Library contamination specific degradation method and kit based on methylation sensitive restriction enzymes

Abstract

This application relates to a method and kit for the specific degradation of library contaminants based on a methylation-sensitive restriction endonuclease. The method includes: determining the recognition site of the methylation-sensitive restriction endonuclease, digesting library contaminants, fragment purification, verification, and quality control. In the library contaminant digestion step, the library reaction system is incubated at 35°C–39°C for 0.5–1.5 hours, followed by incubation at 60°C–70°C for 15–25 minutes to obtain the digested DNA library reaction system. This method selectively removes unmethylated DNA while having almost no effect on highly methylated DNA, avoiding non-specific degradation and effectively improving the purity of the target fragment.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a method and kit for the specific degradation of library contamination based on methylation-sensitive restriction endonucleases. Background Technology

[0002] The principle of library contamination-specific degradation methods based on methylation-sensitive restriction endonucleases in existing technologies is to degrade library contamination by utilizing the specific recognition and cleavage of DNA sequences in specific methylation states by methylation-sensitive restriction endonucleases. First, genomic DNA is extracted and fragmented from the biological sample. Then, the methylation-sensitive restriction endonuclease is activated; this enzyme only cleaves unmethylated DNA sequences without affecting methylated portions. Next, library construction is performed, including end repair and adapter ligation. Then, the target DNA sequence is captured by hybridization with specific probes. Non-specifically bound DNA is removed through elution and purification to obtain a pure library. Finally, the library quality is tested using instruments to ensure it meets requirements.

[0003] However, existing technologies have some limitations, such as enzyme digestion efficiency and kit stability and reproducibility. The digestion efficiency of methylation-sensitive restriction endonucleases needs further optimization to ensure that all unmethylated DNA sequences are effectively cleaved, avoiding the influence of uncut contaminating DNA on subsequent experimental results. Although methylation-sensitive restriction endonucleases are specific to DNA sequences with specific methylation states, in practical applications, misjudgment of methylation states may occur, leading to the cleavage of partially methylated DNA or the unequal cleavage of unmethylated DNA. The methylation-sensitive restriction endonucleases and other reagents in the kit need to be stored and transported under specific conditions to ensure their activity and stability. Improper storage conditions may lead to reduced or inactivated enzyme activity, affecting experimental results. Various factors during the experimental process, such as sample processing and enzyme digestion conditions, may result in poor reproducibility of experimental results. Furthermore, methylation-sensitive detection methods also have certain limitations; they may not be effective in detecting low-abundance methylation sites or contaminating DNA. Compared with other high-throughput DNA methylation detection methods, methods based on methylation-sensitive restriction endonucleases may have limitations in detection throughput.

[0004] Therefore, it is necessary to study a method for the specific degradation of library contamination that has high enzyme digestion efficiency, good stability, high reproducibility, and is suitable for this method, as well as a kit. Summary of the Invention

[0005] The purpose of this application is to provide a library contamination-specific degradation method based on methylation-sensitive restriction endonucleases, which can effectively degrade contaminating DNA and has significant advantages such as high specificity, simple operation, and good retention of target DNA. To achieve the above objective, the following technical solution is proposed.

[0006] A method for the specific degradation of library contamination based on a methylation-sensitive restriction endonuclease includes the following steps:

[0007] (1) Determine the recognition sites of methylation-sensitive restriction endonucleases;

[0008] (2) Enzyme digestion of library contaminants: Prepare a DNA library reaction system, wherein the DNA includes host DNA and target DNA; add the methylation-sensitive restriction endonuclease to the DNA library reaction system and incubate, and obtain the enzyme-digested DNA library reaction system after incubation;

[0009] (3) Fragment purification: The DNA library reaction system after enzyme digestion was selectively purified using magnetic beads to obtain a purified DNA library, wherein the DNA fragments in the purified DNA library are larger than 200 bp;

[0010] (4) Validation and quality control: Amplification and sequencing were used to detect the removal rate of host DNA and the retention rate of target DNA in the purified DNA library;

[0011] The target DNA includes human genomic DNA, and the recognition site of the methylation-sensitive restriction endonuclease includes at least one of the following sites:

[0012] chr11:2,180,310-2,180,313;

[0013] chr6:22,146,094-22,146,097;

[0014] chr17:7,845,201-7,845,204;

[0015] chr1:48,616,494-48,616,497.

[0016] The reference gene for this site is hg38 from the UCSC database.

[0017] Preferably, in the enzyme digestion of library contaminants step, the library reaction system is incubated at 35°C-39°C for 0.5-1.5 hours, followed by incubation at 60°C-70°C for 15-25 minutes.

[0018] Preferably, determining the recognition site of the methylation-sensitive restriction endonuclease includes the following steps:

[0019] (1) Obtain methylation data of the host DNA and the target DNA;

[0020] (2) Analyze the differentially methylated sites based on the methylation data;

[0021] (3) Screening for methylation-sensitive restriction endonuclease recognition sites from the differentially methylated sites. Preferably, the analytical formula for the differentially methylated sites is M = log2((β + α) / (1 - β + α)); where M is the methylation intensity, β is the methylation ratio, and α is a smoothing factor to prevent the denominator from being zero, with the value of α ranging from 0.001 to 0.01.

[0022] Preferably, the screening conditions for the above-mentioned differentially methylated sites are: Δβ ≥ 0.70, β_target ≥ 0.75, and β_host ≤ 0.15 in ≥90% of normal tissues.

[0023] Preferably, the methylation-sensitive restriction endonuclease includes HpaII and HinP1I.

[0024] Preferably, the amount of the methylation-sensitive restriction endonuclease used is 10-20 U / μg DNA.

[0025] Preferably, the volume of the magnetic beads is 0.6-1.0 times that of the library reaction system.

[0026] This application also provides a kit for the specific degradation of library contaminants, which implements the above-described method for the specific degradation of library contaminants based on methylation-sensitive restriction endonucleases.

[0027] Preferably, the kit includes: a methylation-sensitive restriction endonuclease, a reaction buffer, a magnetic bead purification reagent, a positive control, and a negative control.

[0028] Preferably, the reaction buffer solution includes Tris-HCl, NaCl, MgCl2, and DTT.

[0029] Preferably, the positive control is an artificially prepared DNA mixture containing recognition sites for methylation-sensitive restriction endonucleases HpaII and HinP1I, used to verify the correlation between enzyme digestion and methylation status.

[0030] Preferably, the negative control is unmethylated human genomic DNA and contains at least one recognition sequence of the methylation-sensitive restriction endonuclease to ensure the specific detection capability of the enzyme digestion reaction.

[0031] This application has the following beneficial effects:

[0032] By screening sites with significant methylation differences between host DNA and target DNA in the early stages, precise cutting points are provided for subsequent enzyme digestion, fundamentally ensuring the specificity and effectiveness of the digestion reaction. During the digestion process, methylation-sensitive restriction endonucleases such as HpaII and HinP1I are used to cleave only unmethylated sites, selectively removing unmethylated host DNA while having almost no effect on the highly methylated target DNA. This avoids non-specific degradation and effectively improves the purity of the target fragment.

[0033] Furthermore, the enzyme digestion system in this application is rationally designed. The enzyme concentration of 10-20 U / μg DNA, combined with incubation at 35-39℃ for 0.5-1.5 hours and a heat inactivation step at 60-70℃, not only ensures sufficient digestion efficiency but also avoids interference from residual enzyme activity in the library. Following this, selective purification using 0.8× magnetic beads effectively removes enzyme fragments smaller than 200 bp, further improving library quality while preserving the complete target fragment. This physical screening step complements the preceding enzymatic treatment, ensuring maximum removal of host DNA fragments while completely preserving the target sequence.

[0034] In the validation phase, qPCR was used for ΔCt analysis, providing quantitative references for contamination removal rate and target retention rate. A ΔCt ≥ 3 for host DNA indicates a contamination removal rate of over 90%, while a ΔCt ≤ 0.5 for target DNA indicates a target retention rate of over 75%. This balanced approach fully demonstrates the reliability of the method. Finally, high-throughput sequencing was used to assess the decrease in the proportion of host DNA before and after treatment, further proving that this method can significantly reduce the proportion of host DNA in actual libraries, balancing sensitivity and specificity. Therefore, this patented method not only achieves precise removal of contaminating DNA at the molecular level but also possesses comprehensive advantages in practical operation, including strong reproducibility, controllable cost, and good target retention. This greatly improves library quality and the reliability of downstream sequencing data, providing strong technical support for high-purity microbial detection, liquid biopsy, and environmental sample analysis. Attached Figure Description

[0035] The accompanying drawings described below are incorporated in and form part of this specification, illustrating embodiments consistent with this application, and together with the description serve to explain the principles of this application.

[0036] Figure 1 This is the qPCR melting curve from an experimental example in this application.

[0037] Figure 2 This shows the changes in Ct values ​​of the host DNA and target DNA before and after contamination degradation treatment in an experimental example of this application.

[0038] Figure 3This is a comparison chart showing the proportions of host DNA and target DNA before and after pollutant degradation treatment in an experimental example of this application.

[0039] Figure 4 This is the qPCR detection result after purification using magnetic beads of different volumes in one experimental example of this application. Detailed Implementation

[0040] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] In the following implementation, host DNA refers to DNA molecules derived from a contaminated background, commonly introduced exogenously during library construction, such as normal tissue DNA from human samples, human background DNA, or host cell genomic DNA introduced during experimental procedures. Host DNA typically carries CpG sites with low methylation levels, making it easily recognized and cleaved by methylation-sensitive restriction endonucleases. Target DNA refers to the DNA to be sequenced, usually the nucleic acid sequence of the research object itself, such as tumor tissue DNA, microbial DNA, extracellular DNA (cfDNA), or exogenous hypermethylated DNA. The CpG sites of target DNA are often in a hypermethylated state, making them difficult to recognize by methylation-sensitive restriction endonucleases, thus remaining after enzyme digestion.

[0042] This application provides a method for the specific degradation of library contamination based on a methylation-sensitive restriction endonuclease, comprising the following steps:

[0043] (1) Degradation of library contaminants: A two-enzyme system was used to degrade contaminants;

[0044] (2) Fragment purification: Magnetic bead method for selective purification of fragments larger than 200 bp;

[0045] (3) Validation and quality control: qPCR and sequencing were used to detect the contamination removal rate and the retention rate of the target DNA;

[0046] In the library pollutant degradation step, the reaction conditions of the library reaction system are as follows:

[0047] Incubate at 35°C-39°C for 0.75-1.25 hours; then incubate at 60°C-70°C for 15-25 minutes.

[0048] In one optional implementation, the library reaction system includes:

[0049] Library DNA, methylation-sensitive restriction endonuclease, buffer, and water.

[0050] In an optional implementation, the library DNA is pre-fragmented to 300-500 bp.

[0051] In an optional implementation, the concentration of the library DNA in the system is 10-100 ng / μL.

[0052] In an optional embodiment, the methylation-sensitive restriction endonuclease is HpaII and / or HinP1I. This is used to specifically cleave unmethylated contaminating DNA.

[0053] In an optional embodiment, the concentration of the methylation-sensitive restriction endonuclease in the system is 10-20 U / μg DNA.

[0054] In an optional embodiment, the buffer solution comprises Tris-HCl, NaCl, MgCl2, and DTT. The buffer solution is used to provide an optimal environment for enzyme activity.

[0055] In an optional embodiment, the library reaction system further includes serum protein, preferably calf serum protein.

[0056] In an optional implementation, the reaction conditions for the above-described library reaction system are as follows:

[0057] The entire reaction system can achieve efficient enzymatic digestion by incubating at 35°C-39°C for 0.5-1.5 hours. If more thorough digestion is required, the incubation time can be extended to 2-2.5 hours. Subsequently, incubation at 60°C-70°C for 15-25 minutes will completely inactivate the two restriction enzymes, ensuring that subsequent operations are not interfered with. The entire process should be carried out in an enzyme-free environment using ultrapure water free of RNase and DNase.

[0058] In an optional embodiment, the reaction conditions are incubation at 37°C for 1 hour, followed by incubation at 65°C for 20 minutes.

[0059] In an optional implementation, the total volume of the library reaction system is less than 20 μL.

[0060] In an optional implementation, the library contaminants are degraded and then purified to remove the small fragments that have been cleaved by enzymes.

[0061] In an alternative embodiment, the enzyme-digested fragments include host DNA or degraded fragments.

[0062] In an optional implementation, the fragments after enzyme digestion are less than 200 bp.

[0063] In an alternative implementation, magnetic beads are used for fragment purification.

[0064] In an optional implementation, the magnetic bead method includes the following steps:

[0065] Add magnetic beads to the reaction system;

[0066] Allowing the magnetic beads to stand allows them to bind to DNA.

[0067] The magnetic beads are placed on the magnetic rack to attract them.

[0068] Remove the supernatant;

[0069] washing;

[0070] dry;

[0071] Elute the target DNA.

[0072] In an alternative implementation, the supernatant contains fragments less than 200 bp and irrelevant contaminants.

[0073] In one alternative embodiment, the volume of the magnetic beads is 0.6-1.0 times the volume of the library reaction system.

[0074] In an optional implementation, the volume of the magnetic beads is 0.8 times the volume of the library reaction system. This effectively removes short fragments below 200 bp while retaining fragments larger than 200 bp.

[0075] In an alternative implementation, the DNA is allowed to bind at room temperature for 3-8 minutes.

[0076] In an optional implementation, the DNA is allowed to stand at room temperature for 5 minutes. This enhances the binding efficiency of the DNA to the magnetic beads and improves the recovery rate.

[0077] In one alternative implementation, the magnetic beads are adsorbed for 3-8 minutes.

[0078] In an optional implementation, the magnetic beads are washed with 78-85% ethanol.

[0079] In an optional implementation, the magnetic beads are washed with 80% ethanol. This reduces non-specific binding and improves fragment purity.

[0080] In an optional implementation, the washing step is repeated 1-2 times.

[0081] In one alternative implementation, drying can be either air drying or oven drying.

[0082] In one optional implementation, the drying time is 1-2 minutes. Drying can be stopped when the magnetic beads are observed to turn slightly white.

[0083] The above drying method and drying time can prevent the magnetic beads from becoming too dry, which would reduce the elution efficiency.

[0084] In an optional implementation, the target DNA is eluted using a buffer solution.

[0085] In an alternative implementation, the buffer solution is an EB or ET buffer solution.

[0086] In an optional implementation, the elution conditions are incubation at 35-39°C for 1-3 minutes.

[0087] In an optional implementation, the elution condition is incubation at 37°C for 2 minutes. This can improve the target DNA elution rate and facilitate subsequent library amplification.

[0088] In an optional implementation, qPCR is used for the detection and quality control of the library after pollutant degradation.

[0089] In an optional implementation, qPCR is used to quantitatively analyze the contaminant removal rate, ΔCt = Ct 处理 - Ct 未处理 A host DNA ΔCt ≥ 3 indicates a reduction of approximately 90% in contaminating DNA. Wherein, Ct... 处理 This refers to the number of cycles required for a library sample treated with a methylation-sensitive restriction endonuclease to reach a set fluorescence threshold during qPCR amplification; Ct 未处理 This refers to the number of cycles required to reach a set fluorescence threshold when the same library is amplified by qPCR without restriction endonuclease treatment.

[0090] In an optional implementation, qPCR is used to quantitatively analyze the retention rate of the target DNA, ΔCt = Ct 处理 -Ct 未处理 If ΔCt≤0.5 for the target DNA, it means that ≥75% of the target DNA is retained.

[0091] In an alternative implementation, changes in the proportion of sequences in the library are assessed by sequencing alignment analysis in conjunction with qPCR quantitative analysis.

[0092] The methylation differential sites include at least one of the following physical locations:

[0093] chr11: 2,180,310;

[0094] chr6: 22,146,094;

[0095] chr17: 7,845,201;

[0096] chr1: 48,616,494;

[0097] The reference gene for the above sites was selected from hg38 in the UCSC database.

[0098] The aforementioned differentially methylated sites are located on enzyme recognition sites, and the enzyme recognition sites corresponding to these methylated sites are:

[0099] chr11: 2,180,310-2,180,313, HpaII enzyme recognition site;

[0100] chr6: 22,146,094-22,146,097, HinP1I enzyme recognition site;

[0101] chr17: 7,845,201-7,845,204, HpaII enzyme recognition site;

[0102] chr1: 48,616,494-48,616,497, HinP1I enzyme recognition site.

[0103] All of the above chromosomes are human chromosomes, and the reference gene is hg38 from the UCSC database.

[0104] In an optional implementation, the determination of differentially methylated sites includes the following steps:

[0105] (1) Obtain methylation data;

[0106] (2) Analysis of differentially methylated sites;

[0107] (3) Screening for methylation-sensitive restriction endonuclease recognition sites.

[0108] The system identifies contaminants, such as host DNA, and distinguishes them from target DNA by methylation differences at CpG sites, pinpointing sites containing enzyme recognition sequences. These differentially expressed sites are the targets for subsequent enzyme cleavage.

[0109] In an alternative implementation, the host DNA may be derived from potential contaminants in the sample, including at least one of the following tissues or sources: clinical samples such as human peripheral blood, plasma, serum, tissue, saliva, and urine; animal tissue samples (tissues and blood of model animals such as mice and rats); DNA remaining in human cell lines (HEK293, HeLa, A549, etc.) during in vitro culture; and residual DNA from host expression systems (E. coli, insect cells, human CHO cells, etc.) associated with library construction.

[0110] In an optional implementation, the target DNA may be derived from DNA fragments in the sample that are intended to be retained for downstream analysis, including at least one of the following species or tissues: clinical samples such as human peripheral blood, plasma, serum, tissue, saliva, and urine; clinical research-related targets such as tumor tissue DNA, cell-free DNA (cfDNA), ctDNA, and puncture fluid DNA; microbial DNA, including bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, etc.), viruses (such as HBV, HPV, EBV, etc.), and fungi (such as Candida albicans, Aspergillus, etc.); environmental or gut microbiome sample DNA; and non-host exogenous species, such as plant DNA and insect DNA.

[0111] In an optional implementation, the human methylation data is derived from the TCGA database.

[0112] In an alternative implementation, methylation data for other species are derived from the SRA database.

[0113] In an optional embodiment, the methylation-sensitive restriction endonuclease includes HpaII and / or HinP1I. The cleavage site of HpaII is CCGG, and the cleavage site of HinP1I is GCGC.

[0114] In an optional embodiment, the formula for the above-mentioned differential methylation site analysis is M = log2((β + α) / (1 - β + α)); where M is the methylation intensity, β is the methylation ratio, and α is a smoothing factor to prevent the denominator from being zero.

[0115] In an optional implementation, the value of α ranges from 0.001 to 0.01.

[0116] In an alternative implementation, α is 0.001.

[0117] In an optional implementation, the screening criteria for differential methylation site analysis are as follows: in ≥90% of normal tissues, Δβ ≥ 0.70, β_target ≥ 0.75, and β_host ≤ 0.15, where Δβ refers to the differential β value of the differential site (Δβ = β_target - β_host), β_host refers to the β value of the differential site in the host DNA, and β_target refers to the β value of the differential site in the target DNA; and the methylation level of the site fluctuates little in other tissues, has no homologous sequences or restriction enzyme site deletions in other species, and has tissue and species specificity.

[0118] In an alternative embodiment, library contaminants are degraded by incubating them in a prepared library reaction system.

[0119] This application provides a library contamination-specific degradation kit, comprising:

[0120] Methylation-sensitive restriction endonucleases;

[0121] Reaction buffer solution;

[0122] Magnetic bead purification reagent;

[0123] Positive control;

[0124] Negative control.

[0125] In an alternative embodiment, the methylation-sensitive restriction endonuclease includes HinP1I and / or HpaII.

[0126] In an optional implementation, the concentration of HinP1I in the kit is 10-20 U / μL.

[0127] In an optional implementation, the concentration of HinP1I in the kit is 15 U / μL.

[0128] In an optional implementation, the concentration of HpaII in the kit is 10-20 U / μL.

[0129] In an optional implementation, the concentration of HpaII in the kit is 15 U / μL.

[0130] In an optional implementation, the methylation-sensitive restriction endonuclease is mixed with a storage buffer.

[0131] In an alternative embodiment, the storage buffer comprises KCl, Tris-HCl, DTT, EDTA, and glycerol.

[0132] In an optional embodiment, the storage buffer contains 45-55 mM KCl, 8-12 mM Tris-HCl pH 7.2-7.5, 0.8-1.2 mM DTT, 0.05-0.15 mM EDTA, and 45-55% glycerol.

[0133] In an optional embodiment, the storage buffer contains the following components: 50 mM KCl, 10 mM Tris-HCl pH 7.4, 1 mM DTT, 0.1 mM EDTA, and 50% glycerol.

[0134] In an optional embodiment, the reaction buffer comprises Tris-HCl, NaCl, MgCl2, and DTT.

[0135] In an optional embodiment, the reaction buffer contains 90-110 mM Tris-HCl (pH 7.7-8.1), 90-110 mM NaCl, 9-11 mM MgCl2, and 0.8-1.2 mM DTT.

[0136] In an optional embodiment, the reaction buffer contains 100 mM Tris-HCl (pH 7.9), 100 mM NaCl, 10 mM MgCl2, and 1 mM DTT.

[0137] In an alternative embodiment, the magnetic bead purification reagent includes magnetic beads, binding buffer, and elution solution.

[0138] In an optional embodiment, the volume of the magnetic beads used in the purification reagent is 0.6-1.0 times that of the library reaction system.

[0139] In an optional embodiment, the volume of the magnetic beads used in the purification reagent is 0.8 times that of the library reaction system.

[0140] In an alternative embodiment, the binding buffer in the magnetic bead purification reagent includes PEG and NaCl.

[0141] In an alternative implementation, the pH range of the binding buffer is 5.0-6.5.

[0142] In an optional implementation, the pH of the binding buffer is 5.5.

[0143] In an optional embodiment, the PEG in the binding buffer of the magnetic bead purification reagent is PEG8000.

[0144] In an optional embodiment, the NaCl content in the binding buffer of the magnetic bead purification reagent is 2-3M.

[0145] In an optional embodiment, the NaCl content in the binding buffer of the magnetic bead purification reagent is 2.5M.

[0146] In an optional embodiment, the eluent in the magnetic bead purification reagent is Tris-HCl.

[0147] In an optional embodiment, the eluent in the magnetic bead purification reagent contains 8-12 mM Tris-HCl and has a pH of 8.3-8.7.

[0148] In an optional embodiment, the eluent in the magnetic bead purification reagent is 10 mM Tris-HCl, pH 8.5.

[0149] In an optional implementation, the volume of eluent used for each elution is greater than 20 μL.

[0150] In an optional implementation, the sample is incubated at 45-55°C for 1-3 minutes before elution.

[0151] In an optional implementation, incubation at 50°C for 2 minutes before elution can effectively improve the recovery rate.

[0152] In an optional embodiment, the positive control is an artificially prepared DNA mixture composed of 50% fully methylated human genomic DNA and 50% unmethylated human genomic DNA in a specific mass ratio, with a final concentration of 45-55 ng / μL, preferably 50 ng / μL. The methylated DNA may be derived from human genomic DNA treated with SssI methyltransferase, while the unmethylated DNA may be derived from WGA (Whole Genome Amplification) amplification products. The positive control contains at least two methylation-sensitive restriction endonuclease (HpaII, HinP1I) recognition sites to verify the correlation between enzyme digestion and methylation status.

[0153] In an optional embodiment, the negative control is unmethylated human genomic DNA and should contain at least one recognition sequence of the methylation-sensitive restriction endonuclease to ensure the specific detection capability of the enzyme digestion reaction. The final concentration is 45-55 ng / μL, preferably 50 ng / μL.

[0154] In an optional implementation, the kit also includes an instruction manual.

[0155] In an optional implementation, the target DNA retention rate of the above kit is above 75%.

[0156] In an optional implementation, the total amount of DNA in the sample detected by the kit is less than 500 ng.

[0157] In an optional implementation, the total volume of the reaction system for the sample tested by the kit is less than 20 μL.

[0158] This application also provides the use of the above-described kit in the specific degradation of library contaminants.

[0159] In an optional implementation, library contaminants include: residual adapter ligation products, library amplification fragments left over from the previous sequencing round, cross-contaminated library DNA, primer dimers and other non-specific amplification products, synthetic oligonucleotide contaminants, and whole-genome background DNA that has not undergone strict methylation treatment.

[0160] This application also provides the use of the above-mentioned kit in the specific degradation of library contaminants. This kit utilizes the specific recognition and cleavage mechanism of methylation-sensitive restriction endonucleases, combined with magnetic bead purification, to efficiently remove the aforementioned contaminants while maximizing the retention of target DNA, ensuring the accuracy and specificity of subsequent library construction and sequencing results.

[0161] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0162] Example

[0163] Example 1

[0164] Identify differentially methylated sites

[0165] 1. Obtain methylation data

[0166] Humanized methylation differential data:

[0167] Data source: TCGA project, Lung Cancer LUSC, LUAD (human-derived);

[0168] Data type: Illumina HumanMethylation450 BeadChip;

[0169] Download method: Obtain the beta value matrix (0~1, representing the degree of methylation) in .txt or .csv format from UCSC Xena. Use the R package minfi to read the methylation β values.

[0170] Mouse methylation differential data:

[0171] Data source: SRA;

[0172] Data type: Whole Genome Bisulfite Sequencing (WGBS);

[0173] Download method: Download data using sra-tools (fasterq-dump), then use Bismark for alignment and methylation.

[0174] Output: bedGraph: position + percentage of CpG methylation;

[0175] CpG_report.txt: Detailed statistics for each site (chromosome, location, + / - chains, number of methylated and unmethylated chains).

[0176] Use a script or methylKit to merge the CpG reports of multiple samples into a matrix, consistent with the format of the human source matrix.

[0177] This step uses human and mouse methylation difference data from the TCGA and SRA public databases, mainly to analyze and screen for hypomethylated regions that are widely present in different tissue backgrounds, as a design reference for methylation-sensitive enzyme recognition sites.

[0178] 2. Analysis of differentially methylated sites

[0179] Calculate the differential β value (Δβ = β_target - β_host) and FDR for each site using limma or methylKit.

[0180] The M-value conversion formula is: M = log2((β + α) / (1 - β + α)), where β is the methylation ratio and α is a smoothing factor to prevent the denominator from being zero;

[0181] M = methylation intensity, U = non-methylation intensity, α is a constant 0.001;

[0182] Screening criteria: Δβ ≥ 0.70, β_target ≥ 0.75, β_host ≤ 0.15 in ≥90% of normal tissues, and the methylation level of this site fluctuates little in other tissues, has no homologous sequences or restriction enzyme site deletions in other species, and is tissue and species specific.

[0183] The methylation sites need to be tissue-specific. For example, the screening results for detecting lung cancer-related contamination are shown below:

[0184] Table 1. Screening results of differentially methylated sites

[0185]

[0186] In Table 1, β_host refers to the β value of the differentially expressed site in the host DNA, β_target refers to the β value of the differentially expressed site in the target DNA, and β_other tissues is the mean and standard deviation of the population calculated from the β values ​​of all non-lung tissue samples.

[0187] This site is almost completely methylated in normal lung tissue, but nearly unmethylated in lung cancer, showing a very significant difference (Δβ=0.70). Meanwhile, it remains close to 0.5 in other tissues with little fluctuation (SD=0.03), indicating that it only shows a strong difference in lung tissue. Therefore, it can be considered a lung tissue-specific methylation marker site, used to distinguish contamination or determine the source of a sample.

[0188] 3. Screening for methylation-sensitive restriction endonuclease recognition sites

[0189] To achieve efficient recognition of methylation-sensitive restriction endonucleases in library contamination degradation, recognition sites for methylation-sensitive restriction endonucleases were screened based on differentially methylated sites.

[0190] This step not only screens for the classic HpaII enzyme recognition site CCGG, but also introduces the HinP1I enzyme recognition site GCGC to construct a dual-enzyme recognition system, thereby improving coverage and cleavage efficiency.

[0191] The genome reference sequence hg38.fa of the species was downloaded using UCSC, and all positions matching the following identification sequence were extracted:

[0192] HpaII recognition site: CCGG;

[0193] HinP1I recognition site: GCGC.

[0194] The entire genome sequence is scanned using regular expressions combined with the awk tool, and the precise location of the identified sites on the chromosome is output. The output format is a .bed file.

[0195] The obtained recognition sites also need to be species-specific. Taking species specificity relative to mice as an example, the following screening was performed: BLAST was used to compare the obtained sites with mouse DNA (mouse reference gene mm10). The results are shown in the following example:

[0196] Table 2 Comparison results of enzyme recognition site specificity

[0197]

[0198] Analysis showed that these two sites lacked cleavage sites in mice, meaning they could not be recognized and cleaved by HpaII or HinP1I in mice. This indicates that these are human-specific cleavage-sensitive sites, which can improve the species specificity of contaminant identification.

[0199] The screening was continued using the method described above, and the resulting sites are shown in Table 3.

[0200] Table 3 Restriction endonuclease recognition sites

[0201]

[0202] Experimental Example 1

[0203] Degradation of pollutants in the library

[0204] 1. Degradation of contaminants in the library

[0205] (1) Sample processing

[0206] To construct a library of samples containing pollutants, the test samples were prepared using the following method:

[0207] First, genomic DNA extracted from peripheral blood of healthy individuals was used as the target DNA source. Library construction (including fragmentation, adapter ligation, and PCR amplification) was performed using a standard Illumina library construction kit according to the manufacturer's instructions. To simulate library contamination, the following contaminants (i.e., host DNA) were artificially added to the above human library to achieve a contamination rate of approximately 30%:

[0208] Exogenous microbial genome fragments: Incorporation of gut-derived microbial DNA library fragments:

[0209] Sequencing residual library fragments: Library fragments with adapter sequences recovered from other sequencing projects (formed by heat denaturation and then cooling to form double-stranded DNA).

[0210] Unconnected adapter amplification products: Non-specific PCR fragments (approximately 200-600 bp) randomly amplified using primers, without adapters but containing partial primer sequences.

[0211] The final sample was a library reaction system consisting of the above-mentioned contaminated fragments and the target DNA, with the total DNA concentration controlled at 20-30 ng / μL to simulate library contamination that may occur during common sample preparation processes.

[0212] (2) Degradation of library contaminants:

[0213] The library pollutant degradation reaction system (i.e., the library reaction system) was prepared and incubated at 37°C for 1 hour, followed by incubation at 65°C for 20 minutes to terminate the enzymatic digestion. The system is shown in Table 4.

[0214] Table 4 Degradation reaction system of library pollutants

[0215]

[0216] The addition of BSA can improve the enzymatic digestion efficiency of restriction endonucleases (HpaII, HinP1I) in complex reaction systems to a certain extent, especially in samples with high GC background or potential inhibitor residues (feces, blood).

[0217] 2. Fragment purification

[0218] Fragments larger than 200 bp were selectively purified using the AMPure XP magnetic bead method to remove smaller fragments after enzyme digestion. The purification steps are as follows:

[0219] (1) Add 0.8 × volume AMPure XP magnetic beads (i.e., 40 μL) to a 50 μL library reaction system.

[0220] (2) Incubate at room temperature for 5 minutes to bind DNA;

[0221] (3) Place the magnetic rack on the magnet and let it stand for 5 minutes to attract the magnetic beads;

[0222] (4) Remove the supernatant (containing fragments below 200 bp and irrelevant contaminants);

[0223] (5) Wash twice with 80% ethanol (200 μL each time);

[0224] (6) Air dry the magnetic beads for 1-2 minutes (be careful not to over-dry them);

[0225] (7) Elute the target DNA with 20 μL EB buffer (or TE).

[0226] 3. Verification and Quality Control

[0227] (1) qPCR assay for contamination removal and target retention

[0228] qPCR quantitative analysis was performed using target DNA-specific primers, namely primers for human Alu sequences and host DNA primers, namely primers for Escherichia coli 16S rRNA genes, broad-spectrum bacterial 16S rRNA genes, and fungal ITS region genes. The analytical methods are referenced in Bibikova M, Barnes B, Tsan C, et al. High density DNA methylation array with single CpG site resolution. Genomics. 2011;98(4):288-295. GenomeRes. 2002.

[0229] The primers used are shown in Table 5.

[0230] Table 5 qPCR primer sequences

[0231]

[0232] The qPCR system (20 μL) is as follows:

[0233] qPCR Master Mix (Takara TB Green® Premix Ex Taq™ II (Tli RNaseHPlus) Kit): 10 μL;

[0234] Forward / reverse primers: 0.4 μL (10 μM) each;

[0235] Template DNA: 1 μL;

[0236] ddH2O: Add to a final volume of 20 μL.

[0237] The qPCR reaction conditions are as follows:

[0238] Pre-denaturation at 95℃ for 3 minutes;

[0239] 40 cycles: 95℃ for 15 seconds, 60℃ for 30 seconds.

[0240] The melt curve obtained after qPCR is as follows Figure 1 As shown, each amplification product exhibited a single, specific melting peak: Alu sequence product at approximately 82°C, E. coli 16S rRNA product at approximately 88°C, broad-spectrum bacterial 16S rRNA product at approximately 86°C, and fungal ITS product at approximately 90°C. No heterogeneous peaks or cross-amplification were observed, indicating that each primer possesses good specificity and stability, and can be used to simultaneously assess the retention of target DNA and the removal of multiple contaminants.

[0241] Furthermore, the contamination removal rate and target DNA retention rate were calculated based on the Ct values ​​obtained from qPCR, using the following formulas.

[0242]

[0243]

[0244] Where ΔCt = Ct 处理 - Ct 未处理 A ΔCt ≥ 3 for host DNA indicates a reduction of approximately 90% in host DNA; a ΔCt ≤ 0.5 for target DNA indicates a retention of ≥ 75% of target DNA. The analysis results are shown below. Figure 2 The calculation results are shown in Table 7.

[0245] (2) Verify the selective removal effect

[0246] The enzyme-digested library was sequenced using PE150 sequencing on the Illumina NovaSeq 6000 platform, and the proportion of human sequences was analyzed using BWA and Samtools.

[0247] The BWA and Samtools methods for analyzing human sequence proportions are as follows:

[0248] The human genome reference sequence hg38.fa was downloaded using UCSC, an index file was constructed, and the sample sequencing reads were aligned to the human reference genome using BWA-MEM. The reads were then converted to BAM format, sorted, and indexed. The total number of reads and the number of reads aligned to the human genome were counted. For specific methods, refer to: Li H, Durbin R. Fast and accurate shortread alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754-1760.

[0249] The results are as follows Figure 3 As shown, Figure 3 The results showed that the proportion of host DNA decreased from 30% before treatment to 10%, verifying that the scheme effectively preserved the target sequence while removing contaminants.

[0250] Experimental Example 2

[0251] The procedures for this experiment are basically the same as those for Experiment 1, the only difference being the use of respiratory swab samples from healthy subjects.

[0252] The total sample DNA was approximately 200 ng, with the remainder consisting of environmental contaminant sequences (bacteria, viruses, etc.). After library contaminant degradation treatment, the contaminant removal rate and target DNA retention rate were measured and calculated, and the results are shown in Table 6. These results indicate that the Ct value of the target DNA fragment remained stable, suggesting that the contaminants were partially degraded while the target sequence was well preserved.

[0253] Experimental Example 3

[0254] The procedure for this experiment is basically the same as that for Experiment 1, except that the intestinal contents of healthy subjects are used and the samples are obtained after microbial enrichment.

[0255] To verify the applicability of this method in highly contaminated samples, DNA from gut microbiota-enriched samples was selected as the test material. The total DNA amount of the sample was 400 ng. After library contaminant degradation treatment, the contaminant removal rate and target DNA retention rate were measured and calculated, and the results are shown in Table 6. These results indicate that the kit can enrich the target sequence and remove contaminant sequences even in highly contaminated environments.

[0256] Test Example 4

[0257] The procedures for this experiment are basically the same as those for Experiment 1, the only difference being the use of skin swab samples from healthy subjects.

[0258] To verify the applicability of this method in highly contaminated samples, genomic DNA from skin swabs was selected as the test material. The total DNA concentration was approximately 20 ng / μL. After library contaminant degradation treatment, the contaminant removal rate and target DNA retention rate were measured and calculated, and the results are shown in Table 6. These results demonstrate the strong degradation capability of the method proposed in this application for complex exogenous contaminants.

[0259] Experimental Example 5

[0260] The procedures for this experiment are basically the same as those for Experiment 1, the only difference being the use of cell-free circulating DNA samples from the plasma of healthy subjects.

[0261] To verify the applicability of this method in extremely low-volume samples, plasma cfDNA was selected as the test material. The plasma cfDNA concentration was approximately 3.5 ng / μL, with a total volume of less than 50 ng. After library contaminant degradation treatment, the contaminant removal rate and target DNA retention rate were measured and calculated, and the results are shown in Table 6. These results indicate that the method described in this application can effectively reduce contamination and improve library quality even in ultra-low-volume cfDNA samples.

[0262] Experimental Example 6

[0263] The procedures in this experiment were basically the same as in Experiment 1, except that genomic DNA was extracted from fecal samples from healthy subjects.

[0264] To verify the applicability of this method in low-abundance DNA samples, genomic DNA extracted from feces was selected as the test material, whose DNA abundance was much lower than that of conventional samples.

[0265] After the library was treated with contaminant degradation, the contaminant removal rate and target DNA retention rate were measured and calculated, and the results are shown in Table 6. The significant reduction in contaminant sequences indicates good degradation efficiency. These results demonstrate that this method is well-suited for complex samples with low DNA content, such as feces, and can effectively degrade contaminants while preserving target sequences.

[0266] Table 6. qPCR assay results in Experiments 2-6

[0267]

[0268] Comparative Examples 1-6

[0269] Degradation of pollutants in the library

[0270] The experimental methods of Comparative Examples 1-6 below are basically the same as those of Experiment 1 above, except for the enzyme digestion conditions shown in Table 7.

[0271] Table 7 Comparative Enzyme Digestion Conditions and Results

[0272]

[0273] Comparative Example 7

[0274] Library pollutant removal

[0275] In this comparative example, the NEBNext® Microbiome DNA Enrichment Kit, a commercially available kit, was used to enrich and remove contaminants from the contaminated library constructed in Example 1. The kit usage method is referenced in: Thorendel M, Jeraldo PR, Greenwood-Quaintance KE, et al. Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing. JMicrobiol Methods. 2016;127:141-145. The analytical methods for samples treated with the commercially available kit were the same as in Example 1.

[0276] Analysis showed that the host DNA removal rate of the commercially available kit was 75% (ΔCt ≈ 2); the target DNA retention rate was approximately 50% (ΔCt ≈ 1); and the host DNA content was 25.60%.

[0277] Comparing the results of Experiment Example 1 with those of commercially available kits, it can be seen that the library-specific degradation method of this application has better performance.

[0278] Comparative Example 8

[0279] Fragment purification

[0280] In this comparative example, the enzyme-digested library reaction system was used as the original sample (50 μL). Fragment purification was performed by setting the magnetic bead volume ratio to 0.6, 0.8, and 1.0 times (0.6×, 0.8×, and 1.0×) of the library reaction system volume, respectively. The purification method was the same as in Example 1.

[0281] After purification, fragment size distribution was detected using an Agilent Bioanalyzer 2100 (DNA High Sensitivity chip); concentration was detected using a Qubit dsDNA HS Assay; and contaminant retention was detected using qPCR.

[0282] The test results are as follows:

[0283] Group A (0.6×): The retained peaks were concentrated in the 350-500 bp range, and a large number of target fragments were lost; qPCR showed that the Ct value of the contaminated fragments increased only slightly, with ΔCt being 2.1, indicating that some small fragments still remained.

[0284] Group B (0.8×): Fragment distribution was concentrated in 200-500 bp, and contaminant peaks were almost completely cleared; qPCR ΔCt was as high as 5.8, and the library construction peaks were concentrated;

[0285] Group C (1.0×): Almost all fragments were retained, including contaminating fragments below 200 bp; low molecular weight contaminants were clearly visible in the Bioanalyzer pattern, and the qPCR ΔCt was only 1.3.

[0286] Based on the above results and Figure 4 It is evident that 0.8× AMPure XP magnetic beads represent the optimal balance between purification efficiency and retention rate. They effectively remove contaminating fragments smaller than 200 bp from the digested material while retaining the vast majority of the target library fragments. Compared to 1.0× and 0.6× beads, they are more suitable for the enzymatic digestion and decontamination process described in this invention.

[0287] Finally, it should be noted that other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This invention is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein, and is not limited to the precise structures described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A method for library contamination-specific degradation based on methylation-sensitive restriction endonucleases, characterized in that, The method includes the following steps: (1) Determine the recognition sites of methylation-sensitive restriction endonucleases: The methylation-sensitive restriction endonucleases include HpaII and HinP1I; (i) Obtain methylation data for the host DNA and the target DNA; (ii) Determine the differentially methylated sites based on the methylation data; (iii) Screening out the recognition sites of the methylation-sensitive restriction endonuclease from the differentially methylated sites; The differentially methylated sites are determined using the formula M = log2((β + α) / (1 - β + α)), where M is the methylation intensity, β is the methylation ratio, and α is a smoothing factor to prevent the denominator from being zero, with α ranging from 0.001 to 0.

01. The screening criteria for the differentially methylated sites are: in ≥90% of normal tissues, Δβ ≥ 0.70, β_target ≥ 0.75, and β_host ≤ 0.

15. (2) Enzyme digestion of library contaminants: Prepare a DNA library reaction system, wherein the DNA includes host DNA and target DNA; add the methylation-sensitive restriction endonuclease to the DNA library reaction system and incubate it to obtain the enzyme-digested DNA library reaction system; (3) Fragment purification: The enzyme-digested DNA library reaction system is selectively purified using magnetic beads to obtain a purified DNA library, wherein the DNA fragments in the purified DNA library are larger than 200 bp; (4) Validation and quality control: The host DNA removal rate and target DNA retention rate in the purified DNA library were detected by amplification and sequencing. The target DNA includes human genomic DNA, and the recognition site of the methylation-sensitive restriction endonuclease is as follows: chr11:2,180,310-2,180,313; chr6:22,146,094-22,146,097; chr17:7,845,201-7,845,204; chr1:48,616,494-48,616,497; The reference gene for this site is hg38 from the UCSC database.

2. The method according to claim 1, characterized in that, In the enzyme digestion of library contaminants step, the library reaction system is incubated at 35°C-39°C for 0.5-1.5 hours, followed by incubation at 60°C-70°C for 15-25 minutes to obtain the enzyme-digested DNA library reaction system.

3. The method according to claim 1, characterized in that, The amount of the methylation-sensitive restriction endonuclease used is 10-20 U / μg DNA.

4. The method according to claim 1, characterized in that, The volume of the magnetic beads used in the magnetic bead method is 0.6-1.0 times the volume of the library reaction system.

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