223> n is a, c, g, t or u <400> 11 aatgatacgg cgaccaccga gatctacacn nnnnnnnaca ctctttccct acacgacgct 60 cttccgatct 70 <210> 12 <211> 55 <212> DNA / RNA <213> Artificial Sequence <220> <221> misc_feature <222> (25)..(32) <223> n is a, t, c or g <220> <221> misc_feature <222> (25)..(25) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (26)..(26) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (27)..(27) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (28)..(28) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (29)..(29) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (30)..(30) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (31)..(31) <223> n is a, c, g, t or u <220> <221> misc_feature <222> (32)..(32) <223> n is a, c, g, t or u <400> 12 caagcagaag acggcatacg agatnnnnnn nngtgactgg agttcagacg tgtgc 55 <210> 13 <211> 17 <212> DNA / RNA <213> Artificial Sequence <400> 13 tgtgagccaa ggagttg 17 <210> 14 <211> 17 <212> DNA / RNA <213> Artificial Sequence <400> 14 gaacgacatg gctacga 17

Claims

1. A method for library construction of cell-free DNA methylation, characterized in that, The method comprises the following steps: 1) extracting and separating a free DNA sample; 2) performing double-end enzyme cutting trimming on the free DNA sample to obtain free DNA fragments; 3) connecting a linker sequence Ad1 containing methylation modification to the 5' end of the free DNA fragments, and connecting a linker sequence Ad2 containing biotin modification to the 3' end of the free DNA fragments; The linker sequence Ad1 and the linker sequence Ad2 are sequences 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; Or, the linker sequence Ad1 and the linker sequence Ad2 are sequences designed based on the 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 as SEQ ID NO 7, and the nucleotide sequence of Ad2 is shown as SEQ ID NO 8; 4) capturing and purifying the free DNA fragments connected with the linker sequences by using magnetic beads to obtain a product; 5) performing bisulfite conversion on the product obtained in step 4) by using a bisulfite conversion solution; 6) performing double-strand extension on the product after bisulfite conversion; 7) performing PCR amplification on the double-strand extended product to obtain a DNA methylation library.

2. The method of claim 1, wherein the method is performed by the system of claim 1. In step 2), the enzyme used for double-end enzyme cutting trimming is one of MspI, AluI, BstNI, HpyCH4V, HaeIII, HpyCH4III, ApekI, BanII, SphI, BssSI, BglII, BamHI, KpnI.

3. The micro free DNA methylation library construction method according to claim 1, characterized in that, In step 5), after bisulfite conversion, Ad2 is bound to the magnetic beads and separated from the free DNA fragments, and the free DNA fragments connected with Ad1 are transferred to the bisulfite conversion solution.

4. The microcell-free DNA library construction method according to any one of claims 1-3, characterized in that, The amount of the free DNA sample extracted and separated in step 1) is 0.3-2 ng.

5. The method of claim 1-3, wherein, In step 6), the reagent for performing double-strand extension includes a DNA polymerase, and the DNA polymerase is one of T4 DNA Polymerase, BstDNA polymerase, Klenow Frgment (3'-5'exo-), PCR polymerase, Hemo KlenTaq (NEB), Amplitaq DNA polymerase, Stoffel Fragment (Life), and Deep / Vent® (exo-) DNA polymerase, Deep / Vent® DNA polymerase of NEB. 6.The microcell-free DNA library construction method according to any one of claims 1-3, characterized in that, In step 7), after PCR amplification of the double-stranded extension product, a quality detection step is further included: detecting the length of the amplified product fragment, and when the length of the product fragment is 150-300 bp, the obtained product is a DNA methylation library.

7. A microcellular free DNA methylation library kit, characterized in that, The microcell-free DNA methylation library construction method according to any one of claims 1-6; The kit comprises a sample extraction and separation reagent, an enzyme cutting reagent, a linker sequence containing methylation and biotin modification, a linker sequence ligation reagent, a sulfite conversion reagent, a double-stranded extension reagent, and a PCR amplification reagent.

8. A method of sequencing cell-free DNA, comprising: The kit is used for library construction by the microcell-free DNA methylation library construction method according to any one of claims 1-6, and then sequencing.

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