阅读说明：本技术 氧化含锯齿末端核酸分子的方法及还原方法与文库构建法 (Method for oxidizing nucleic acid molecules containing jagged ends, reduction method and library construction method ) 是由 刘佳慧 沈璐 赵美茹 李俊 钟文星 赵蔷 刘青峰 黄毅 易鑫 于 2021-09-28 设计创作，主要内容包括：一种氧化含锯齿末端核酸分子的方法及还原方法与文库构建法,氧化含锯齿末端核酸分子的方法包括：提供待测样本,所述待测样本中的至少部分核酸分子具有锯齿末端,使所述待测样本中核酸分子的5-甲基胞嘧啶残基(5mC)、5-羟甲基胞嘧啶残基(5hmC)中的至少一种氧化,获得氧化产物。通过对未进行末端修复的待测样本中的核酸分子锯齿末端进行氧化,得到可用于还原的核酸分子,使得待测样本中核酸分子锯齿末端的甲基化水平真实还原,从而提高甲基化检测的准确性。(A method for oxidizing nucleic acid molecules containing jagged ends, a reduction method and a library construction method are disclosed, wherein the method for oxidizing the nucleic acid molecules containing jagged ends comprises the following steps: providing a sample to be tested, wherein at least part of nucleic acid molecules in the sample to be tested have sawtooth ends, and oxidizing at least one of 5-methylcytosine residues (5mC) and 5-hydroxymethylcytosine residues (5hmC) of the nucleic acid molecules in the sample to be tested to obtain an oxidation product. The nucleic acid molecules which can be used for reduction are obtained by oxidizing the sawtooth tail ends of the nucleic acid molecules in the sample to be detected without tail end repair, so that the methylation level of the sawtooth tail ends of the nucleic acid molecules in the sample to be detected is really reduced, and the accuracy of methylation detection is improved.)

1. A method of oxidizing a nucleic acid molecule comprising a jagged end, comprising:

providing a test sample, wherein at least part of nucleic acid molecules in the test sample have sawtooth ends, oxidizing at least one of 5-methylcytosine residues (5mC) and 5-hydroxymethylcytosine residues (5hmC) of the nucleic acid molecules in the test sample to obtain an oxidation product, wherein the oxidation product contains at least one of oxidized 5-methylcytosine residues and 5-hydroxymethylcytosine residues, and at least one of the 5-methylcytosine residues and 5-hydroxymethylcytosine residues of the sawtooth ends of the nucleic acid molecules in the oxidation product is also oxidized.

2. The method of claim 1, wherein the nucleic acid molecules of the test sample contain 5-methylcytosine residues and 5-hydroxymethylcytosine residues at positions comprising at least one of CpG positions, CHG positions, and CHH positions, wherein H is a non-G base;

and/or the oxidized 5-methylcytosine comprises at least one of 5-carboxycytosine (5caC), 5-aldehyde cytosine (5fC), and 5-hydroxymethylcytosine.

3. The method of claim 1, wherein 5-methylcytosine at the CpG sites of the nucleic acid molecule in the test sample is enzymatically oxidized.

4. The method of claim 3, wherein the enzyme used comprises a ten-eleven translocation (TET) enzyme.

5. The method of claim 1, wherein the oxidation reaction system comprises TET enzyme and Fe²⁺Sulfate, oxidation buffer of (a);

and/or, said Fe-containing²⁺The sulfate salt of (A) includes Fe (NH)₄)₂(SO₄)₂、Fe(SO₄)₂At least one of;

and/or the oxidation buffer comprises at least one of a buffer, NaCl, alpha-ketoglutarate, ascorbic acid, Adenosine Triphosphate (ATP), dithiothreitol;

and/or the pH value of the oxidation reaction system is 4.3-8, preferably 4.3-7.5, and more preferably 6;

and/or, the nucleic acid molecule comprises DNA;

and/or the test sample comprises at least one of a cfDNA sample, a disrupted genomic DNA sample;

and/or the sample to be tested comprises at least one of a body fluid sample and a tissue sample;

and/or the body fluid sample comprises at least one of blood, plasma, serum, saliva, pleural fluid, cerebrospinal fluid, semen, urine.

6. A method of reducing a nucleic acid molecule comprising a jagged end, comprising: contacting the oxidation product obtained by the method of any one of claims 1 to 5 with an organoborane to convert at least one of said oxidized 5-methylcytosine residues, 5-hydroxymethylcytosine residues to dihydrouracil residues to provide a nucleic acid molecule comprising dihydrouracil residues, i.e., a reduction product.

7. The method of claim 6, wherein the organoborane comprises a complex selected from the group consisting of boranes and nitrogen containing compounds selected from the group consisting of nitrogen heterocycles and tertiary amines;

and/or, the organoborane comprises a complex of a borane and a nitrogen heterocycle;

and/or the nitrogen heterocycle comprises pyridine optionally substituted with 1-4 lower alkyl groups;

and/or, the nitrogen heterocycle comprises pyridine, 2-methylpyridine, or 5-ethyl-2-methylpyridine;

and/or the organoborane is at least one of pyridine borane and 2-methyl pyridine borane;

and/or, purifying the oxidation product obtained by the method of any one of claims 1 to 5, and then contacting the purified oxidation product with an organoborane to convert the oxidized 5-methylcytosine residue to a dihydrouracil residue to obtain DNA containing the dihydrouracil residue;

and/or, the purification comprises at least one of magnetic bead purification and column purification;

and/or the reduction system contains organoborane and organic acid salt;

and/or the organic acid salt comprises sodium acetate;

and/or the pH value of the organic acid salt in the reduction system is 4.0-4.5.

8. The method of claim 6, further comprising a repair step comprising end repairing the reduced product to obtain a repaired product.

And/or in the repairing step, the repairing product is subjected to an A reaction, and then a joint is connected to the tail end of the product to obtain a connection product;

and/or, further comprises purifying the reduction product, and then performing end repair on the purified reduction product;

and/or, the method of purifying the reduction product comprises column purification;

and/or, further comprising purifying the ligation product;

and/or, the method for purifying the ligation product comprises magnetic bead purification.

9. A library construction method, comprising amplifying the ligation product obtained by the method of claim 8 to obtain a library that can be used for on-machine sequencing.

10. The library construction method of claim 9, further comprising performing hybrid capture using the probe after amplification, and performing amplification again to obtain a library that can be used in-machine sequencing.

Technical Field

The invention relates to the technical field of gene detection, in particular to a method for oxidizing nucleic acid molecules containing jagged ends, a reduction method and a library construction method.

Background

As early as 1925, DNA methylation modifications were discovered prior to the identification of DNA duplex structure. The predominant form of DNA methylation modification is 5mC and its derivative modifications, considered the "fifth base" of DNA. DNA methylation plays an important role in the physiological and pathological processes of gene regulation, genetic imprinting, aging, inflammation, tumor and the like. Recent studies have shown that methylation characteristics of cell-free DNA (hereinafter referred to as cfDNA) are important markers for early tumor screening.

Bisulfite (e.g., sodium bisulfite) deaminates cytosine (C) to uracil (U), which is recognized as thymine (T) during subsequent PCR using a U-tolerant polymerase to effect a C to T conversion, thereby separating unmodified C from methylated C. Whole Genome Bisulfite Sequencing (WGBS) converts unmethylated cytosine into thymine (about 95% of C can be converted into T), can cover 70-99% of cytosine in a whole genome, and realizes single base resolution methylation detection in a whole genome level. Whole genome bisulfite sequencing is also a "gold standard" for methylation detection. However, bisulfite treatment degrades DNA in large quantities, requires high DNA input, and is not friendly for samples with low DNA content, such as plasma free DNA NA. In addition, bisulfite converts unmethylated cytosine to T, and unmethylated cytosine accounts for more than 95% of all cytosines in the genome, so the second-generation sequencing library constructed after bisulfite treatment has poor base balance (AT ratio > 80%), and 15% of bacteriophage PhiX DNA needs to be added for balancing during sequencing, which results in a great waste of sequencing data. Finally, the sequencing quality of data generated by WGBS is poor, special software (such as Bismar k) is needed to be used when the genome is aligned, the alignment rate is poor, and is usually only about 70%, and further data waste is caused.

Chinese patent publication No. CN 110820050 a shows that under certain conditions, methylated C can be converted into carboxylated C and aldehyde C by 10-11 transposase (TET enzyme), and carboxylated C can be converted into dihydrouracil by organoborane (e.g., pyridine borane), and can be identified as T by sequencing, thereby distinguishing methylated C from unmethylated C. The whole genome methylation non-sulfite library construction technology based on the principle effectively overcomes the defects of low library complexity and low sequencing data depth, and can realize methylation detection on various second-generation sequencing platforms.

Double-stranded cfDNA is reported in the literature to exist in both blunt-ended and jagged-ended forms. The jagged ends are single-stranded (see FIG. 1, ref: Detection and characterization of jagged ends of double-stranded DNA in plasma, DOI:10.1101/gr.261396.120), and the proportion in cfDNA can reach 87.8%; moreover, in circulating tumor DNA (ctDNA), the proportion of jagged ends is relatively increased. For such jagged-end structures, the existing methods of transformation after library construction resulted in a decrease in jagged-end signal during methylation sequencing of cfDNA (see FIG. 2, ref: Detection and characterization of jagged ends of double-stranded DNA in plasma, DOI: 10.1101/gr.261396.120). The existing library construction method cannot truly reflect the methylation state of nucleic acid molecules containing jagged ends such as cfDNA.

Disclosure of Invention

According to a first aspect, in one embodiment, there is provided a method of oxidizing a nucleic acid molecule comprising a jagged end, comprising:

providing a test sample, wherein at least part of nucleic acid molecules in the test sample have jagged ends, oxidizing at least one of 5-methylcytosine residues (5mC) and 5-hydroxymethylcytosine residues (5hmC) of the nucleic acid molecules in the test sample to obtain an oxidized product, wherein the oxidized product contains at least one of oxidized 5-methylcytosine residues and 5-hydroxymethylcytosine residues, and at least one of the oxidized 5-methylcytosine residues and 5-hydroxymethylcytosine residues (mainly 5-methylcytosine residues at CpG sites) of the jagged ends of the nucleic acid molecules in the oxidized product is also oxidized.

According to a second aspect, in one embodiment, there is provided a method of reducing a nucleic acid molecule comprising a jagged end, comprising:

contacting said oxidation product obtained by the method of the first aspect with an organoborane to convert at least one of said oxidized 5-methylcytosine residues, 5-hydroxymethylcytosine residues to dihydrouracil residues to provide a nucleic acid molecule comprising dihydrouracil residues, i.e., a reduction product; the organoborane reduces, deaminates and decarboxylates or dealdehydes the oxidized 5-methylcytosine residues, thereby converting at least one of the oxidized 5-methylcytosine residues, 5-hydroxymethylcytosine residues in the nucleic acid molecule to dihydrouracil residues, resulting in a reduction product, i.e., a product useful for end repair, in which the oxidized 5-methylcytosine residues in the CpG sites of the zigzag ends can be reduced to dihydrouracil residues.

According to a third aspect, in one embodiment, there is provided a library construction method, comprising amplifying the ligation products of the method of the first aspect to obtain a library that can be used for on-machine sequencing.

According to the method for oxidizing the nucleic acid molecule containing the sawtooth tail end, the reduction method and the library construction method of the embodiment, the sawtooth tail end of the nucleic acid molecule in the sample to be detected without tail end repair is oxidized to obtain the nucleic acid molecule which can be used for reduction, so that the methylation level of the sawtooth tail end of the nucleic acid molecule in the sample to be detected is really reduced, and the methylation detection accuracy is improved.

Drawings

Fig. 1 is a diagram of a conventional cfDNA saw-tooth end structure.

Fig. 2 is a diagram showing the results of conventional cfDNA sequencing.

FIG. 3 is a schematic diagram of conventional detection of methylation of non-bisulfite.

FIG. 4 is a schematic diagram of detection of methylation of non-bisulfite in an embodiment of the present invention.

FIG. 5 is a library fragment distribution diagram of example 1.

Fig. 6 is a graph of cfDNA methylation level results calculated based on existing non-bisulfite methylation detection methods.

Fig. 7 is a graph showing the calculated results of cfDNA methylation levels based on the non-bisulfite methylation detection method according to an embodiment of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Wherein like elements in different embodiments are numbered with like associated elements. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.

The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning. The term "connected" and "coupled" when used in this application, unless otherwise indicated, includes both direct and indirect connections (couplings).

Herein, unless otherwise specified, "CpG site" refers to a cytosine-phosphate-guanine site, i.e., a site in a DNA sequence immediately following a cytosine to a guanine.

As used herein, the term "cfDNA" refers to DNA circulating in the peripheral blood of a patient, also known as "circulating cell-free DNA", "cell-free DNA", used interchangeably. The median size of the DNA molecules in cell-free DNA may be less than 1kb (e.g., in the range of 50bp to 500bp, 80bp to 400bp, or 100bp to 1,000 bp), and fragments having median sizes outside this range may also be present. Cell-free DNA may contain circulating tumor DNA (ctdna), i.e., tumor DNA or circulating fetal DNA that circulates freely in the blood of a cancer patient (if the subject is a pregnant female). cfDNA can be highly fragmented, and in some cases, the average fragment size can be about 165bp to 250bp (Newman et al, Nat med.201420: 548-54). cfDN a is obtained by centrifuging whole blood to remove all cells and then separating DNA from the remaining plasma or serum. Such methods are well known (see, e.g., Lo et al, Am J Hum Genet 1998; 62: 768-75). Circulating cell-free DNA is usually double-stranded, but can be rendered single-stranded by denaturation. The circulating cell free DNA comprises complete double-stranded DNA molecules and incomplete double-stranded DNA molecules, and the complete double-stranded DNA molecules and the incomplete double-stranded DNA molecules are mainly distinguished in that the ends of the complete double-stranded DNA are complementary paired complete double strands, the ends of the incomplete double strands are provided with outstanding single strands, the DNA molecules of an original sample do not have another single strand which can be complementary with the single strands, and the DNA molecules are also called as sawtooth ends.

Herein, "jagged end" refers to a single-stranded overhang in a DNA molecule, which jagged end is typically present in an original cfDNA sample, a broken genomic DNA sample, or cfDNA sample, etc., taken from an organism, unless otherwise specified.

Herein, the chemical structure of cytosine residues on DNA is schematically shown as follows:

herein, the chemical structure of the 5-methylcytosine (5mC) residue on DNA is schematically shown below:

as used herein, "methylated C" includes primarily 5-methylcytosine, and may typically further include 5-hydroxymethylcytosine.

Herein, the chemical structure of the 5-hydroxymethylcytosine (5hmC) residue on DNA is schematically shown below:

here, the chemical structure of the 5-carboxycytosine (5caC) residue on DNA is schematically shown below:

here, the chemical structure of the 5-formylcytosine (5fC, also known as 5-formylcytosine) residue on DNA is schematically shown below:

herein, the chemical structure of the Dihydrouracil (DHU) residue on DNA is schematically shown below:

in chinese patents publication nos. CN 110820050 a and CN 111971386 a, cfDNA is first subjected to end repair, a addition and a linker, followed by a transformation reaction of TET enzyme and pyridine borane. Because the ends of the cfDNA serrations are single-stranded, if the CpG sites at the ends of the serrations are methylated, the CpG sites in the newly generated strands are not methylated after the ends are repaired. FIG. 3 is a schematic diagram showing a conventional non-bisulfite methylation detection method, where in FIG. 3 lines 1a, 2a represent cfDNA original sequences, lines 1a, 2a form double strands that are complementary in the middle, the double strands have jagged ends at both ends, lines 1b, 2b represent newly generated sequences with end-repair, and lines 1c, 1d, 2c, 2d represent sequencing adaptors. The dashed box indicates the final detectable methylation signal, and it can be seen that the newly generated sequences (region of lines 1b, 2 b) with end-repair did not detect methylation signal, eventually leading to a decrease in the methylation level at the cfDNA 3' end. Resulting in a significant decrease in its 3' end methylation level (fig. 6), resulting in a decrease in the overall strand methylation level, which does not truly reflect the methylation status of cfDNA.

According to a first aspect, in one embodiment, there is provided a method of oxidizing a nucleic acid molecule comprising a jagged end, comprising:

providing a test sample, wherein at least part of nucleic acid molecules in the test sample have jagged ends, oxidizing at least one of 5-methylcytosine residues (5mC) and 5-hydroxymethylcytosine residues (5hmC) of the nucleic acid molecules in the test sample to obtain an oxidized product, wherein the oxidized product contains at least one of oxidized 5-methylcytosine residues and 5-hydroxymethylcytosine residues, and at least one of the oxidized 5-methylcytosine residues and 5-hydroxymethylcytosine residues (mainly 5-methylcytosine residues at CpG sites) at the jagged ends of the nucleic acid molecules in the oxidized product is also oxidized; the step is used for oxidizing 5-methylcytosine residues in CpG sites on almost all DNA molecules in a sample to be detected, and particularly oxidizing the 5-methylcytosine residues of the CpG sites at the sawtooth tail ends (single-stranded DNA with the protruded tail ends) of the DNA molecules.

In one embodiment, 5-methylcytosine residues and 5-hydroxymethylcytosine residues in terminal and non-terminal CpG sites, CHG sites, CHH sites and the like of the nucleic acid molecules in the sample to be tested are oxidized and can be used for subsequent reduction, and the 5-methylcytosine residues and 5-hydroxymethylcytosine residues including the serrated terminal are reduced into DHU, so that the methylation level of the serrated terminal is really reduced. Compared with the prior art, the oxidation step obviously enhances the methylation signals of the sawtooth ends, thereby improving the accuracy of methylation sequencing of nucleic acid molecules containing the sawtooth ends.

In one embodiment, the site in the nucleic acid molecule comprising a 5-methylcytosine residue (5mC), a 5-hydroxymethylcytosine residue (5hm C) comprises at least one of a CpG site, a CHG site, a CHH site, and H is a non-G base, i.e., a base other than guanine. The 5-methylcytosine residue (5mC) is predominantly located at CpG sites. The methylation levels at various sites can be obtained by analysis of the sequencing data.

In one embodiment, the sites in the nucleic acid molecule that contain 5-methylcytosine residues (5mC), 5-hydroxymethylcytosine residues (5hm C) include all of CpG sites, CHG sites, CHH sites.

In one embodiment, the oxidation process mainly comprises converting 5mC into 5hmC, 5fC, and 5caC in sequence. The amount of 5mC in the sample to be tested is typically about 10 times as much as 5 hmC.

In one embodiment, the present invention is primarily used to detect the methylation level of cytosine, which can be generally calculated by software.

In one embodiment, the oxidized 5-methylcytosine comprises at least one of 5-carboxycytosine (5caC), 5-formylcytosine (5f C), and 5-hydroxymethylcytosine.

In one embodiment, 5-methylcytosine at CpG sites of the nucleic acid molecules in the test sample are enzymatically oxidized. Both terminal and non-terminal methylated CpG will be oxidized.

In one embodiment, the enzyme used includes, but is not limited to, Ten-Eleven Translocation (T ET) enzyme. TET enzymes include, but are not limited to, TET1, TET2, TET3, and the like TET family enzymes. TET enzymes are generally commercially available, for example, from NEB.

In one embodiment, in the oxidation step, the oxidation reaction system contains TET enzyme and Fe²⁺Sulfate, oxidation buffer.

In one embodiment, the Fe-containing compound²⁺Sulfates of (A) include, but are not limited to, Fe (NH)₄)₂(SO₄)₂、Fe(SO₄)₂And so on. For example, the document "distributing Processing by the Iron (II)/α -Ketoglutarate-Dependent Ca catalysis of the TET Enzymes Is a component with an epitaxial Roles for Oxidized 5-M ethylene glycol Bases" (doi:10.1021/jacs.6b03243) discloses that Fe (SO) can be used₄)₂。

In one embodiment, the oxidation buffer contains at least one of buffer, NaCl, alpha-ketoglutarate, ascorbic acid, Adenosine Triphosphate (ATP), Dithiothreitol (DTT).

In one embodiment, the oxidation buffer contains all of buffer, NaCl, alpha-ketoglutarate, ascorbic acid, Adenosine Triphosphate (ATP), Dithiothreitol (DTT).

In one embodiment, the pH of the oxidation buffer is 4.3 to 8, preferably 4.3 to 7.5, and more preferably 6.

In one embodiment, the oxidation reaction can be performed using an existing oxidation reaction system, for example, the oxidation reaction system disclosed in "genome-wide methylation non-bisulfite sequencing library and construction" of Chinese patent publication No. CN 110820050A.

In one embodiment, the nucleic acid molecule comprises DNA.

In one embodiment, the test sample includes, but is not limited to, cfDNA samples, disrupted genomic DNA samples, and the like. Genomic DNA can be disrupted by methods such as sonication.

In one embodiment, the sample to be tested includes, but is not limited to, at least one of a body fluid sample and a tissue sample.

In one embodiment, the bodily fluid sample includes, but is not limited to, at least one of blood, plasma, serum, saliva, pleural fluid, cerebrospinal fluid, semen, urine, and the like.

In one embodiment, the tissue sample comprises various tissue samples of a human or animal body.

According to a second aspect, in one embodiment, there is provided a method of reducing a nucleic acid molecule comprising a jagged end, comprising:

contacting said oxidation product obtained by the method of the first aspect with an organoborane to convert at least one of said oxidized 5-methylcytosine residues, 5-hydroxymethylcytosine residues to dihydrouracil residues to provide a nucleic acid molecule comprising dihydrouracil residues, i.e., a reduction product; the organoborane reduces, deaminates and decarboxylates or dealdehydes at least one of the oxidized 5-methylcytosine residues, 5-hydroxymethylcytosine residues, thereby converting at least one of the oxidized 5-methylcytosine residues, 5-hydroxymethylcytosine residues in the nucleic acid molecule to a dihydrouracil residue to yield a reduced product, i.e., a product useful for terminal repair.

In one example, in the reduction product, the 5-methylcytosine residue, the 5-hydroxymethylcytosine residue oxidized in the CpG site, CHG site, CHH site, etc. at the terminal of the serration is reduced to a dihydrouracil residue. The method can truly reflect the methylation level of cfDNA, firstly, a sample to be detected is oxidized and reduced, methylated C at the end of a sawtooth can be converted into DHU, the DHU can be used for subsequent repairing steps, and after subsequent end repairing, complementary bases of DHU sites in a newly generated chain correspond to T; in the final sequencing data, the double-stranded 3' end is read as a or T, thereby realizing double-stranded detection of methylation signals and making cfDNA methylation level calculation more realistic.

In one embodiment, the organoborane comprises a complex selected from the group consisting of boranes and nitrogen containing compounds selected from the group consisting of nitrogen heterocycles and tertiary amines.

In one embodiment, the organoborane comprises a complex of a borane and a nitrogen heterocycle.

In one embodiment, the nitrogen heterocycle comprises pyridine optionally substituted with 1-4 lower alkyl groups.

In one embodiment, the nitrogen heterocycle comprises pyridine, 2-methylpyridine, or 5-ethyl-2-methylpyridine.

In one embodiment, the organoborane is at least one of pyridine borane and 2-methyl pyridine borane.

In one embodiment, the oxidized product of the first aspect is purified and then the purified oxidized product is contacted with an organoborane to convert the oxidized 5-methylcytosine residue to a dihydrouracil residue to yield DNA comprising a dihydrouracil residue.

In one embodiment, the purification includes, but is not limited to, at least one of magnetic bead purification and column purification, and conventional magnetic bead purification and/or column purification may be used for purification.

In one embodiment, the reduction system comprises an organoborane and an organic acid salt.

In one embodiment, the organic acid salt includes, but is not limited to, sodium acetate. The organic acid salt mainly provides an acidic environment for the organoborane to promote the reduction reaction.

In one embodiment, the pH of the organic acid salt in the reduction system may be 4.0 to 4.5, and is preferably 4.3.

In one embodiment, the concentration of the organic acid salt in the reduction system may be 3M.

In one embodiment, the method further comprises a repairing step, wherein the repairing step comprises performing end repairing on the reduction product to obtain a repaired product. In the repair product, the corresponding base is A on the complementary strand of the DHU, which can be detected in the subsequent sequencing data analysis step, so that the site of the DHU is accurately identified as the 5-methylcytosine residue site in the original sequence.

In one embodiment, the repairing step further comprises performing an "a" reaction on the repaired product, and then attaching a linker to the end of the product to obtain a ligation product.

In one embodiment, the method further comprises purifying the reduction product and then performing end repair on the purified reduction product.

In one embodiment, the method of purifying the reduction product may be column purification. Purification can be carried out by conventional column purification.

In one embodiment, further comprising purifying the ligation product.

In one embodiment, the method for purifying the ligation product may be magnetic bead purification. The magnetic bead purification method is a conventional purification method.

According to a third aspect, in one embodiment, there is provided a library construction method, comprising amplifying the ligation products of the method of the first aspect to obtain a library that can be used for on-machine sequencing.

In one embodiment, after amplification, hybridization capture can be performed using the probe, and amplification can be performed again to obtain a library that can be used for on-machine sequencing.

In one embodiment, the invention innovatively constructs a method for identifying methylated bases, converting the methylated bases and then building a double-chain library.

In one embodiment, the method of the invention can reduce the true methylation state of cfDNA at the genome, whereas the prior art cannot detect the methylation state of the ends of the cfDNA either based on conventional bisulfite methylation detection methods or non-bisulfite methylation detection methods.

In one embodiment, a method for non-bisulfite cell-free DNA saw tooth end methylation detection is provided, comprising the steps of:

step A: 30ng of human cfDNA was taken.

And B: carrying out TET enzyme oxidation on the DNA fragment to obtain a DNA fragment oxidized by the TET enzyme;

and C: taking DNA treated by TET enzyme, and carrying out organoborane reduction treatment to obtain an organoborane reduction product;

step D: performing column purification on the product of pyridine borane reduction and recovering;

step E: carrying out end repair and A addition on the DNA obtained in the previous step;

step F: using ligase to connect the repair-plus A product, the adaptor and the molecular barcode (index) to obtain adaptor-plus d sDNA (double-stranded DNA);

step G: and carrying out PCR amplification on the DNA template to complete library construction.

In one embodiment, in step B, the composition of the TET enzymatic oxidation reaction system is as follows: 50mM HEPES (pH 6.0), 100 mM NaCl, 1mM alpha-ketoglutarate (also known as alpha-KG), 2mM ascorbic acid (as cobic acid), 1.2mM ATP, 1mM DTT, 100. mu.M Fe (NH)₄)₂(SO₄)₂. The buffer system comprises HE PES (pH 6.0) salt solution. Alpha-ketoglutarate and Fe (NH)₄)₂(SO₄)₂The salt is a coenzyme for TET.

In one embodiment, in step C, the organoborane reduction reaction system comprises: 35 μ L of DNA, 600mM sodium acetate (pH 4.3) buffer, 1M pyridine borane.

In one embodiment, the reaction conditions in steps E and F can be performed according to existing dsDNA end repair and addition A methods, as well as existing linker ligation reactions, e.g., can be usedUltra^TMII DNA Library Prep Kit reagent.

In one embodiment, the PCR reaction in step G requires a specific amplification enzyme, for example, KAPA HIFI Urac il polymerase.

In one embodiment, the library prepared in step G is subjected to second-generation sequencing, and according to the adaptor and index used in step F, the corresponding Illumina platform or MGI, Gene + Seq platform can be adopted.

In one embodiment, step B, after obtaining TET enzyme-oxidized DNA fragments, step F, after obtaining adaptor-added dsDNA, and step G, after obtaining amplification products, independently comprise a purification step. Purification can be performed using magnetic beads (e.g., Axygen magnetic beads).

Example 1

In this example, unless otherwise specified, the magnetic beads used were those commercially available from Axygen, USA, also known as Axyg en magnetic beads, product nameAxyPrep Magnetic Bead Purification Kits, cat # MAG-PCR-CL-250.

In the present example, "room temperature" means 23 ℃. + -. 2 ℃ unless otherwise specified.

In the present embodiment, unless otherwise specified, "80% ethanol" refers to 80% ethanol by volume, and is also referred to as "80% V/V ethanol" and is composed of ethanol and NF-H₂O (Nuclease-Free Water, also known as Nuclear-Free Water) is prepared according to the volume ratio of 80: 20.

In this example, "low TE" refers to TE Buffer, also known as TE Buffer, available from Invitrogen, cat # 12090015. The composition is as follows: 10mM Tris-HCl (pH 8.0), 0.1mM EDTA.

This example provides a method for detecting methylation of the jagged ends of non-bisulfite cfDNA, comprising the following steps:

(1) 30ng of human cfDNA sample is taken (the amount of the cfDNA sample can be 30-100 ng, in this embodiment 30 ng).

(2) TET enzymatic oxidation

1) Preparing the following TET enzyme oxidation reaction system on ice:

TABLE 1

Note: TET enzyme was purchased from NEB.

"-" indicates no corresponding data.

The composition of the TET enzyme oxidation buffer was as follows: 167mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid, pH6.0), 333 mM NaCl, 3.3mM alpha-KG (alpha-ketoglutarate), 6.67mM ascorbic acid, 4mM Adenosine Triphosphate (ATP), 8.33mM DTT.

2) Reaction conditions are as follows: 37 ℃ for 80 min.

3) mu.L proteinase K (0.8U) was added to the oxidation reaction and incubated at 50 ℃ for 1 h.

4) The product was added to 1.8 × magnetic beads.

5) Shaking, mixing and centrifuging.

6) And (5) incubating at room temperature for 5-10 min to combine the DNA with the magnetic beads.

7) The EP tube was placed on a magnetic stand for 2min and the solution was clarified.

8) The supernatant was aspirated.

9) 200 μ L of 80% V/V ethanol was added.

10) And standing for 30 s.

11) The ethanol was aspirated off. The rinsing with 80% V/V ethanol was repeated once.

12) The ethanol was evaporated at room temperature and the beads did not visibly brighten or crack.

13) mu.L of enzyme-free water was eluted.

(3) Pyridine borane reduction reaction

1) The following reduction reaction system was prepared:

TABLE 2

Components	Volume of
		3M sodium acetate (pH 4.3)	10μL
10M pyridine borane	5μL
		DNA	35μL
Total volume	50μL

The concentrations of the components in Table 2 are initial concentrations, and after mixing to form a 50. mu.L reaction system, the concentrations of the components are correspondingly reduced.

2) Reaction conditions are as follows: incubate at 37 ℃ and 1200rpm for 16 h.

3) DNA was purified using a spin column (Zymo-IC spin column in this example). Elution volume 52. mu.L.

(4) End repair and add a and add joint

1) The end repair and A addition reaction system is as follows:

TABLE 3

Mixture component	Volume of
		cfDNA	50μL
Tip repair&Adding A-buffer solution	7μL
		Tip repair&Adding A-enzyme mixture	3μL
Total volume	60μL

Repairing reaction conditions:

TABLE 4

Step (ii) of	Temperature of	Time
			Tip repair	20℃	30min
Adding A	65℃	30min
			HOLD	4℃	∞

Note: the hot lid temperature was 85 ℃.

2) And (3) connection reaction:

TABLE 5

Components	Volume of
		Tip repair&Addition of A products	60μL
Joint liquid storage	3μL
		PCR grade water	16μL
Connection enhancing liquid	1μL
		DNA ligase premix	30μL
Total volume	110μL

The ligation enhancement solution and the DNA ligase premix are reagents carried in a NEBNext terminal repair/dA tail adding module.

The linker was purchased from Chiense, MGIEasy DNA Adapters-96(Plate) kit, cat #: 1000005282.

incubation conditions were as follows: at 20 deg.C for 30 min.

3) Post-ligation purification

The purification system was as follows:

TABLE 6

The purification steps are as follows:

a) shaking, mixing and centrifuging.

b) And (5) incubating at room temperature for 5-10 min to combine the DNA with the magnetic beads.

c) The EP tube was placed on a magnetic stand for 2min and the solution was clarified.

d) The supernatant was aspirated.

e) 200 μ L of 80% ethanol was added.

f) And standing for 30 s.

g) The ethanol was aspirated off. The rinsing with 80% ethanol was repeated once.

h) The ethanol was evaporated at room temperature or 37 ℃ without significant brightening or cracking of the magnetic beads.

i) The DNA was eluted by adding 25. mu.L of enzyme-free water.

(5) Library amplification

1) Preparing the following PCR amplification reaction system:

TABLE 7

Components	Volume of
		KAPA HiFi HotStart Uracil+ReadyMix(2X)	25μL
Primer of MGI	2.5μL
		Pyridine borane-treated DNA	22.5μL
Total volume	50μL

The final concentration of the primer in the recommended system is 0.5-2 μ M, and the final concentration of the primer in the embodiment is specifically 0.75 μ M.

2) The reaction conditions were as follows:

TABLE 8

3) The PCR product was purified with 0.9X Axygen magnetic beads in the following system:

TABLE 9

Components	Volume of
		Amplification products of the previous step	50μL
Axygen magnetic bead	45μL
		Total volume	95μL

The purification steps are as follows

A. Shaking, mixing and centrifuging.

B. And (5) incubating at room temperature for 5-10 min to combine the DNA with the magnetic beads.

C. The EP tube was placed on a magnetic stand for 2min and the solution was clarified.

D. The supernatant was aspirated.

E. 200 μ L of 80% ethanol was added.

F. And standing for 30 s.

G. The ethanol was aspirated off.

H. The ethanol was evaporated at room temperature or 37 ℃ without significant brightening or cracking of the magnetic beads.

I. The library DNA was eluted by adding 25. mu.L of low TE. And the concentration was measured at about 100 ng/. mu.L using LIFE Qubit dsDNA HS Assay Kit (cat # Q32854).

J. The library fragment distribution was examined using Qsep 100 (taiwan photoding), and fig. 5 is a library fragment distribution graph.

(6) Sequencing and Credit analysis to calculate methylation levels

90G of each sample was sequenced using MGI T7 platform PE150 mode, referring to the method and apparatus for simultaneously detecting methylation level, genomic variation and insert fragment of Chinese patent publication No. CN111755072A (specifically referring to paragraphs 56-61 of the patent specification, namely steps S2 and S4), to calculate the methylation level of cfDNA, and the calculation result is shown in FIG. 7.

Comparative example 1

In this comparative example, the method, reagents and component concentrations used for the respective steps were the same as in example 1 unless otherwise specified.

This comparative example provides a conventional non-bisulfite cfDNA saw-tooth-end methylation assay, with the following detection steps:

(1) 30ng of the same human cfDNA sample as in example 1, specifically, plasma DNA of the same human was taken.

(2) End repair and addition of A

1) The end repair and A addition reaction system is as follows:

watch 10

The repair reaction conditions were as follows:

TABLE 11

Step (ii) of	Temperature of	Time
			Tip repair	20℃	30min
Adding A	65℃	30min
			HOLD	4℃	∞

Note: the hot lid temperature was 85 ℃.

2) The ligation reaction system is as follows:

TABLE 12

Components	Volume of
		Tip repair&Addition of A products	60μL
Joint liquid storage (15 mu M)	3μL
		PCR grade water	16μL
Connection enhancing liquid	1μL
		DNA ligase premix	30μL
Total volume	110μL

The ligation enhancement solution and the DNA ligase premix are reagents carried in a NEBNext terminal repair/dA tail adding module.

Incubation conditions were as follows: at 20 deg.C for 30 min.

3) Post-ligation purification

The purification system was as follows:

watch 13

Components	Volume of
		JointLigation product	110μL
Magnetic bead	88μL
		Total volume	198μL

The purification steps are as follows:

a) shaking, mixing and centrifuging.

b) And (5) incubating at room temperature for 5-10 min to combine the DNA with the magnetic beads.

c) The EP tube was placed on a magnetic stand for 2min and the solution was clarified.

d) The supernatant was aspirated.

e) 200 μ L of 80% ethanol was added.

f) And standing for 30 s.

g) The ethanol was aspirated off. The rinsing with 80% ethanol was repeated once.

h) The ethanol was evaporated at room temperature or 37 ℃ without significant brightening or cracking of the magnetic beads.

i) DNA was eluted by adding 22. mu.L of enzyme-free water.

(3) TET enzymatic oxidation

1) Preparing the following TET enzyme oxidation reaction system on ice:

TABLE 14

Components	Volume of	Final concentration
			Adaptor-ligated DNA	20μL	0.6ng/μL
TET enzyme oxidation buffer (pH 6.0)	15μL	1×
			1.5mM Fe(NH4)2(SO4)2	3.33μL	100μM
TET enzyme	4μL
			Enzyme-free water	Make up to 50. mu.L
Total volume	50μL

TET enzyme was the same as in example 1.

2) Reaction conditions are as follows: 37 ℃ for 80 min.

3) mu.L proteinase K (0.8U) was added to the oxidation reaction and incubated at 50 ℃ for 1 h.

4) The product was added to 1.8 × magnetic beads.

5) Shaking, mixing and centrifuging.

6) And (5) incubating at room temperature for 5-10 min to combine the DNA with the magnetic beads.

7) The EP tube was placed on a magnetic stand for 2min and the solution was clarified.

8) The supernatant was aspirated.

9) 200 μ L of 80% ethanol was added.

10) And standing for 30 s.

11) The ethanol was aspirated off. The rinsing with 80% ethanol was repeated once.

12) The ethanol is volatilized at room temperature, and the magnetic beads do not obviously brighten or crack.

13) Elution was performed with 37. mu.L of enzyme-free water.

(4) Pyridine borane reduction reaction

1) The following reduction reaction system was prepared:

watch 15

Components	Volume of
		3M sodium acetate (pH 4.3)	10μL
10M pyridine borane	5μL
		DNA	35μL

2) Reaction conditions are as follows: incubate at 37 ℃ and 1200rpm for 16 h.

3) DNA was purified using a spin column (Zymo-IC spin column in this comparative example). Elution volume 25. mu.L.

(5) Library amplification

1) Preparing the following PCR amplification reaction system:

TABLE 16

Components	Volume of
		KAPA HiFi HotStart Uracil+ReadyMix(2×)	25μL
Primer of MGI	2.5μL
		Pyridine borane-treated DNA	22.5μL
Total volume	50μL

Note: final concentration of primers in recommended system was 0.75 μ M.

2) The reaction conditions were as follows:

TABLE 17

3) The PCR product was purified using 0.9 XAxygen magnetic beads. The purification system was as follows:

watch 18

Components	Volume of
		Amplification products of the previous step	50μL
Axygen magnetic bead	45μL
		Total volume	95μL

The purification steps are as follows:

A. shaking, mixing and centrifuging.

B. And (5) incubating at room temperature for 5-10 min to combine the DNA with the magnetic beads.

C. The EP tube was placed on a magnetic stand for 2min and the solution was clarified.

D. The supernatant was aspirated.

E. 200 μ L of 80% ethanol was added.

F. And standing for 30 s.

G. The ethanol was aspirated off. The rinsing with 80% ethanol was repeated once.

H. The ethanol was evaporated at room temperature or 37 ℃ without significant brightening or cracking of the magnetic beads.

I. The library DNA was eluted by adding 25. mu.L of low TE. And the concentration was measured at about 100 ng/. mu.L using LIFE Qubit dsDNA HS Assay Kit (cat # Q32854).

J. The library fragment distribution was examined using Qsep 100 (taiwan photodinghy).

(6) Sequencing and Credit analysis to calculate methylation levels

Each sample was sequenced by using MGI T7 platform PE150 mode 90G, and the methylation level of cf DNA was calculated by referring to the method and apparatus for simultaneously detecting methylation level, genomic variation and insert fragment of chinese patent publication No. CN111755072A (specifically, see paragraphs 56 to 61 of this patent specification, i.e., the detection method for DNA sequencing data of a sample to be detected in steps S2 and S4), and the calculation result is shown in fig. 6, and it can be seen that the methylation level at the end of cf DNA 3' was significantly reduced.

In FIGS. 6 and 7, the abscissa indicates the site on the Reads (Reads) (in the 5 'to 3' direction) and the ordinate indicates the methylation level of the CG site. In this example, 5hmC was not blocked, and thus, "methylation level at CG site" refers to the methylation level of both 5mC and 5hmC, mainly 5 mC. "CG site" includes CpG sites, CHG sites, CHH sites, mainly CpG sites, H is a non-G base, i.e., a base other than guanine.

In one embodiment, the present invention provides a method that reduces the methylation level at the end of a serration, which truly reflects the true methylation level of cfDN a. The specific principle is as follows: cfDNA is firstly oxidized by TET enzyme and reduced by organic borane (such as pyridine borane), methylated C at the sawtooth end can be converted into DHU, and after the end is repaired, complementary base at the DHU site in a newly generated chain can correspond to T; in the final sequencing data, the double strand 3' end was read as a or T, thus achieving a double strand detection methylation signal (fig. 4), making cfDNA methylation level calculations more realistic (fig. 7).

If the A is firstly repaired and added, then TET oxidation and organoborane reduction are carried out, the methylation levels of the 5' end and the middle part of the reading are consistent, and the reflected result is the real methylation level of the genome; however, the level of methylation at the 3 'end was significantly reduced by analysis of the sequencing data from the library constructed in comparative example 1 (see FIG. 6), indicating that this method does not render the level of genomic methylation calculated at the 3' end authentic. Whereas the method of example 1 is able to restore the true methylation status (see figure 7, consistent 5 'to 3' methylation levels).

In fig. 4, lines 1a, 2a represent cfDNA original sequences, lines 1b, 2b represent newly generated sequences for end-repair, and lines 1c, 1d, 2c, 2d represent sequencing adapters. The dashed box represents the final detectable methylation signal, and it can be seen that the newly generated sequence of end-repair can detect the methylation signal and can reduce the true methylation level of cfDNA.

The conventional non-bisulfite sulfite method for detecting cfDNA methylation can cause the methylation level at the 3' end to be significantly reduced, and can not truly reflect the methylation state of cfDNA. In one embodiment, the invention can reduce the true methylation state of the 3' end of cfDNA on the genome, and can accurately calculate the methylation level of cfDNA.

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.