Quality control method for rapid detection of trace gdna contamination in cfdna on basis of fragmentomics

Abstract

The present invention relates to a quality control method for the rapid detection of trace gDNA contamination in a cfDNA sample on the basis of fragmentomics. On the basis of the difference in fragment sizes of cfDNA and gDNA, cfDNA and gDNA in a cfDNA sample are subjected to nucleic acid amplification, and a molar proportion of cfDNA of the sample is calculated, so as to evaluate the degree of contamination of gDNA in the cfDNA sample, and provide guidance for bioinformatic analysis of the cfDNA sample. The method requires a small sample size, can rapidly determine the degree of contamination of gDNA in cfDNA samples at the source, and provides timely guidance for bioinformatic analysis, thereby effectively saving on time and economic costs.

Description

A quality control method for rapid detection of trace gDNA contamination in cfDNA based on fragmentomics

[0001] Cross-reference to related applications

[0002] This application claims the benefit and priority of patent application No. 202411719130.0, filed on November 28, 2024, entitled “A quality control method for rapid detection of trace gDNA contamination in cfDNA based on fragmentomics”, the entire contents of which, including any sequence listings and figures, are incorporated herein by reference in their entirety.

[0003] Reference to electronic sequence listing

[0004] This disclosure contains an electronic sequence listing (created using the software "WIPOSequence" according to WIPO standard ST.26), which is incorporated herein by reference in its full text. According to WIPO standard ST.26, the symbol "t" is used to represent both T in DNA and U in RNA. Therefore, in a sequence listing prepared according to ST.26, in any case where the sequence is RNA, T in the sequence should be considered as U. Technical Field

[0005] This invention belongs to the field of biotechnology, specifically relating to a quality control method for rapid detection of trace gDNA contamination in cfDNA based on fragment omics. Background Technology

[0006] Circulating biomarkers have become important indicators for clinical disease diagnosis. In particular, the discovery that DNA from various human tissues exists in blood and other extracellular fluids (cell-free deoxyribonucleic acid, cfDNA) has driven numerous blood-based cfDNA tests to query specific genomic abnormalities, such as chromosomal copy number aberrations for non-invasive prenatal testing or tumor-derived mutations for targeted cancer therapy selection. A key application of blood-based cfDNA or other circulating analytes is the early detection of common cancer signals in various cancers through blood tests. Sample quality and cfDNA purity play a crucial role in the accuracy of the tests. Genomic contamination can obfuscate biological inferences and lead to erroneous conclusions regarding evolutionary relationships and lateral gene transfer; therefore, high-quality genomic data is essential for cross-disciplinary biological data analysis.

[0007] During transportation, bumps and vibrations can cause hemolysis, leading to the release of gDNA. When cfDNA is contaminated with gDNA, its sequence characteristics are diluted, and biological indicators are altered, significantly impacting subsequent detection. Identifying this problem at its source would greatly improve the accuracy of subsequent tests. Existing quality control methods include Qsep fragment detection, Qubit quantification, and sequencing data analysis. These methods can detect contamination indirectly and with a certain lag, and are limited to small sample volumes. Therefore, there is a need to develop a more sensitive quality control method that allows for earlier intervention, facilitating efficient decision-making in experimental quality control.

[0008] In summary, existing methods cannot directly identify whether cfDNA samples contain gDNA after nucleic acid extraction. There is no effective quality control method for gDNA contamination before cfDNA NGS sequencing detection, which usually results in a large amount of data waste.

[0009] Therefore, there is an urgent need to establish an efficient quality control method for gDNA contamination, so as to treat contamination before detection, reduce cost waste, reduce data waste, improve the accuracy of sequencing pooling, and increase the sample sequencing qualification rate. Summary of the Invention

[0010] The purpose of this invention is to establish a rapid and efficient quality control method for gDNA contamination in cfDNA samples based on the size difference between cfDNA and gDNA fragments.

[0011] In a first aspect of the present invention, a method for determining the degree of gDNA contamination in a cfDNA sample is provided, the method comprising the following steps:

[0012] (Z1) Provide a cfDNA sample, which is a cfDNA sample derived from bodily fluids;

[0013] (Z2) Amplify the cfDNA and gDNA in the cfDNA sample to obtain a first amplification product with a length of L1 corresponding to the cfDNA and a second amplification product with a length of L2 corresponding to the gDNA.

[0014] (Z3) Quantitatively detect the concentration or level of the first amplification product, designated as Y1; and quantitatively detect the concentration or level of the second amplification product, designated as Y2;

[0015] (Z4) Based on the concentration or level Y1 of the first amplification product and the concentration or level Y2 of the second amplification product, the relative proportion R of cfDNA in the cfDNA sample is obtained, thereby determining the degree of gDNA contamination in the cfDNA sample.

[0016] In another preferred embodiment, the body fluid is blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, ascites, serous fluid, sputum, tears, urine, saliva, breast milk, gastric juice, or pancreatic juice.

[0017] In another preferred embodiment, in step (Z2), the target sequence for amplifying the cfDNA in the cfDNA sample is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or a combination thereof.

[0018] In another preferred embodiment, in step (Z2), the target sequence for amplifying the cfDNA in the cfDNA sample is preferably SEQ ID NO:15.

[0019] In another preferred embodiment, in step (Z2), the primer pairs for amplifying the cfDNA in the cfDNA sample are selected from: SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:12 or combinations thereof.

[0020] In another preferred embodiment, in step (Z2), the primer pair used to amplify the cfDNA in the cfDNA sample is SEQ ID NO:1 and SEQ ID NO:2.

[0021] In another preferred embodiment, in step (Z2), the target sequence for amplifying the gDNA in the cfDNA sample is the housekeeping gene of the gDNA.

[0022] In another preferred embodiment, the housekeeping genes of the gDNA include (but are not limited to): β-Actin, GAPDH, and α-Tublin.

[0023] In another preferred embodiment, the housekeeping gene of the gDNA is preferably β-Actin.

[0024] In another preferred embodiment, in step (Z2), the target sequence for amplifying gDNA in the cfDNA sample is selected from the group consisting of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or a combination thereof.

[0025] In another preferred embodiment, in step (Z2), the target sequence for amplifying the gDNA in the cfDNA sample is preferably SEQ ID NO:16.

[0026] In another preferred embodiment, in step (Z2), the primer pairs for amplifying the gDNA in the cfDNA sample are selected from: SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:13 and SEQ ID NO:14 or combinations thereof.

[0027] In another preferred embodiment, in step (Z2), the primer pair used to amplify the gDNA in the cfDNA sample is preferably SEQ ID NO:3 and SEQ ID NO:4.

[0028] In another preferred embodiment, in step (Z2), the primer set for amplifying the cfDNA and gDNA in the cfDNA sample includes: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0029] In another preferred embodiment, the primer pair for amplifying the cfDNA is SEQ ID NO:5 and SEQ ID NO:6; and

[0030] The primer pair for amplifying gDNA is SEQ ID NO:7 and SEQ ID NO:8.

[0031] In another preferred embodiment, L1 is 50-200bp, more preferably 80-180bp, and most preferably 100-150bp.

[0032] In another preferred embodiment, the L2 is 300-600bp, more preferably 320-500bp, and most preferably 350bp-450bp.

[0033] In another preferred embodiment, the ΔL of L2-L1 is 100-450bp, more preferably 150-400bp, and even more preferably 200-350bp.

[0034] In another preferred embodiment, L2 / L1 ≥ 1.5, and more preferably, L2 / L1 ≥ 3.

[0035] In another preferred embodiment, the concentration is a molar concentration; or the level is a molar quantity.

[0036] In another preferred embodiment, in step (Z3), the mass concentration or mass number C1 and length L1 of the first amplification product are determined to obtain the molar concentration or molar quantity Y1 of the first amplification product; and the mass concentration or mass number C2 and length L2 of the second amplification product are determined to obtain the molar concentration or molar quantity Y2 of the second amplification product.

[0037] In another preferred embodiment, in step (Z3), the mass concentration C1 and length L1 of the first amplification product are measured to obtain the molar concentration Y1 of the first amplification product; and the mass concentration C2 and length L2 of the second amplification product are measured to obtain the molar concentration Y2 of the second amplification product.

[0038] In another preferred embodiment, the method for determining the mass concentration includes (but is not limited to): ultraviolet spectrophotometry, fluorescence spectrophotometry, qPCR, and Qubit method.

[0039] In another preferred embodiment, the mass concentration is determined by the Qubit method.

[0040] In another preferred embodiment, in step (Z3), the mass number C1 and length L1 of the first amplification product are determined to obtain the molar quantity Y1 of the first amplification product; and the mass number C2 and length L2 of the second amplification product are determined to obtain the molar quantity Y2 of the second amplification product.

[0041] In another preferred embodiment, the length is determined by capillary electrophoresis.

[0042] In another preferred embodiment, the unit of the mass number is nanogram, and the unit of the mole quantity is nanomolar.

[0043] In another preferred embodiment, the molar quantity of the first amplification product Y1 = (C1 / 1000*10^6) / (660*L1)*1000.

[0044] In another preferred embodiment, when SEQ ID NO:1 and SEQ ID NO:2 are used, the L1 of the first amplification product is 139.

[0045] In another preferred embodiment, the molar quantity of the first amplification product Y1 = (C1 / 1000*10^6) / (660*139)*1000.

[0046] In another preferred embodiment, the molar quantity of the second amplification product Y2 = (C2 / 1000*10^6) / (660*L2)*1000.

[0047] In another preferred embodiment, when SEQ ID NO:3 and SEQ ID NO:4 are used, the L2 of the second amplification product is 420.

[0048] In another preferred embodiment, the molar quantity of the second amplification product Y2 = (C2 / 1000*10^6) / (660*420)*1000.

[0049] In another preferred embodiment, in step (Z4), the relative proportion R is compared with a predetermined reference value to determine the degree of gDNA contamination in the cfDNA sample.

[0050] In another preferred embodiment, the relative proportion R is a relative proportion based on molar concentration or molar number.

[0051] In another preferred embodiment, the relative proportion of the cfDNA is R = Y1 / (Y1+Y2)×100%.

[0052] In another preferred embodiment, the determination is made according to the following criteria:

[0053] (a) If R < 38%, then the gDNA in the cfDNA sample is considered to be severely contaminated;

[0054] (b) If R < 50%, the gDNA in the cfDNA sample is considered to be moderately contaminated;

[0055] (c) If 50% ≤ R < 55%, then the gDNA in the cfDNA sample is considered to be slightly contaminated;

[0056] (d) If 55% ≤ R < 70%, then the gDNA in the cfDNA sample is determined to be uncontaminated;

[0057] (e) If R ≥ 70%, the cfDNA sample acquisition or nucleic acid amplification process is deemed unqualified.

[0058] In another preferred embodiment, step (Z4) further includes: providing the following recommendations for bioinformatics analysis based on the degree of gDNA contamination:

[0059] When a cfDNA sample is determined to be severely contaminated or unqualified, it is not recommended to perform bioinformatics analysis on the cfDNA; the cfDNA sample must be extracted again.

[0060] cfDNA samples that are determined to be slightly contaminated or uncontaminated can undergo cfDNA bioinformatics analysis, requiring 6G of data.

[0061] cfDNA samples identified as moderately contaminated can undergo cfDNA bioinformatics analysis, requiring 8G of data.

[0062] In another preferred embodiment, the method is a non-diagnostic, non-treatment method.

[0063] In a second aspect of the invention, a cfDNA quality control system for determining the degree of gDNA contamination in cfDNA samples is provided, the system comprising the following modules:

[0064] (S1) Sample acquisition module, the sample acquisition module is configured to: acquire cfDNA samples from body fluids;

[0065] (S2) Nucleic acid amplification module, wherein the nucleic acid amplification module is configured to amplify cfDNA and gDNA in cfDNA sample to obtain a first amplification product of length L1 corresponding to cfDNA and a second amplification product of length L2 corresponding to gDNA.

[0066] (S3) Quantitative detection module, wherein the quantitative calculation module is configured to: quantitatively detect the concentration or level of the first amplification product, defined as Y1; and quantitatively detect the concentration or level of the second amplification product, defined as Y2;

[0067] (S4) Contamination level analysis module, which is configured to: obtain the relative proportion R of cfDNA in cfDNA sample based on the concentration or level Y1 of the first amplification product and the concentration or level Y2 of the second amplification product, thereby giving the assessment result of the contamination level of gDNA in cfDNA sample.

[0068] (S5) Output module, the output module is configured to output the assessment result of the contamination level of the gDNA.

[0069] In another preferred embodiment, the analysis module is further configured to: determine the degree of gDNA contamination in the cfDNA sample based on the relative ratio R, and provide a recommended value for the amount of data required for bioinformatics analysis of the qualified cfDNA sample.

[0070] In another preferred embodiment, the quantitative detection module includes:

[0071] (S3-1) Mass concentration detection submodule, which is configured to: quantitatively detect the mass concentration or mass number C1 of the first amplification product and the mass concentration or mass number C2 of the second amplification product;

[0072] (S3-2) Amplification product length detection submodule, configured to: determine the length L1 of the first amplification product and determine the length L2 of the second amplification product; and

[0073] (S3-3) The molar relative abundance quantification submodule is configured to: obtain the molar concentration or molar quantity Y1 of the first amplified product based on its mass concentration or mass number C1 and length L1; and obtain the molar concentration or molar quantity Y2 of the second amplified product based on its mass concentration or mass number C2 and length L2; and

[0074] Based on the molar concentration or molar quantity Y1 of the first amplification product and the molar concentration or molar quantity Y2 of the second amplification product, the relative proportion R (i.e., molar relative abundance) of cfDNA in the cfDNA sample is determined.

[0075] In another preferred embodiment, the quantitative detection module includes:

[0076] (S3-1) Mass concentration detection submodule, which is configured to: quantitatively detect the mass number C1 of the first amplification product and the mass number C2 of the second amplification product;

[0077] (S3-2) Amplification product length detection submodule, configured to: determine the length L1 of the first amplification product and determine the length L2 of the second amplification product; and

[0078] (S3-3) The molar relative abundance quantification submodule is configured to: obtain the molar concentration or molar quantity Y1 of the first amplified product based on the mass number C1 and length L1 of the first amplified product; and obtain the molar concentration or molar quantity Y2 of the second amplified product based on the mass number C2 and length L2 of the second amplified product; and

[0079] Based on the molar concentration or molar quantity Y1 of the first amplification product and the molar concentration or molar quantity Y2 of the second amplification product, the relative proportion R (i.e., molar relative abundance) of cfDNA in the cfDNA sample is determined.

[0080] In another preferred embodiment, the target sequence for amplifying the cfDNA in the cfDNA sample during the amplification is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, or a combination thereof.

[0081] In another preferred embodiment, the target sequence for amplifying the cfDNA in the cfDNA sample is preferably SEQ ID NO:15.

[0082] In another preferred embodiment, the nucleic acid amplification module is configured to amplify cfDNA using cfDNA primer pairs selected from: SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:11 and SEQ ID NO:12, or combinations thereof.

[0083] In another preferred embodiment, the cfDNA primer pair is SEQ ID NO:1 and SEQ ID NO:2.

[0084] In another preferred embodiment, the nucleic acid amplification module is configured to amplify gDNA using gDNA primer pairs selected from: SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:13 and SEQ ID NO:14, or combinations thereof.

[0085] In another preferred embodiment, the gDNA primer pair is SEQ ID NO:3 and SEQ ID NO:4.

[0086] In another preferred embodiment, the nucleic acid amplification module is configured to amplify cfDNA using primer pairs shown in SEQ ID NO:1 and SEQ ID NO:2, and to amplify gDNA using primer pairs shown in SEQ ID NO:3 and SEQ ID NO:4.

[0087] In another preferred embodiment, the analysis module is judged according to the following criteria:

[0088] (a) If R < 38%, then the gDNA in the cfDNA sample is considered to be severely contaminated;

[0089] (b) If R < 50%, the gDNA in the cfDNA sample is considered to be moderately contaminated;

[0090] (c) If 50% ≤ R < 55%, then the gDNA in the cfDNA sample is considered to be slightly contaminated;

[0091] (d) If 55% ≤ R < 70%, then the gDNA in the cfDNA sample is determined to be uncontaminated;

[0092] (e) If R ≥ 70%, the cfDNA sample acquisition or nucleic acid amplification process is deemed unqualified.

[0093] In another preferred embodiment, when a cfDNA sample is determined to be heavily contaminated or unqualified, it is not recommended to perform bioinformatics analysis on the cfDNA and the cfDNA sample needs to be extracted again.

[0094] In another preferred embodiment, for qualified cfDNA samples, the recommended amount of data required for bioinformatics analysis of the cfDNA samples is:

[0095] cfDNA samples that are determined to be slightly contaminated or uncontaminated can undergo cfDNA bioinformatics analysis, requiring 6G of data.

[0096] cfDNA samples identified as moderately contaminated can undergo cfDNA bioinformatics analysis, requiring 8G of data.

[0097] In a third aspect of the invention, a primer pair combination is provided, the primer pair combination comprising:

[0098] A pair of cfDNA primers for amplifying cfDNA, the cfDNA primer pair comprising the primers shown in SEQ ID NO:1 and SEQ ID NO:2; and

[0099] gDNA primer pairs for amplifying gDNA, the gDNA primer pairs including the primers shown in SEQ ID NO:3 and SEQ ID NO:4.

[0100] In a fourth aspect of the invention, the use of the primer pair combination described in the third aspect of the invention is provided for preparing a kit for determining the degree of gDNA contamination in cfDNA samples before bioinformatics analysis, so as to avoid severely contaminated samples from entering the bioinformatics analysis step, and to provide sequencing data volume requirements for other contamination levels of samples that can enter the bioinformatics analysis step.

[0101] In a fifth aspect of the invention, a kit is provided, the kit comprising:

[0102] (a) A pair of cfDNA primers for amplifying cfDNA, the cfDNA primers comprising the primers shown in SEQ ID NO:1 and SEQ ID NO:2;

[0103] (b) a pair of gDNA primers for amplifying gDNA, said gDNA primer pair comprising the primers shown in SEQ ID NO:3 and SEQ ID NO:4; and

[0104] (c) Optional reagents for extracting cfDNA.

[0105] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0106] Figure 1 shows the primer design principle for cfDNA and gDNA.

[0107] Figure 2 shows the sequencing results of the cfDNA1 mixed primer amplification product.

[0108] Figure 3 shows the sequencing results of the gDNA1 mixed primer amplification products.

[0109] Figure 4 shows the capillary electrophoresis results of the cfDNA1 mixed primer amplification products.

[0110] Figure 5 shows the capillary electrophoresis results of the gDNA1 mixed primer amplification products.

[0111] Figure 6 shows the criteria for assessing the degree of gDNA contamination.

[0112] Figure 7 shows the comparison of cfDNA and gDNA mass concentrations in samples collected from different sampling tubes.

[0113] Figure 8 shows a schematic diagram of the decision support method of the present invention. Detailed Implementation

[0114] Through extensive and in-depth research and numerous screenings, the inventors unexpectedly discovered for the first time that the content of certain specific cfDNA sequences in cfDNA samples not only has an excellent correlation with the quality of cfDNA, but also that the relative abundance of these sequences with specific gDNA sequences can rapidly, efficiently, and accurately characterize the degree of gDNA contamination in cfDNA samples. Based on this discovery, the present invention was completed.

[0115] Specifically, based on the cfDNA sequence and the housekeeping gene of gDNA, the inventors designed corresponding cfDNA and gDNA amplification primers to amplify cfDNA and gDNA in cfDNA solutions with known contamination levels. This allows for the determination of the molar percentage of cfDNA in solutions with different contamination levels, and based on this, a contamination percentage assessment standard is established. By comparing the molar percentage of cfDNA in the cfDNA sample to be tested with the assessment standard, the contamination level of gDNA in the cfDNA sample to be tested can be obtained quickly, accurately, and efficiently.

[0116] The method of this invention can rapidly (e.g., within 1 hour) determine the degree of gDNA contamination in cfDNA samples, controlling sample quality at the source of cfDNA detection and preventing severely contaminated samples from entering the bioinformatics analysis step, thus saving economic and time costs. Simultaneously, it provides the required sequencing data volume for samples with other contamination levels that can proceed to the bioinformatics analysis step, avoiding batch effects and time costs introduced by additional testing. Furthermore, the method of this invention is simple to operate and requires a sample volume as low as 1 μL.

[0117] Fragmentomics based on specific amplification products

[0118] Fragmentomics refers to the analysis of cell-free nucleic acid fragments circulating in the body to reveal an individual's bioinformatics characteristics. In the human body, cells release many different types of cell-free nucleic acid fragments, including cell-free DNA, RNA, and micronon-coding RNA. These fragments can reflect information such as cellular state, genetic variation, and epigenetic modifications, thus having broad research application value.

[0119] Fragmentomics research methods typically include advanced sequencing technologies, mass spectrometry, and bioinformatics analysis. These technologies can efficiently isolate, identify, and quantify specific fragments and extract valuable information from large amounts of data.

[0120] As used herein, the term "short amplicon" refers to the amplification product of cfDNA, and the term "long amplicon" refers to the amplification product of gDNA.

[0121] Through extensive screening, the inventors unexpectedly obtained short amplicon based on a specific cfDNA species (i.e., the first amplification product corresponding to the length L1 of the cfDNA) and long amplicon based on a specific gDNA target sequence (i.e., the second amplification product corresponding to the length L2 of the gDNA).

[0122] In this invention, the yield of the first amplification product and the yield of the second amplification product can be calculated to obtain the relative abundance R, thereby characterizing the severity of gDNA contamination.

[0123] In this invention, the relative abundance R can be calculated using a variety of different methods. For example, qPCR can calculate the relative abundance using the Ct value, while conventional PCR can calculate the relative abundance based on primer amplification efficiency.

[0124] In this invention, a particularly preferred method is to obtain relative abundance based on the number of moles.

[0125] cfDNA

[0126] cfDNA, or cell-free DNA, typically exists primarily as fragments of approximately 170 bp. It is DNA that exists freely in bodily fluids such as blood and does not bind to cells. cfDNA detection is of great significance. For example, in the field of oncology, it can be used for early diagnosis, treatment monitoring, and prognostic assessment of tumors. Because tumor cells release cfDNA into the bloodstream, detecting tumor-specific mutations and other information within it can help detect tumors at an early stage. In prenatal diagnosis, cfDNA testing can be used to screen for fetal chromosomal abnormalities and other diseases, offering the advantage of being non-invasive.

[0127] Existing methods for cfDNA extraction mainly include phenol-chloroform extraction, silica membrane adsorption, magnetic bead extraction, and commercially available reagent kits. Because the release of gDNA during sample transport or extraction can contaminate the sample, the cfDNA content is usually low. Therefore, achieving high-accuracy extraction and detection is challenging, requiring advanced pretreatment techniques and stringent experimental conditions to improve the reliability of the detection results.

[0128] gDNA

[0129] gDNA, or genomic DNA, is the sum of all genetic material within an organism's cells. Human gDNA consists of approximately 3 billion base pairs. gDNA carries genes that encode proteins, as well as various sequence elements that regulate gene expression. These genes and regulatory elements collectively determine an organism's various biological characteristics, including morphology, physiological functions, and behavior. gDNA contains almost all of an organism's genetic information. During hemolysis, gDNA is released into the blood plasma, existing in large fragments or degraded into smaller fragments, contaminating cfDNA.

[0130] The method of the present invention

[0131] The method of this invention refers to a quality control method for rapid detection of trace gDNA contamination in cfDNA based on fragment omics. Specifically, based on the difference in fragment size between cfDNA and gDNA, the degree of gDNA contamination in the cfDNA solution is evaluated by amplifying cfDNA and gDNA in the sample using nucleic acid and calculating the molar percentage of cfDNA.

[0132] cfDNA sample

[0133] In this invention, the cfDNA sample is a cfDNA sample derived from bodily fluids.

[0134] Representative bodily fluids include (but are not limited to): blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, ascites, serous fluid, sputum, tears, urine, saliva, breast milk, gastric juice, or pancreatic juice.

[0135] In another preferred embodiment, the body fluid is blood, cerebrospinal fluid, and lymph.

[0136] In another preferred embodiment, the bodily fluid is blood.

[0137] In another preferred embodiment, methods for obtaining cfDNA samples from body fluids include (but are not limited to): phenol-chloroform extraction, silica membrane adsorption extraction, magnetic bead extraction, and commercially available kit methods.

[0138] In another preferred embodiment, the method for obtaining cfDNA samples from bodily fluids also includes extraction using a formulation reagent with performance comparable to commercially available cfDNA extraction kits.

[0139] In another preferred embodiment, the comparable performance means that the cfDNA yields extracted from the same sample are similar, and the purity and stability are comparable.

[0140] cfDNA and gDNA in PCR amplification samples

[0141] In this invention, amplification primers for cfDNA and gDNA are designed on the housekeeping gene based on the difference in fragment size between cfDNA and gDNA. Short amplicons represent cfDNA, and long amplicons represent gDNA.

[0142] Among them, housekeeping genes refer to a class of genes that are expressed in all cells and whose products are essential for maintaining basic cellular life activities.

[0143] An amplicon is a product obtained by amplifying a specific DNA fragment using techniques such as polymerase chain reaction (PCR). Its size depends on the selected amplification region and generally ranges from tens to thousands of base pairs.

[0144] In another preferred example, the housekeeping gene has the following characteristics:

[0145] (1) It is continuously expressed in different types of cells, and the expression level is relatively stable;

[0146] (2) Housekeeping genes usually encode proteins that maintain the basic structure and function of cells, such as proteins involved in the composition of ribosomes, enzymes involved in cell metabolism, and proteins that maintain the integrity of cell structure.

[0147] (3) The expression of housekeeping genes is not greatly affected by external environmental factors and can maintain a certain expression level under a wide range of physiological conditions to ensure the normal survival of cells and the progress of basic metabolic activities.

[0148] In another preferred embodiment, the housekeeping genes include (but are not limited to): β-Actin, GAPDH, and α-Tublin.

[0149] In another preferred embodiment, the housekeeping gene is preferably β-Actin.

[0150] In another preferred embodiment, the primer pair for amplifying cfDNA in the PCR amplification is SEQ ID NO:1 and SEQ ID NO:2.

[0151] In another preferred embodiment, the primer pair for amplifying cfDNA in the PCR amplification is SEQ ID NO:5 and SEQ ID NO:6.

[0152] In another preferred embodiment, the primer pair for amplifying cfDNA in the PCR amplification is SEQ ID NO:11 and SEQ ID NO:12.

[0153] In another preferred embodiment, the primer pair for amplifying cfDNA in the PCR amplification is SEQ ID NO:1 and SEQ ID NO:2.

[0154] In another preferred embodiment, the primer pair for amplifying gDNA in the PCR amplification is SEQ ID NO:3 and SEQ ID NO:4.

[0155] In another preferred embodiment, the primer pair for amplifying gDNA in the PCR amplification is SEQ ID NO:7 and SEQ ID NO:8.

[0156] In another preferred embodiment, the primer pair for amplifying gDNA in the PCR amplification is SEQ ID NO:9 and SEQ ID NO:10.

[0157] In another preferred embodiment, in the PCR amplification, the primer pair for amplifying gDNA is SEQ ID NO:13, and the downstream primer is SEQ ID NO:14.

[0158] In another preferred embodiment, the primer pairs for amplifying cfDNA and the primer pairs for amplifying gDNA can be used in any combination during the PCR amplification.

[0159] Quantification and proportion of cfDNA and gDNA

[0160] In this invention, the ratio of cfDNA to gDNA is a molar ratio. The amplified cfDNA and gDNA are quantified, and the molar ratio of cfDNA in the sample is calculated.

[0161] The methods that can be used for quantification include (but are not limited to): gel imaging analysis, ultraviolet spectrophotometry, quantitative real-time PCR, Qubit method, and biochip method.

[0162] In another preferred embodiment, the quantification method used is the Qubit method. The principle of Qubit quantitative detection is based on the property of fluorescent dyes specifically binding to dsDNA. When the fluorescent dye binds to dsDNA, it emits fluorescence under excitation light of a specific wavelength, and the instrument determines the concentration of dsDNA by detecting the intensity of the fluorescence signal.

[0163] Calculate the number of moles based on the concentration:

[0164] The number of moles of cfDNA = (mass number / 1000 * 10^6) / (660 * length of the first amplification product) * 1000.

[0165] Number of gDNA moles = (mass number / 1000 * 10^6) / (660 * length of the second amplification product) * 1000.

[0166] In another preferred embodiment, the mass number is obtained by mass concentration and volume.

[0167] In another preferred embodiment, the molar numbers of cfDNA and gDNA are quantified as Y1 and Y2, respectively.

[0168] In another preferred embodiment, the proportion of cfDNA in the cfDNA sample is Y1 / (Y1+Y2)×100%, and the proportion of gDNA is Y2 / (Y1+Y2)×100%.

[0169] Determination of the degree of contamination by trace amounts of gDNA in cfDNA samples

[0170] In this invention, samples with known contamination levels obtained from comprehensive bioinformatics index analysis are used to set thresholds for the proportion of cfDNA under different contamination conditions. By comparing the proportion of cfDNA in the cfDNA sample with the set thresholds, the contamination level of gDNA in the cfDNA sample to be tested can be obtained.

[0171] In another preferred embodiment, by analyzing samples with known contamination levels obtained from comprehensive bioinformatics index analysis, setting thresholds for the proportion of gDNA under different contamination conditions, and comparing the proportion of gDNA with the set thresholds, the contamination level of gDNA in the cfDNA solution to be tested can also be obtained.

[0172] In another preferred embodiment, the pollution level assessment criteria are as follows:

[0173] (a) If Y1 / (Y1+Y2)×100%<38%, then the cfDNA sample is determined to be severely contaminated with gDNA;

[0174] (b) If 38% ≤ Y1 / (Y1+Y2)×100% < 50%, then the gDNA in the cfDNA sample is determined to be moderately contaminated;

[0175] (c) If 50% ≤ Y1 / (Y1+Y2)×100% < 55%, then the gDNA in the cfDNA sample is determined to be slightly contaminated;

[0176] (d) If 55% ≤ Y1 / (Y1+Y2)×100% < 70%, then the gDNA in the cfDNA sample is determined to be uncontaminated;

[0177] (e) If Y1 / (Y1+Y2)×100%≥70%, then the cfDNA sample extraction or amplification process is deemed unqualified.

[0178] In another preferred embodiment, for samples determined to be slightly contaminated or uncontaminated, subsequent bioinformatics analysis can be performed, and the amount of data required for bioinformatics analysis is 6G;

[0179] For samples determined to be moderately contaminated, subsequent bioinformatics analysis can be performed, which requires 8G of data.

[0180] For samples determined to be severely contaminated or unqualified, bioinformatics analysis of cfDNA is not recommended; the samples must be extracted again.

[0181] Detection of amplification product length

[0182] In this invention, various methods can be used to determine the lengths of the first and second amplification products. A preferred method is to determine the length of the amplification products by capillary electrophoresis.

[0183] The main advantages of this invention include:

[0184] (a) This invention provides a simple and rapid quality control method for trace genomic nucleic acid contamination in trace amounts of cell-free nucleic acids in trace amounts of plasma.

[0185] (b) The method of the present invention has low sample size requirements and can quickly detect the proportion of contamination in trace samples.

[0186] (c) The method of the present invention controls the quality of samples from the source of detection and can process samples as soon as possible, thereby avoiding waste of resources.

[0187] (d) The method of the present invention can calculate the required amount of sequencing data by assessing the contamination percentage, thus avoiding the batch effect and time cost introduced by additional testing.

[0188] (e) The method of the present invention is stable and efficient, and has good data repeatability.

[0189] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0190] Example 1: Sample extraction and calculation of the relative proportion of cfDNA

[0191] 1. cfDNA Nucleic Acid Extraction. High-quality cfDNA samples were obtained using a dedicated cfDNA extraction kit (Novizan Cyclic Cell-Free Nucleic Acid Extraction Kit N913).

[0192] 2. Calculation of the relative proportion of cfDNA

[0193] 2.1 Amplification of cfDNA and gDNA

[0194] According to the formulas in Tables 1 and 2, the corresponding primer sequences of different primer sets shown in Table 3 were used as primers for amplification of cfDNA and gDNA, respectively. The cfDNA and gDNA in the sample obtained in step (a) were amplified (PCR / qPCR), and the amplification reaction procedure is shown in Table 4.

[0195] In this context, cfDNA mixed primers refer to a mixture of the upstream primer (_F) and downstream primer (_R) of cfDNA in the corresponding primer set, and gDNA mixed primers refer to a mixture of the upstream primer (_F) and downstream primer (_R) of gDNA in the corresponding primer set. The primer design principles for cfDNA and gDNA are shown in Figure 1.

[0196] The target sequence for cfDNA1 amplification is:

[0197] The target sequence for gDNA1 amplification is:

[0198] The target sequence for cfDNA2 amplification is:

[0199] The target sequence for gDNA2 amplification is:

[0200] The target sequence for cfDNA3 amplification is:

[0201] The target sequence for gDNA3 amplification is:

[0202] Table 1 cfDNA amplification system

[0203] Table 2 gDNA amplification system

[0204] Table 3 Primer pairs used for amplification

[0205] Table 4. Amplification reaction procedure

[0206] 2.2 Characterization of amplification products

[0207] The sequencing results of the amplification products of the cfDNA1 mixed primer and the gDNA1 mixed primer are shown in Figure 2 and Figure 3, respectively.

[0208] Capillary electrophoresis was used to analyze the fragment size of the amplification products. 1 μL of PCR amplification product was pipetted, thoroughly mixed with Dilution Buffer, and briefly centrifuged. The sample was collected at the bottom of the tube, ensuring there were no air bubbles in the solution. The tube was then placed in the sample detection position on the Qsep400 instrument for detection. The capillary electrophoresis results of the amplification products with mixed cfDNA and gDNA primers are shown in Figures 4 and 5, respectively.

[0209] 2.3 cfDNA Proportion Analysis

[0210] The amplification products were purified and quantified using Qubit. The relative proportion of cfDNA in the sample was calculated using the quantitative data of cfDNA and gDNA PCR products. The relevant formula is as follows:

[0211] cfDNA moles = (mass number / 1000 * 10^6) / (660 * length of the first amplification product) * 1000;

[0212] Number of gDNA moles = (mass number / 1000 * 10^6) / (660 * length of the second amplification product) * 1000;

[0213] cfDNA percentage = (moles of cfDNA / (moles of cfDNA + moles of gDNA)) * 100%.

[0214] Example 2: Development of standards for assessing gDNA contamination levels

[0215] Three samples of known different levels of contamination were selected, and cfDNA and gDNA were amplified and quantified using different primer pairs. The proportion of cfDNA was calculated.

[0216] (a) cfDNA1+gDNA1 primer set

[0217] Using SEQ ID NO:1 and SEQ ID NO:2 as upstream and downstream primers for cfDNA, and SEQ ID NO:3 and SEQ ID NO:4 as upstream and downstream primers for gDNA, cfDNA and gDNA in samples with known different levels of contamination were amplified and quantified, and the proportion of cfDNA in samples with known contamination levels was calculated.

[0218] The calculation results are shown in Table 5, and the corresponding bar chart and line graph results are shown in Figure 6. The comparison of the mass concentrations of cfDNA and gDNA is shown in Figure 7.

[0219] Among the three samples with the same level of contamination, the mass concentrations of cfDNA and gDNA fluctuated significantly, but the molar percentage of cfDNA remained relatively stable, and the CV values ​​of each group were relatively small.

[0220] The results showed that the calculated percentage of cfDNA was linearly correlated with the actual contamination level of the EDTA tube.

[0221] Table 5. Standards for assessing gDNA contamination levels

[0222] Based on this, thresholds for severe pollution, moderate pollution, mild pollution, and no pollution can be defined as evaluation criteria for the proportion of gDNA pollution, as shown in Table 6.

[0223] Table 6. Evaluation Criteria for gDNA Contamination Level

[0224]

[0225] When the calculated percentage of cfDNA Y1 / (Y1+Y2)×100% is ≥55% and <70%, it indicates that the sample has mild contamination or no contamination, and subsequent testing can be carried out normally. The required data volume is 6G(2x).

[0226] When the calculated percentage of cfDNA Y1 / (Y1+Y2)×100% is ≥38% and <50%, it indicates that the sample has moderate contamination, and the required sequencing data volume is 8G (2.7x).

[0227] When the calculated percentage of cfDNA Y1 / (Y1+Y2)×100% < 38%, it indicates that the sample is severely contaminated and cannot be further tested; resampling is required.

[0228] When the cfDNA percentage is higher than 70%, it indicates that there are many gDNA fragments in the blood that have been degraded into small fragments, suggesting abnormal library preparation or severe gDNA degradation, requiring resampling.

[0229] (b) cfDNA2+gDNA2 primer set

[0230] Using SEQ ID NO:5 as the upstream primer for cfDNA, SEQ ID NO:6 as the downstream primer for cfDNA, and SEQ ID NO:7 as the upstream primer for gDNA, SEQ ID NO:8 as the downstream primer for gDNA, cfDNA and gDNA in samples with known levels of contamination were amplified and quantified, and the proportion of cfDNA in samples with known levels of contamination was calculated.

[0231] The results are shown in Table 7. As the contamination level increased from no contamination to severe contamination, the proportion of cfDNA in the samples decreased, indicating that the cfDNA2+gDNA2 primer set can be used to distinguish samples with different levels of contamination.

[0232] However, the average percentage of cfDNA in severely contaminated, moderately contaminated, lightly contaminated, and uncontaminated tubes was between 60% and 80%, and the differentiation between the different levels of contamination was low compared with the results of the cfDNA1+gDNA1 primer set.

[0233] Table 7. Degree of gDNA contamination in cfDNA after primer amplification using cfDNA2+gDNA2 group.

[0234] (c) cfDNA3+gDNA3 primer set

[0235] Using SEQ ID NO:11 as the upstream primer for cfDNA, SEQ ID NO:12 as the downstream primer for cfDNA, SEQ ID NO:13 as the upstream primer for gDNA, and SEQ ID NO:14 as the downstream primer for gDNA, cfDNA and gDNA in samples with known levels of contamination were amplified and quantified, and the proportion of cfDNA in samples with known levels of contamination was calculated.

[0236] The results are shown in Table 8. As the level of gDNA contamination in the EDTA sampling tube increased, the proportion of cfDNA in the sample gradually decreased.

[0237] However, when there was no gDNA contamination in the sampling tube, the proportion of cfDNA in the sampling tube was actually lower than that in the severely contaminated samples. This is because the uncontaminated amplification group samples deviated from the linear relationship due to low primer amplification efficiency and low amplification product yield. In addition, the proportion of cfDNA in samples with the same level of contamination fluctuated significantly.

[0238] Table 8. Degree of gDNA contamination in cfDNA after amplification using primers from the cfDNA3+gDNA3 group.

[0239] Example 3: PCR detection of clinical samples

[0240] The "gDNA contamination level assessment criteria" described in Example 2 were used to assess 10 clinical samples, and the assessment results are shown in Table 9.

[0241] The results showed that most samples were of high quality, being either uncontaminated or slightly contaminated.

[0242] One sample was moderately contaminated, and the amount of sequencing data required for subsequent testing was 8G (2.7x).

[0243] Two samples were found to be substandard, and one sample was severely contaminated with cfDNA content below 38%. All of these samples required resampling and testing.

[0244] Table 9. PCR test results of clinical samples

[0245] Example 4: Comparison of the results of the present invention's method detection and library Qsep detection in clinical samples

[0246] The "gDNA contamination level assessment criteria" described in Example 2 were used to assess 10 clinical samples and compared with the results of Qsep library detection.

[0247] The results are shown in Table 10. Using the method of this invention, the degree of gDNA contamination in the samples was determined. Of the 10 clinical samples, 1 was uncontaminated, 2 were slightly contaminated, and 9 were moderately contaminated. The uncontaminated and slightly contaminated samples could proceed with subsequent testing normally, requiring 6G (2x) of data. The moderately contaminated samples required 8G (2.7x) of sequencing data for subsequent testing.

[0248] Furthermore, the PCR detection method of this invention shows a high degree of consistency with the Qsep detection method, approximately 86.4%.

[0249] The PCR detection method of this invention can more accurately determine the degree of contamination from four dimensions, including uncontaminated, slightly contaminated, moderately contaminated, and severely contaminated samples. It can meet the requirements of different biomarker combinations on the degree of gDNA contamination, and build a quality control process based on the degree of contamination and various detection process factors, providing guidance for the amount of sequencing data required for subsequent experiments.

[0250] Meanwhile, compared to the Qsep detection cycle of approximately 8 hours, the method of this invention takes only 1 hour, making it faster and less expensive.

[0251] Qsep fragment detection utilizes the principle of capillary electrophoresis to determine fragment size and concentration by analyzing the migration behavior of nucleic acid molecules in an electric field. Specifically, during the detection process, the sample is injected into a capillary. Under the influence of the electric field, nucleic acid fragments of different sizes migrate at different speeds. By detecting the migration time of fluorescently labeled nucleic acid fragments in the capillary, the fragment size can be accurately determined; simultaneously, the fragment concentration can be quantitatively analyzed based on the intensity of the fluorescence signal.

[0252] In this embodiment, if there are three spurious peaks between 450-1000 bp in the Qsep result, it indicates severe sample contamination; otherwise, it indicates mild contamination. Although Qsep results have high resolution and can accurately distinguish minute fragment differences, the judgment result is qualitative or semi-quantitative. Factors such as the number, position, and height of spurious peaks have a certain impact on the interpretation.

[0253] Table 10 Comparison results of the detection method of the present invention and Qsep detection.

[0254] Example 5: Comparison of PCR detection results for samples collected from different sampling tubes

[0255] The proportion of cfDNA in the samples was calculated and the degree of contamination was determined using the method of this invention. The determination results were further verified by bioinformatics analysis.

[0256] The results are shown in Table 11.

[0257] The results showed that the bioinformatics analysis results and the PCR experimental results were highly consistent and could be interpreted in the same way. This indicates that the method of the present invention has high accuracy in predicting the degree of contamination.

[0258] Table 11 Comparison of the judgment results between the method of the present invention and the bioinformatics analysis method.

[0259] As demonstrated by the embodiments of this invention, based on the lengths of cfDNA and gDNA fragments, the cfDNA1+gDNA1 primer set is selected to amplify cfDNA and gDNA in the sample, respectively. Subsequently, the proportion of cfDNA in the sample is quantified and calculated. By comparing the results, the degree of sample contamination can be obtained, guiding subsequent bioinformatics analysis of the sample (as shown in Figure 8). This achieves quality control of trace gDNA contamination in cfDNA based on fragment omics detection. This primer set exhibits good stability, high accuracy, and rapid detection in the quality control of trace gDNA contamination in cfDNA; this effect is not readily apparent.

[0260] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for determining the degree of gDNA contamination in a cfDNA sample, characterized in that, The method includes the following steps: (Z1) Provide a cfDNA sample, which is a cfDNA sample derived from bodily fluids; (Z2) Amplify the cfDNA and gDNA in the cfDNA sample to obtain a first amplification product with a length of L1 corresponding to the cfDNA and a second amplification product with a length of L2 corresponding to the gDNA. (Z3) Quantitatively detect the concentration or level of the first amplification product, designated as Y1; and quantitatively detect the concentration or level of the second amplification product, designated as Y2; (Z4) Based on the concentration or level Y1 of the first amplification product and the concentration or level Y2 of the second amplification product, the relative proportion R of cfDNA in the cfDNA sample is obtained, thereby determining the degree of gDNA contamination in the cfDNA sample.

2. The method as described in claim 1, characterized in that, In step (Z2), the target sequence for amplifying cfDNA in the cfDNA sample is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or a combination thereof.

3. The method as described in claim 1, characterized in that, In step (Z2), the target sequence for amplifying gDNA in the cfDNA sample is selected from the group consisting of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or a combination thereof.

4. The method as described in claim 1, characterized in that, In the amplification process, the primer pair for amplifying cfDNA is SEQ ID NO:1 and SEQ ID NO:2; and The primer pair for amplifying gDNA is SEQ ID NO:3 and SEQ ID NO:

4.

5. The method as described in claim 1, characterized in that, In the amplification process, the primer pair for amplifying cfDNA is SEQ ID NO:5 and SEQ ID NO:6; and The primer pair for amplifying gDNA is SEQ ID NO:7 and SEQ ID NO:

8.

6. The method as described in claim 1, characterized in that, In step (Z3), the mass concentration or mass number C1 and length L1 of the first amplification product are determined to obtain the molar concentration or molar number Y1 of the first amplification product; and the mass concentration or mass number C2 and length L2 of the second amplification product are determined to obtain the molar concentration or molar number Y2 of the second amplification product.

7. The method as described in claim 6, characterized in that, The mass concentration was determined using the Qubit method.

8. The method as described in claim 6, characterized in that, The molar quantity of the first amplified product Y1 = (C1 / 1000*10^6) / (660*L1)*1000.

9. The method as described in claim 6, characterized in that, The molar quantity of the second amplification product Y2 = (C2 / 1000*10^6) / (660*L2)*1000.

10. The method as described in claim 1, characterized in that, The relative proportion of cfDNA is R = Y1 / (Y1+Y2)×100%.

11. The method as described in claim 1, characterized in that, In step (Z4), the relative proportion R of cfDNA is compared with a predetermined reference value to determine the degree of gDNA contamination in the cfDNA sample.

12. The method as described in claim 11, characterized in that, The following criteria shall be used for judgment: (a) If R < 38%, then the gDNA in the cfDNA sample is considered to be severely contaminated; (b) If 38% ≤ R < 50%, then the gDNA in the cfDNA sample is considered to be moderately contaminated; (c) If 50% ≤ R < 55%, then the gDNA in the cfDNA sample is considered to be slightly contaminated; (d) If 55% ≤ R < 70%, then the gDNA in the cfDNA sample is determined to be uncontaminated; (e) If R ≥ 70%, the cfDNA sample is deemed unqualified.

13. The method as described in claim 12, characterized in that, Step (Z4) also includes providing the following recommendations based on the level of gDNA contamination: When a cfDNA sample is determined to be severely contaminated or unqualified, it is not recommended to perform bioinformatics analysis on the cfDNA; the cfDNA sample must be extracted again. cfDNA samples that are determined to be slightly contaminated or uncontaminated can undergo cfDNA bioinformatics analysis, requiring 6G of data. cfDNA samples identified as moderately contaminated can undergo cfDNA bioinformatics analysis, requiring 8G of data.

14. A cfDNA quality control system for determining the degree of gDNA contamination in cfDNA samples, characterized in that, The system includes the following modules: (S1) Sample acquisition module, the sample acquisition module is configured to: acquire cfDNA samples from body fluids; (S2) Nucleic acid amplification module, wherein the nucleic acid amplification module is configured to amplify cfDNA and gDNA in cfDNA sample to obtain a first amplification product of length L1 corresponding to cfDNA and a second amplification product of length L2 corresponding to gDNA. (S3) Quantitative detection module, wherein the quantitative calculation module is configured to: quantitatively detect the concentration or level of the first amplification product, defined as Y1; and quantitatively detect the concentration or level of the second amplification product, defined as Y2; (S4) Contamination level analysis module, which is configured to: obtain the relative proportion R of cfDNA in cfDNA sample based on the concentration or level Y1 of the first amplification product and the concentration or level Y2 of the second amplification product, thereby giving the assessment result of the contamination level of gDNA in cfDNA sample. (S5) Output module, the output module is configured to output the assessment result of the contamination level of the gDNA.

15. The use of a primer pair combination, characterized in that, This kit is used to prepare a kit for determining the degree of gDNA contamination in cfDNA samples before bioinformatics analysis, so as to avoid severely contaminated samples from entering the bioinformatics analysis step, and to provide the sequencing data volume requirements for other contamination levels of samples that can enter the bioinformatics analysis step. The primer pair comprises: A pair of cfDNA primers for amplifying cfDNA, the cfDNA primer pair comprising the primers shown in SEQ ID NO:1 and SEQ ID NO:2; and gDNA primer pairs for amplifying gDNA, the gDNA primer pairs including the primers shown in SEQ ID NO:3 and SEQ ID NO:4.

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