CN111961729A - Kit for detecting content of 5-hydroxymethylcytosine and application thereof - Google Patents
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Abstract
The invention provides a kit for detecting the content of 5-hydroxymethylcytosine and application thereof, wherein the kit is used for detecting the content of 5-hydroxymethylcytosine in a gene marker and judging the canceration condition of a liver cirrhosis patient by using the detection result of the content of 5-hydroxymethylcytosine in the gene marker; the kit has higher accuracy when being applied to liver cancer detection, can quickly react, and has higher sensitivity and specificity on liver cirrhosis detection and dynamic monitoring of liver cancer conversion from liver cirrhosis; the operability is strong, and detection can be carried out only by adopting peripheral blood of veins; compared with the conventional tissue biopsy technology, the invention has no invasiveness and can carry out frequent and multiple detections; the kit disclosed by the invention is low in detection cost and high in user acceptability of a subject.
Description
Technical Field
The invention relates to the technical field of kit detection, in particular to a kit for detecting the content of 5-hydroxymethylcytosine and application thereof.
Background
Cirrhosis is a clinically common chronic progressive disease, and the incidence of cirrhosis is mainly caused by cirrhosis after hepatitis virus infection. Complications such as upper gastrointestinal hemorrhage, hepatic encephalopathy, secondary infection, splenic hyperfunction, ascites, canceration and the like are usually accompanied, and the probability of liver cirrhosis transforming into liver cancer is as high as 70 percent, so that liver cirrhosis is not only considered as a risk factor of tumor etiology, but also is early/middle stage of tumor development. According to the statistics of the world health organization, the number of the death people caused by liver cirrhosis is only second to that of malignant tumors, cardiovascular and cerebrovascular diseases and accidents, and is at the 5 th position of the death reason. At present, serum tumor markers are mainly clinically used for detecting liver cancer high-risk groups such as liver cirrhosis patients, such as detecting alpha-fetoprotein (AFP) content, abnormal prothrombin (DCP) and the like in blood, but the sensitivities of the alpha-fetoprotein (AFP) content and the abnormal prothrombin (DCP) content and the abnormal prothrombin content are respectively less than 60% and 44%. Also, AFP levels are susceptible to pregnancy or other disease factors, such as elevated serum AFP in some chronic hepatitis patients, which can lead to false positive test results. In addition, currently, diagnostic methods such as ultrasound examination and CT examination are clinically used, but the diagnosis missing rate of simple B-ultrasound examination or CT examination is high, the imaging operation is easily affected by the experience of operators, the equipment dependence is high, the cost is high, and the diagnostic method is not suitable for frequent use. This leaves most patients with the opportunity to find early treatment early, losing the best treatment period.
Therefore, a new liver cirrhosis marker is searched, particularly a diagnosis marker for a high risk group with advanced liver cirrhosis conversion to liver cancer, so that the diagnosis rate of early liver cancer can be effectively improved, early intervention treatment is realized, and the significance for reducing the fatality rate is achieved.
Disclosure of Invention
The invention aims to provide a kit which has high detection diagnosis and low detection cost and can be applied to liver cancer detection and application thereof.
In order to achieve the purpose, the invention provides a kit for detecting the content of 5-hydroxymethylcytosine, which comprises a reagent for detecting a gene marker 5-hydroxymethylcytosine.
Further, the genetic markers include GPR132, LDAH, MSL2, LFNG, MAD1L1, GATAD2A, CASZ1, SUFU, RAD51B, and/or CAMTA 1.
Further, the reagent comprises glucosyltransferase T4-beta-GT, diphenyl cyclooctyne-tetrapolyethylene glycol-Biotin DBCO-PEG4-Biotin, streptomycin avidin immunomagnetic beads, uridine diphosphate glucose UDP-6-N3-Glu with azide modification group, spikein nucleic acid, HIFIDDNA polymerase mix, salmon sperm DNA, NGS universal amplification primer and buffer solution.
Further, the buffer solution is a buffer solution containing Tris-HCl, EDTA, NaCl and a surfactant Tween 20.
The invention also provides application of the kit for detecting the content of the 5-hydroxymethylcytosine, which comprises the steps of detecting the content of the gene marker 5-hydroxymethylcytosine and judging the canceration condition of a cirrhosis patient by detecting the content of the 5-hydroxymethylcytosine in the gene marker.
Further, the detection of the content of the gene marker 5-hydroxymethylcytosine comprises the following steps:
step 1: extracting cfDNA of patient sample plasma;
step 2: performing fragment length detection on the extracted cfDNA; performing end repair and completion on the screened cfDNA;
and step 3: connecting the DNA with the filled end with a sequencing joint to obtain a connection product;
and 4, step 4: transferring the sugar UDP-6-N3-glucose modifying group containing an azide modifying group to the hydroxymethyl group of 5-hydroxymethylcytosine by glucosyltransferase T4-beta-GT;
and 5: adding a molecule of biotin diphenyl cyclooctyne-tetraethylene glycol-biotin to 5-hydroxymethyl cytosine marked by an azide group by a click chemistry method;
step 6: combining a DNA fragment containing a 5-hydroxymethyl cytosine label on a solid phase material streptomycin avidin immunomagnetic bead through a solid phase affinity reaction;
and 7: removing unbound DNA fragments by multiple washes using a buffer containing Tris-HCl, EDTA, NaCl and surfactant Tween 20;
and step 9: performing PCR amplification by using DNA combined on streptavidin immunomagnetic beads as a template to prepare a sequencing library; the preparation process of the sequencing library comprises a plurality of purification steps, and a magnetic bead method is selected for purification;
step 10: performing quality inspection on the sequencing library;
step 11: and uniformly mixing a certain number of libraries containing different barcode according to the same molar concentration, and performing on-machine sequencing by using a standard method according to a second-generation sequencing instrument to obtain a sequencing result.
Step 12: and (3) carrying out data analysis on the sequencing result to obtain an FPKM value corresponding to reads in a differential gene region, and comparing an AD-wdscore threshold value by using an AD-wdscore calculation formula in combination with clinical AFP and DCP indexes to finally judge that the current patient is in a liver cirrhosis or liver cancer state.
Further, the method for judging the canceration of the cirrhosis patients by utilizing the detection of the content of the 5-hydroxymethylcytosine in the gene marker comprises the following steps:
analyzing sequencing data, carrying out homogenization treatment on sequencing depth by using FPKM (fragment Per Kilobase Per Million), and determining the content of a gene marker 5-hydroxymethylcytosine;
step b: performing mathematical correlation on the 5-hydroxymethylcytosine content of the gene marker in the step a to obtain a diagnosis score wdscore;
combining the diagnosis value wdscore with clinical serological detection results AFP and DCP to obtain AD-wdscore;
step d: obtaining the detection result of a subject by taking the threshold value of the diagnostic score AD-wdscore of the 5-hydroxymethylcytosine content of the gene marker in the kit which can distinguish the liver cancer and the cirrhosis sample as the standard;
further, an Ad-wdscore value greater than 3.382203 indicates that the subject has liver cancer.
Compared with the prior art, the invention has the advantages that: according to the invention, by designing the kit for detecting the content of 5-hydroxymethylcytosine, the detection means for detecting the cirrhosis is realized by the content of 5-hydroxymethylcytosine on the gene marker, and the source range of the used DNA sample is wide. Therefore, the detection method of the invention has the following advantages: (1) the accuracy is high, the rapid reaction can be realized, and the sensitivity and the specificity for the liver cirrhosis detection and the dynamic monitoring of the liver cirrhosis converted into the liver cancer are high; (2) the operability is strong, and the detection can be carried out only by adopting peripheral blood; (3) the method is non-invasive, and the acceptability of a subject user is high; (4) the cost is low, and frequent and multiple detections can be carried out.
According to the kit detection, based on the characteristic that 5-hydroxymethylcytosine can be subjected to glycosylation modification by T4-beta-glucosyltransferase, an N3 modified glucose group is transferred to the 5-hydroxymethylcytosine, then a sequence containing glycosylation modification is captured by adopting a click chemistry method, the characteristic of high reaction efficiency of click chemistry is utilized, the capture of a target sequence with great efficiency is realized, and the efficiency is far higher than that of a capture method using an antibody and the like. The method is particularly suitable for detecting the nucleic acid with extremely low sample starting amount, namely cfDNA.
5-hydroxymethylcytosine is an epigenetic modification, and the change of the modification abundance is proved to be closely related to the occurrence and development of various diseases, including cancer, and the change of epigenetic modification generally occurs at the early stage of cancer development, so that the diagnosis at the early stage of cancer is more suitable than the mutation related to cancer.
Drawings
FIG. 1 is a line graph of forest acuity and specificity for differentiation of liver cancer and cirrhosis using differential markers obtained by screening in combination with clinical markers
FIG. 2 is a graph showing the result distribution of AD-wdscore diagnostic model for differentiating patients with liver cancer and liver cirrhosis.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be further described below.
The invention aims to provide a kit which has high detection diagnosis and low detection cost and can be applied to liver cancer detection and application thereof.
In order to achieve the purpose, the invention provides a kit for detecting the content of 5-hydroxymethylcytosine, which comprises a reagent for detecting a gene marker 5-hydroxymethylcytosine.
In this example, the gene markers include GPR132, LDAH, MSL2, LFNG, MAD1L1, GATAD2A, CASZ1, SUFU, RAD51B, and/or CAMTA 1.
In this example, reagents include glucosyltransferase T4- β -GT, diphenylcyclooctyne-tetrapolyethylene glycol-Biotin DBC0-PEG4-Biotin, streptavidin magnetic beads, uridine diphosphate glucose UDP-6-N3-Glu with azide modification, spikein nucleic acid, HIFIDDNA polymerase mix, salmon sperm DNA, NGS universal amplification primers, and buffers.
In this example, the buffer is a buffer containing Tris-HCl, EDTA, NaCl and the surfactant Tween 20.
The invention also provides application of the kit for detecting the content of the 5-hydroxymethylcytosine, which comprises the steps of detecting the content of the gene marker 5-hydroxymethylcytosine and judging the canceration condition of a cirrhosis patient by detecting the content of the 5-hydroxymethylcytosine in the gene marker.
In this embodiment, the detection of the content of the gene marker 5-hydroxymethylcytosine comprises the following steps:
step 1: extracting cfDNA of patient sample plasma;
step 2: performing fragment length detection on the extracted cfDNA; performing end repair and completion on cfDNA;
and step 3: connecting the DNA with the filled end with a sequencing joint to obtain a connection product;
and 4, step 4: transferring the sugar UDP-6-N3-glucose modifying group containing an azide modifying group to the hydroxymethyl group of 5-hydroxymethylcytosine by glucosyltransferase T4-beta-GT;
and 5: adding a molecule of biotin diphenyl cyclooctyne-tetraethylene glycol-biotin to 5-hydroxymethyl cytosine marked by an azide group by a click chemistry method;
step 6: the DNA fragment containing the 5-hydroxymethyl cytosine label is combined on the solid phase material streptomycin avidin immunomagnetic bead through a solid phase affinity reaction. And 7: removing unbound DNA fragments by multiple washes using a buffer containing Tris-HCl, EDTA, NaCl and surfactant Tween 20;
and 8: performing PCR amplification by using DNA combined on streptavidin immunomagnetic beads as a template to prepare a sequencing library; the preparation process of the sequencing library comprises a plurality of purification steps, and a magnetic bead method is selected for purification;
step 10: performing quality inspection on the sequencing library;
step 11: uniformly mixing a certain number of libraries containing different barcode according to the same molar concentration, and performing on-machine sequencing by using a standard method according to a second-generation sequencing instrument to obtain a sequencing result;
step 12: and (3) carrying out data analysis on the sequencing result to obtain an FPKM value corresponding to reads in a differential gene region, and comparing an AD-wdscore threshold value by using an AD-wdscore calculation formula in combination with clinical AFP and DCP indexes to finally judge that the current patient is in a liver cirrhosis or liver cancer state.
In this embodiment, the determination of the carcinogenesis of a cirrhosis patient by detecting the content of 5-hydroxymethylcytosine in the gene marker comprises the following steps:
analyzing sequencing data, carrying out homogenization treatment on sequencing depth by using FPKM (fragment Per Kilobase Per Million), and determining the content of a gene marker 5-hydroxymethylcytosine;
step b: performing mathematical correlation on the 5-hydroxymethylcytosine content of the gene marker in the step a to obtain a diagnosis score wdscore;
combining the diagnosis value wdscore with clinical serological detection results AFP and DCP to obtain AD-wdscore;
step d: obtaining the detection result of a subject by taking the threshold value of the diagnostic score AD-wdscore of the 5-hydroxymethylcytosine content of the gene marker in the kit which can distinguish the liver cancer and the cirrhosis sample as the standard;
in this example, an Ad-wdscore value greater than 3.382203 indicates that the subject has liver cancer. The invention will be further explained with reference to experimental data and examples below:
in this example, the content of 5-hydroxymethylcytosine (5hmC) is first detected by a kit:
(1) extracting plasma cfDNA:
10ng of plasma cfDNA was extracted from 127 cirrhosis patient samples, which can be extracted by any method well known to those skilled in the art to be suitable for extracting plasma cfDNA.
(2) Add a to the cfDNA end and ligate with sequencing adapters:
a system containing 10ng of plasma free DNA, 15. mu.L of End-Prep mix4, 1. mu.L of spike in and supplemented with nucleic-free water to a total volume of 50. mu.L was prepared in PCR tubes according to the Vazyme DNA Library Prep Kit instructions, incubated at 20 ℃ for 30 minutes and then at 65 ℃ for 15 minutes. To the reaction mixture were added 25. mu.L of Rapid Ligation buffer2, 5. mu.L of Rapid DNA Ligase, 1. mu.L of adapter, and a system supplemented with nucleic-freewater to a total volume of 100. mu.L, incubated at 20 ℃ for 15 minutes, and then maintained at 4 ℃. The reaction product was purified using AmpureXP beads and eluted with 21. mu.L of nucleic-free water to give the final DNA ligation sample.
(3) 5-hydroxymethylcytosine labeling:
a total volume of 25. mu.L of labeling reaction mixture was prepared: t4 phage β -glucosyltransferase (β -GT), uridine diphosphate glucose with azide modification (UDP-N3-Glu), 10 × Buffer, and 21 μ L of the purified product described above. Incubating the mixture at 37 ℃ for 2 hours; adding 2.5 μ L of diphenylcyclooctyne-tetraethylene glycol biotin to the reaction product, and incubating at 37 ℃ for 2 hours; 10. mu.g of shredded salmon sperm DNA (salmon sperm DNA) was added to the reaction mixture, and the reaction mixture was purified using Micro Bio-spin 30 column from Bio-Rad, and the volume of the purified product was 50. mu.L.
(4) Solid phase enrichment of DNA fragments containing labeled 5-hydroxymethylcytosine:
firstly, a magnetic bead preparation step is performed: 5 μ L of streptavidin immunomagnetic beads C1 streptadvin beads (life technology) were removed and blown uniformly and placed on a magnetic frame, after clarification the supernatant was aspirated, 50 μ L of 2 buffer1(1M pH7.5 Tris, 0.5M EDTA, 5M NaCl, Tween20) was added to the frame and incubated for 3min, after clarification the supernatant was aspirated, and 50 μ L of 2 buffer1 was added to blow uniformly resuspend the beads.
Then, the magnetic beads were mixed with the above-mentioned purified labeled product at a volume ratio of 1:1 (50. mu.L each), and the mixture was mixed in a rotary mixer for 30 minutes to allow sufficient binding.
Then, the column was eluted with 100. mu.L of buffer1(1X), buffer2(1X), buffer3, and buffer4, respectively, and each buffer was washed twice, each for 5min on a rotating rack (spin-off immediately after spinning to avoid loss of the cover liquid).
(5) And (3) PCR amplification:
a total volume of 50. mu.L of a nucleic-free water reaction system containing 25. mu.L of VAHTS HiFi amplification Mix, 2. mu.L of PCR Primer Mix 3 for Illumina, and 23. mu.L was prepared, and the reaction mixture was added to the washed magnetic beads for amplification according to the following PCR reaction conditions:
the amplification product was purified using AmpureXP beads to obtain the final sequencing library.
(6) High throughput sequencing after quality inspection of the sequencing library:
the obtained sequencing library was subjected to concentration determination using Qubit and the size content of the library DNA fragments was determined using LabChip GX Touch. The sequencing library by quality inspection can be used for high-throughput sequencing, a certain number (1-96) of libraries containing different barcode are uniformly mixed according to the same molar concentration, and the on-machine sequencing is carried out by using a standard method according to a second-generation sequencing instrument.
Sequencing data analysis determined the weighting coefficients of the gene markers and the diagnostic model AD-wdscore:
and performing primary quality control on the obtained 5hmC sequencing result, cutting off a linker sequence and a low-quality sequencing site, and comparing sequencing data qualified in quality control to a human standard reference genome by using bowtie 2. Reads perfectly matched to the reference genome were extracted with samtools and the duplicate was removed with picard. 5hmC enriched site information is obtained through macs2 callpeak, and a 5hmC enriched region with a fixed length of 201bp is obtained by amplifying 100bp before and after summit. The 5hmC modification level (FPKM) was then counted for each 5hmC enriched region using the bedtools. And modeling and analyzing the data by using a partial least square method and an elastic network to obtain a weighting coefficient of each model marker and scoring the sample to obtain a weighted-diagnostic score (weighted-diagnostic score). Combining wdscore with clinical serological indicators AFP and DCP to obtain a diagnostic score (AD-wdscore) that distinguishes between cirrhosis and liver cancer, and using the detection of the score to distinguish between cirrhosis and liver cancer patients.
AD-wdscore formula:
AD-wdscore＝0.528*log2(AFP)+0.3935*log2(DCP)+1.8577*wdscore
the 10 early stage liver cirrhosis canceration early stage markers capable of distinguishing liver cirrhosis and liver cancer are used. As shown in FIG. 1, the use of the marker of the present invention showed a sensitivity of about 0.9756 and a specificity of about 0.9167 in samples of 117 patients with liver cirrhosis and 134 patients with liver cancer. As shown in FIG. 2, the differentiation of liver cirrhosis and liver cancer by AD-wdscore (threshold 3.382203) is significantly better than that by the existing clinical indexes of AFP (threshold 40mg/mL) and DCP (threshold 40 mg/mL).
The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any way. It will be understood by those skilled in the art that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. A kit for detecting the content of 5-hydroxymethylcytosine is characterized by comprising a reagent for detecting a gene marker 5-hydroxymethylcytosine.
2. The kit for detecting 5-hydroxymethylcytosine according to claim 1, wherein the gene markers include GPR132, LDAH, MSL2, LFNG, MAD1L1, GATAD2A, CASZ1, SUFU, RAD51B and/or CAMTA 1.
3. The kit for detecting 5-hydroxymethylcytosine content according to claim 1, wherein the reagents comprise glucosyltransferase T4- β -GT, diphenylcyclooctyne-tetrapolyethylene glycol-Biotin DBCO-PEG4-Biotin, magnetic strepavidin beads, uridine diphosphate glucose UDP-6-N3-Glu with azide modification group, spike in nucleic acid, HIFIDNA polymerase mix, salmon sperm DNA, NGS universal amplification primers and buffer.
4. The kit for detecting the content of 5-hydroxymethylcytosine according to claim 3, wherein the buffer is a buffer containing Tris-HCl, EDTA, NaCl and a surfactant Tween 20.
5. The application of the kit for detecting the content of 5-hydroxymethylcytosine is characterized by comprising the steps of detecting the content of 5-hydroxymethylcytosine serving as a gene marker and judging the canceration condition of a cirrhosis patient by using the detection of the content of 5-hydroxymethylcytosine in the gene marker.
6. The use of the kit for detecting the content of 5-hydroxymethylcytosine according to claim 5, wherein the detection of the content of the gene marker 5-hydroxymethylcytosine comprises the following steps:
step 1: extracting cfDNA of patient sample plasma;
step 2: performing fragment length detection on the extracted cfDNA; performing end repair and completion on the screened cfDNA;
and step 3: connecting the DNA with the filled end with a sequencing joint to obtain a connection product;
and 4, step 4: transferring the modification group of uridine diphosphate glucose UDP-6-N3-Glu with an azide modification group to the hydroxymethyl group of 5-hydroxymethylcytosine by glucosyltransferase T4-beta-GT;
and 5: adding a molecule of Biotin diphenyl cyclooctyne-tetraethylene glycol-Biotin DBCO-PEG4-Biotin on 5-hydroxymethyl cytosine marked by an azide group by a click chemistry method;
step 6: combining a DNA fragment containing a 5-hydroxymethyl cytosine label on a solid phase material streptomycin avidin immunomagnetic bead through a solid phase affinity reaction;
and 7: removing unbound DNA fragments by multiple washes using a buffer containing Tris-HCl, EDTA, NaCl and surfactant Tween 20;
and 8: performing PCR amplification by using DNA combined on streptavidin immunomagnetic beads as a template to prepare a sequencing library; the preparation process of the sequencing library comprises a plurality of purification steps, and a magnetic bead method is selected for purification;
and step 9: performing quality inspection on the sequencing library;
step 10: and uniformly mixing a certain number of libraries containing different barcode according to the same molar concentration, and performing on-machine sequencing by using a standard method according to a second-generation sequencing instrument to obtain a sequencing result.
7. The use of the kit for detecting the content of 5-hydroxymethylcytosine according to claim 5, wherein the detection of the content of 5-hydroxymethylcytosine in the gene markers for judging the canceration of the cirrhosis patients comprises the following steps:
analyzing sequencing data, carrying out homogenization treatment on sequencing depth by using FPKM (fragment Per Kilobase Per Million), and determining the content of a gene marker 5-hydroxymethylcytosine;
step b: performing mathematical correlation on the 5-hydroxymethylcytosine content of the gene marker in the step a to obtain a diagnosis score wdscore; combining the diagnosis value wdscore with clinical serological detection results AFP and DCP to obtain AD-wdscore; (ii) a
Step d: the detection result of the subject is obtained by taking the threshold value of the diagnostic score AD-wdscore of the 5-hydroxymethylcytosine content of the gene marker which can distinguish the liver cancer and the cirrhosis sample and is provided by the kit of the invention as the standard.
8. The use of the kit according to claim 7, wherein an AD-wdscore value greater than 3.382203 indicates that the subject has liver cancer.
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Cited By (7)
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| CN113061652A (en) * | 2021-03-30 | 2021-07-02 | 成都泰莱医学检验实验室有限公司 | Method for determining 5hmC content in gene marker based on glucose modification |
| CN113151482A (en) * | 2021-05-21 | 2021-07-23 | 深圳泰莱生物科技有限公司 | Method for identifying benign and malignant lung nodules based on monochromatic multiple fluorescence quantitative PCR |
| CN113249446A (en) * | 2021-04-13 | 2021-08-13 | 中山大学 | Quantitative method of 5hmC level of whole genome based on nucleic acid isothermal amplification and application thereof |
| CN113430255A (en) * | 2021-07-19 | 2021-09-24 | 深圳泰莱生物科技有限公司 | Lung cancer detection method based on 5hmC click chemistry high-throughput sequencing technology |
| CN113528616A (en) * | 2021-07-26 | 2021-10-22 | 深圳泰莱生物科技有限公司 | Detection method for capturing cfDNA5hmC fragment |
| WO2023040997A1 (en) * | 2021-09-17 | 2023-03-23 | 北京大学 | Single gene test method and application thereof |
| CN115992203A (en) * | 2022-07-26 | 2023-04-21 | 生工生物工程（上海）股份有限公司 | Method for constructing genome-wide hydroxymethylation capture sequencing library |
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- 2020-09-01 CN CN202010902373.3A patent/CN111961729A/en active Pending
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| CN113061652A (en) * | 2021-03-30 | 2021-07-02 | 成都泰莱医学检验实验室有限公司 | Method for determining 5hmC content in gene marker based on glucose modification |
| CN113249446A (en) * | 2021-04-13 | 2021-08-13 | 中山大学 | Quantitative method of 5hmC level of whole genome based on nucleic acid isothermal amplification and application thereof |
| CN113151482A (en) * | 2021-05-21 | 2021-07-23 | 深圳泰莱生物科技有限公司 | Method for identifying benign and malignant lung nodules based on monochromatic multiple fluorescence quantitative PCR |
| CN113430255A (en) * | 2021-07-19 | 2021-09-24 | 深圳泰莱生物科技有限公司 | Lung cancer detection method based on 5hmC click chemistry high-throughput sequencing technology |
| CN113528616A (en) * | 2021-07-26 | 2021-10-22 | 深圳泰莱生物科技有限公司 | Detection method for capturing cfDNA5hmC fragment |
| WO2023040997A1 (en) * | 2021-09-17 | 2023-03-23 | 北京大学 | Single gene test method and application thereof |
| CN115992203A (en) * | 2022-07-26 | 2023-04-21 | 生工生物工程（上海）股份有限公司 | Method for constructing genome-wide hydroxymethylation capture sequencing library |
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