A method and system for assessing the mutagenicity of a compound

Abstract

The application provides a compound mutagenicity evaluation method and system, belonging to the field of molecular biology and chemical technology, comprising: culturing human cells meeting the requirements, and then treating the human cells with different concentrations of the compound; extracting genomic DNA from the human cells and sequencing; obtaining total base data and mutant base data based on the sequencing data, and calculating the mutation frequency; evaluating the mutagenicity of the system based on the mutation frequency and the minimum mutagenic concentration. For human cells, through quality control of sequencing process data and specific screening methods in the base model, the final evaluation method and system can identify ultra-low frequency mutations induced by short-term exposure of compounds, especially can detect 10 ‑8 ultra-low frequency mutations induced by 24h exposure of the compound.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology and chemical technology, and in particular to a method and system for evaluating the mutagenicity of a compound. BACKGROUND

[0002] In the process of drug research and production, by-products such as ethyl methanesulfonate (EMS), nitrosamines (such as NDMA) often appear. These compounds have strong mutagenicity and can cause damage to the patient's genome, leading to genetic mutations, increasing the risk of cancer or the probability of occurrence of other genetic diseases. In addition, long-term exposure to drugs containing EMS can also affect germ cells and pass on mutations to the next generation. Therefore, in the process of drug research, production and safety evaluation, great attention and strict control of the content are needed.

[0003] Conventional in vitro mutagenicity tests for evaluating compounds include bacterial reverse mutation test (Ames test), mouse lymphoma assay (MLA), etc. However, the in vitro test system has certain limitations. For example, the metabolic activation system specific to bacteria is not suitable for evaluating nitroimidazole compounds. In addition, for N-nitroso compounds, positive results can only be obtained under special metabolic activation conditions. The MLA has a high false positive rate. The in vivo Pig-a gene mutation test using normal animals is a mutagenicity evaluation method that has entered the field of safety evaluation in recent years and has been widely recognized. However, this method can only detect gene mutations occurring in red blood cells. Although transgenic animal tests have the advantage of being able to detect gene mutations and mutation profiles in different target tissues such as liver and kidney after exposure to the test substance, the animal models used in this method are difficult to obtain and the reagents used for late-stage detection are expensive. All the above conventional mutagenicity evaluation methods use phenotypic changes (increase in colonies, clones or cell surface antigen changes, etc.) as the observation index of mutation occurrence, and do not directly investigate the changes in gene sequences caused by drugs, so the results may be biased.

[0004] Pig-a / PIG-A gene mutation test is a detection method based on endogenous phosphatidylinositol glycan class-A gene (PIG-A / Pig-a). The test principle is that after the mutation of PIG-A / Pig-a gene on the X chromosome, the synthesis of cell surface glycosylphosphatidylinositol (GPI) anchor is hindered and the anchor protein is lost. Therefore, the expression level of the lost anchor protein can be quantitatively detected by flow cytometry and other means to evaluate the mutagenic risk of the test substance. At present, a large number of tests have verified the sensitivity and specificity of the in vivo Pig-a gene mutation test system based on the analysis of peripheral blood of rodents. The peripheral blood cell doubling time is short, and the distribution of GPI anchor is uniform, which is suitable for detecting the mutagenic risk of test substances based on cell phenotype.

[0005] Next-generation sequencing (NGS) is based on polymerase chain reaction (PCR) and gene chip technology, which can simultaneously perform millions to billions of individual sequencing reactions in large-scale parallel sequencing. The proportion of the number of DNA fragments obtained after conventional NGS PCR amplification may vary, and PCR amplification will introduce base mismatches and sequence preferences, resulting in changes in the relative frequency and abundance of DNA fragments after amplification. And the error rate of conventional NGS technology is about 10 -4 ~10 -2 , which is much higher than the background mutation frequency of the human body (10 -9 ~10 -7 ).

[0006] In the prior art, a kind of method and reagent for detecting and evaluating genotoxicity disclosed in Chinese invention patent with publication number CN111836905A directly measures mutation induced by compound using double sequencing technology, extracts DNA fragments of subjects or test animals for double sequencing, generates error-corrected consensus sequence, identifies mutation spectrum, calculates mutation frequency, to evaluate genotoxicity potential and carcinogenic risk. But there are corresponding problems, that is, it is a general method, the error rate is large, it is difficult to identify ultra-low frequency mutations induced by short-term exposure of compounds, and the technology is mainly based on transgenic animal models, resulting in long test cycle and high reagent cost. SUMMARY

[0007] To solve the above problems, the application provides a compound mutagenicity evaluation method and system. For human cells, through quality control of sequencing process data and specific screening in the base model, the final evaluation method and system can identify ultra-low frequency mutations induced by short-term exposure of compounds, especially ultra-low frequency mutations (10 -8 level) induced by 24h exposure of compounds.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0009] The application provides a compound mutagenicity evaluation method, which comprises the following steps: culturing human cells meeting the requirements, and then treating the human cells with different concentrations of the compound; extracting genomic DNA from the human cells and performing sequencing; obtaining total base data and mutation base data based on the sequencing data, and calculating the mutation frequency; the mutation frequency meets the following three conditions: (1) using the Mann-Kendall method to perform trend test on different compound concentration treatment groups, meeting p-value 1<0.05; (2) using ANOVA single factor variance analysis to test the mutation frequency of the sample of each concentration treatment group and the control group, meeting p-value 2<0.05; (3) obtaining the change fold FC of each concentration treatment group relative to the control group by comparing the mutation frequency of the sample of each concentration treatment group with the mutation frequency of the sample of the control group, meeting FC≥α, wherein the value of α is related to whether the processing soft cutoff is used and the number of terminal filtered bases; the lowest concentration meeting the above three conditions is the minimum mutagenic concentration of the compound system.

[0010] Further, the sequencing comprises that the concentration A260 / A280 ratio of the genomic DNA of the human cells is 1.8-2.0, and the size of the DNA fragments is controlled to be 180-220bp;

[0011] Then, the DNA fragments are filled, and molecular identifiers are introduced in the linking process; the DNA library is hybridized in a liquid phase environment using a biotin probe and a blocking solution, after eluting the captured library, the target fragments are enriched through PCR amplification, and the final library is obtained; the final library is sequenced to obtain raw data, and the raw data is quality controlled, aligned and consistency analyzed, and finally the sequencing data is obtained.

[0012] Further, for the sequencing data, first, the total base data of the sample is obtained; then, the mutation bases common to the samples treated with different concentrations of the compound and the control samples are removed, next, the sample mutation base data is obtained, and the sample mutation frequency is calculated.

[0013] Further, the total base data is obtained in the following manner: the sequencing data is screened, and the data meeting the following conditions is counted:

[0014] (1) the number of clusters of each sequence in the consistency analysis is greater than or equal to 1, excluding unclustered sequences; (2) the MAQ of each sequence after the consistency analysis is greater than or equal to 60; based on the sequences screened according to the above rules, the molecules are selected according to the following rules: (1) if either strand of a double helix of a DNA molecule is covered by a sequencing molecule, the molecule is retained; (2) if both strands of a double helix of a DNA molecule are covered by a sequencing molecule, only one of the molecules is retained; finally, the number of bases contained in the molecules satisfying the above rules is counted as the total base data of the sample.

[0015] Further, the mutant base data is obtained by screening the molecules satisfying the following conditions: (1) at least one single nucleotide mutation is contained, without considering short insertion and deletion base mutations; (2) the MAQ of each sequence after the consistency analysis is greater than or equal to 60; (3) both strands of a double helix are covered by a sequencing molecule; (4) the number of clusters of each sequence in the consistency analysis is greater than or equal to 1; (5) soft clipping is processed; (6) the mutant bases at the 5'-end and 3'-end are filtered; the number of single nucleotide mutation bases of the molecules satisfying the above conditions is counted as the mutant base data of the sample.

[0016] Further, whether the soft clipping is processed or not, the number of filtered bases n located at the end satisfies the following formula:

[0017]

[0018] Wherein, if the soft clipping is processed, C is 1, and if the soft clipping is not processed, C is 0.

[0019] The application further provides a compound mutagenicity evaluation system, comprising: a human cell culture and processing module, which is used to culture human cells meeting the requirements, and then treat the human cells with different concentrations of the compound; a sequencing module, which is used to extract genomic DNA from the human cells and perform sequencing; a calculation module, which is used to obtain total base data and mutant base data of a sample based on the sequencing data, and calculate the mutant frequency of the sample; and an evaluation module, which is used to evaluate the mutagenicity of the compound system and determine the minimum mutagenic concentration according to the following condition evaluation system: (1) the Mann-Kendall method is used to perform trend test on different compound concentration treatment groups, and p-value 1<0.05 is met; (2) ANOVA single factor variance analysis is used to test the mutant frequency of each concentration treatment group and the control group, and p-value 2<0.05 is met; (3) the change fold FC of the mutant frequency of each concentration treatment group relative to the control group is obtained, and FC≥α is met, wherein the value of α is related to whether the soft clipping is processed or not and the number of end filtering bases; the minimum concentration meeting the above three conditions is the minimum mutagenic concentration of the compound system.

[0020] Further, the sequencing comprises: the concentration of the human cell genomic DNA is 1.8-2.0 at A260 / A280 ratio, and the size of the DNA fragments is controlled at 180-220 bp; then the DNA fragments are filled up, and molecular identifiers are introduced in the process of ligation; the DNA library is hybridized in a liquid phase environment using a biotin probe and a blocking solution, after the captured library is eluted, the target fragments are enriched by PCR amplification to obtain a final library; the final library is sequenced to obtain raw data, and the raw data is quality controlled, aligned and consistency analyzed to finally obtain sequencing data.

[0021] Further, for the sequencing data, first, total base data of the sample is obtained; then, mutant bases common to the samples treated with different concentrations of compounds and the control sample are removed; next, sample mutant base data is obtained, and sample mutation frequency is calculated.

[0022] Further, the total base data of the sample is obtained in the following manner: the sequencing data is screened, and the following conditions are met for statistics: (1) the number of clusters of each sequence in the consistency analysis is ≥1, and sequences not clustered are excluded; (2) the MAQ of each sequence after consistency analysis is ≥60; based on the sequences screened above, molecular selection is performed according to the following rules: (1) if either strand of a DNA molecule double helix is covered by a sequencing molecule, the molecule is retained; (2) if both strands of a DNA molecule double helix are covered by a sequencing molecule, only one of the molecules is retained; finally, the number of bases contained in the molecules meeting the above rules is counted as the total base data of the sample.

[0023] Further, the sample mutant base data is obtained in the following manner: molecules meeting the following conditions are screened: (1) containing at least one single nucleotide mutation, without considering short insertion and deletion base mutations; (2) the MAQ of each sequence after consistency analysis is ≥60; (3) both strands of the double helix are covered by a sequencing molecule (double strand coverage); (4) the number of clusters of each sequence in the consistency analysis is ≥1; (5) soft truncation is processed; (6) mutant bases located at the 5'-end and the 3'-end are filtered; for the molecules meeting the above conditions, the number of single nucleotide mutant bases is calculated as the mutant base data of the sample.

[0024] Further, whether the soft truncation is processed or not, the number of filtered bases n located at the end satisfies the following formula:

[0025]

[0026] Wherein, if the soft truncation is processed, C is 1, and if the soft truncation is not processed, C is 0.

[0027] The embodiment of the present application also provides a computer readable storage medium, characterized in that the computer readable storage medium has instructions stored therein, which, when executed by one or more processors, perform the above-mentioned evaluation method for evaluating the mutagenicity and the minimum mutagenic concentration of a system.

[0028] The embodiment of the present application also provides a computer system, comprising: at least one computer having a processor, a memory and a database, wherein the processor is configured to execute a computer program stored in the processor to perform the above-mentioned evaluation method.

[0029] The technical scheme provided by the embodiment of the present application has the following beneficial effects:

[0030] (1) The present application proposes a method of directly analyzing the mutation of cell DNA after the test substance is exposed to human cells, which is more accurate than the traditional drug mutagenicity evaluation method based on the phenotype detection of bacterial, cell or rodent test system, and the correlation between the results and the prediction of human carcinogenic risk is higher; (2) The current gold standard for drug mutagenicity evaluation is the mutagenicity evaluation test based on transgenic animal models, which is also a phenotype-based detection method, but the animal resources are scarce, the test period is long (about 3 months), and the reagent cost is high, with a quotation of about 400,000 US dollars from foreign companies. Under the premise of meeting the 3R principle of animal experiments, the present application saves cost and obtains more accurate data; (3) In addition to obtaining mutation rate data, the detection method can also analyze the mutation spectrum of different test substances and compare the mutation characteristics to provide data support for the mutagenic mechanism of the test substances. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0032] Figure 1 Base model v201, v202, v203 results provided for embodiment 1 of the present application;

[0033] Figure 2 Base model v201, v207, v213, v219 results provided for embodiment 1 of the present application;

[0034] Figure 3 Base model v204, v210, v216, v222 results provided for embodiment 1 of the present application;

[0035] Figure 4The base model v207 mutation spectrum characteristic result provided for the embodiment 1 of the present application;

[0036] Figure 5 The EMS mutation rate result of different concentrations provided for the comparative example 1 of the present application by flow cytometry;

[0037] Figure 6 The molecular model v101 to v104 result provided for the comparative example 2 of the present application. DETAILED DESCRIPTION

[0038] The present application will be further described in detail by specific embodiments. However, those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific technology or condition is not specified in the examples, it is carried out according to the technology or condition described in the literature in the art or according to the product instruction. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained by purchase.

[0039] The word "comprise", "contain", "have" or any other variant thereof used in this text is intended to cover non-exclusive inclusion. For example, a process, method, article or equipment including listed elements is not necessarily limited to those elements, but can include other elements not explicitly listed or inherent to such a process, method, article or equipment. Unless the context clearly specifies, the singular form "one" and "said" includes the plural discussion object.

[0040] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, equipment, materials used in the examples, any method, equipment and material of the prior art similar or equivalent to the method, equipment and material described in the examples of the present application can also be used to realize the present application according to the master of the prior art of those skilled in the art and the description of the present application.

[0041] Unless otherwise specified, the experimental methods, detection methods, preparation methods disclosed in the present application all adopt the conventional molecular biology, immunology, inspection, gene sequencing technology, bioinformatics technology and related conventional technology in the field.

[0042] Example 1

[0043] The embodiment of the present application provides a compound mutagenicity evaluation method, comprising:

[0044] S1, culturing human cells meeting the requirements, and then treating the human cells with different concentrations of the compound;

[0045] S2, extracting genomic DNA from the human cells and sequencing;

[0046] S3, obtaining total base data and mutation base data of the sample based on the sequencing data, and calculating mutation frequency of the sample;

[0047] S4, evaluating mutagenicity of the compound and determining the minimum mutagenic concentration by using the following condition evaluation system,

[0048] (1) using Mann-Kendall method to perform trend test on the mutation frequency of the sample and the concentration of the compound in each concentration treatment group and the control group, and meeting p-value 1<0.05, that is, the mutation frequency has a dose-dependent effect on the compound;

[0049] (2) using ANOVA single factor variance analysis to test the mutation frequency of the sample in each concentration treatment group and the mutation frequency of the sample in the control group, and meeting p-value 2<0.05, that is, the mutation frequency of the sample in the treatment group has a significant difference from the mutation frequency of the sample in the control group;

[0050] (3) obtaining the change fold of the mutation frequency of the sample in each concentration treatment group relative to the control group, denoted as FC (Fold Change), by comparing the mutation frequency of the sample in each concentration treatment group with the mutation frequency of the sample in the control group; meeting FC≥α, wherein the value of α is related to whether the treatment is soft truncated or not and the number of terminal filtered bases,

[0051] the minimum concentration of the compound in the treatment group meeting the above three conditions is the minimum mutagenic concentration of the compound system.

[0052] Specifically, the value of α meets the following formula:

[0053] α=A1n 3 -A2n 2 +A3n+2.1+0.33C

[0054] In the above formula, A1, A2 and A3 are correction coefficients, n is the number of filtered bases, C is 1 when the treatment is soft truncated, and C is 0 when the treatment is not soft truncated. In the embodiment of the present application, the value of A1 is 0.0011, the value of A2 is 0.028, and the value of A3 is 0.21.

[0055] The closer the calculated α value is to the actual calculated value FC, the more stable it is, and preferably, the difference between the actual calculated value FC and the calculated α value is less than 0.5.

[0056] Specifically, in the embodiments of the present application, the human cell adopts TK6 cell line, the cell p53 function of human lymphoblast TK6 cell is sound, which can reduce the false positive rate, and the doubling time is shorter than that of mouse-derived cell line (such as L5178Y), and the prediction degree of human body mutagenicity risk is more reliable. The human lymphoblast (TK6) is from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences, and is stored in liquid nitrogen. After thawing, it is passaged for less than 10 times. RPMI1640 complete culture medium containing 10% inactivated horse serum, 2% sodium pyruvate and 1% penicillin-streptomycin is used, and the culture is carried out at 37°C, 5% CO2. The GPI(-) cell rate of the TK6 cell used should be controlled within the range of 5×10 -6 ~7.5×10 -5 . After the TK6 cell reaches the logarithmic growth phase, it is centrifuged at room temperature for 5 min, and the cell density is adjusted to about 4×10 5 ~5×10 5 cells / mL in 10 ml of RPMI1640 complete culture medium. EMS with concentrations of 0 μg / mL, 10 μg / mL, 100 μg / mL, 120 μg / mL and 150 μg / mL is added respectively, and the culture is carried out at 37°C, 5% CO2 for 24 h. After the culture is completed, the cells are centrifuged at room temperature for 5 min, and cell counting is carried out, and the cells are washed with PBS solution for 2 times, and are resuspended in 10 mL of RPMI1640 complete culture medium.

[0057] The group with 0 μg / mL EMS added is the control group, and the control group does not add the compound, but only adds the same volume of pure water. The groups with 10 μg / mL, 100 μg / mL, 120 μg / mL and 150 μg / mL EMS added are different concentration treatment groups. Each group of samples is subjected to 3 parallel experiments.

[0058] The sequencing process included: extracting genomic DNA from cell line samples; firstly, accurately determining the DNA concentration using a Qubit 4.0 quantitative PCR instrument, requiring an A260 / A280 ratio of 1.8–2.0; then, based on the quantification results, taking 700–1200 ng of DNA sample and adding TE buffer to bring the reaction system to 130 μL (the upper limit of Covaris microtube volume); next, randomly fragmenting the DNA using a Covaris M220 ultrasonic disruptor, with the specific fragmentation time set according to the amount of DNA input (as shown in Table 1). For example, if the DNA extraction volume exceeded 1200 ng, 1200 ng was fragmented for 220 seconds. Finally, the fragment size was confirmed to be strictly controlled within the range of 180–220 bp using an Agilent 4200 TapeStation. After purification and recovery of the fragmented products using 1.8X XP magnetic beads, library construction was performed using the xGen Prism DNA Library Prep Kit, employing an optimized blunt-end ligation method to improve ligation efficiency and reduce adapter dimer formation. A unique molecular identifier (UMI) was introduced during the ligation process to facilitate subsequent data deduplication and error correction, thereby improving the accuracy of low-frequency mutation detection. Next, the DNA library was hybridized for 2 hours in a liquid phase using biotinylated probes and blocking buffer. After eluting the captured library, the target fragment was enriched by PCR amplification to obtain the final library. The final library was then subjected to high-throughput paired-end sequencing on a next-generation sequencer (GENETRON S2000, Chongqing Pan-Genomics Co., Ltd.), with a read length of 2 × 150 bp, yielding raw data in FASTQ format.

[0059] Table 1: Selection of DNA input amount and interruption time

[0060] Amount X (ng) Time of break (s) 700≤X≤900 200 900<X<1100 210 1100≤X≤1200 220

[0061] Raw sequencing data quality control and alignment: The raw cell line sample data was filtered using Trimmomatic (http: / / www.usadellab.org / cms / ?page=trimmomatic) software to remove sequences containing adapters and low-quality sequences. The low-quality sequence filtering parameters were: TRAILING:3, SLIDINGWINDOW:4:15, MINLEN:36. After filtering, valid data of acceptable quality was obtained. The valid data was then aligned to the human reference genome (GRCh37, 2009-02-27) using BWA-MEM (https: / / sourceforge.net / projects / bio-bwa / ) software to obtain raw alignment result files in BAM format.

[0062] Consensus analysis of molecular label: the raw alignment result file obtained in the previous step is corrected by IndelRealigner using GATK software (http: / / www.broadinstitute.org / gatk, version: v3.1-0-g72492bb), and sequences not aligned to the reference genome are removed to obtain the corrected BAM file. The consensus software (v1.0, internal software of Pansheng) is used for consensus analysis of UMI. The rules for consensus analysis are: the number of sequences supporting the consensus base is ≥2 or the proportion is ≥80%, and if the above conditions are not met, the consensus base is N (A, T, C, G bases are not determined, represented by N). The number of clusters of each sequence of a sequencing molecule is ≥1 when consensus analysis is performed, and sequences that do not form clusters are excluded to reduce noise caused by sequencing errors. After UMI (molecular identifier) consensus analysis, noise can be removed to a greater extent, and the mutated bases of the sample can be more accurately detected. The final sequence obtained after consensus analysis is stored as a BAM format file. The mpileup file is obtained by analyzing the BAM file using the mpileup function of the samtools (v1.7, http: / / www.htslib.org / doc / samtools.html) software, and the parameters are: -A -B-C 0-Q20-q 20. The obtained mpileup file is processed to obtain a VCF format file containing only mutation sites.

[0063] For the sequencing data, first, the total base data of the sample is obtained, and then the mutation bases common to the samples treated with different concentrations of compounds and the control samples are removed; next, the sample mutation base data is obtained, and the sample mutation frequency is calculated. Specifically, after obtaining the vcf file of the cell line sample treated with different concentrations of EMS, the mutation bases common to the samples treated with different concentrations of compounds and the control samples are removed. This part of the base includes both genetic mutations and random mutations generated during the culture of the cell line. The remaining mutations after removing the common mutation bases are the background mutations of the sample and the mutations caused by compound treatment.

[0064] The total base data of the sample is obtained in the following way:

[0065] The sequencing data is screened, and the following conditions are met for statistics:

[0066] (1) The number of clusters of each sequence during consensus analysis is ≥1, and sequences that do not form clusters are excluded to reduce noise;

[0067] (2) The MAQ of each sequence after consensus analysis is ≥60 to ensure high confidence alignment;

[0068] Based on the sequences screened above, molecular selection is carried out according to the following rules:

[0069] (1) If either strand of the double helix of a DNA molecule is covered by the sequencing molecule (i.e. single strand coverage), the molecule is retained;

[0070] (2) If both strands of the double helix of a DNA molecule are covered by the sequencing molecule (i.e. double strand coverage), only one of the molecules is retained;

[0071] The number of bases contained in the molecules satisfying the above rules is finally counted as the total base data of the sample.

[0072] The sample mutation base data is obtained in the following manner:

[0073] Molecules satisfying the following conditions are screened:

[0074] (1) Containing at least one single nucleotide mutation, without considering short insertion and deletion base mutations;

[0075] (2) MAQ of each sequence after consistency analysis is ≥ 60, ensuring high confidence alignment;

[0076] (3) Both strands of the double helix are covered by the sequencing molecule, improving the accuracy of mutation base detection and reducing noise;

[0077] (4) The minimum cluster support threshold of each sequence during consistency analysis is set to 1, 2 and 3 respectively. This threshold is a value that needs to be carefully weighed. A large value can improve accuracy, but it may also filter out real mutations (especially in low amplification efficiency or high GC content regions). A small value has high sensitivity, which can detect low frequency mutations, especially for low coverage regions, but there is a risk of retaining false positive signals caused by sequencing errors;

[0078] (5) Soft clipping is processed. Soft clipping has a dual impact on NGS data analysis: on the one hand, it can lead to missed detection of variations (e.g. when the truncated region contains a real SNV) or bias in allele frequency calculation; on the other hand, soft clipping can automatically remove low-quality ends, reducing the risk of misjudgment of sequencing errors, and helping to identify variations in complex or repetitive regions. Reasonable processing of soft clipping is crucial to balance detection sensitivity and specificity;

[0079] (6) The mutation bases located at the 5'-end and 3'-end are filtered, and the DNA is randomly broken by using an ultrasonic disruptor in the library construction step, in which process, the DNA ends may be oxidatively damaged due to the generation of reactive oxygen species (ROS), and for the oxidative damage that may occur, the application adopts a dynamic filtering mechanism to filter the mutation bases (at intervals of 5 bp) within a range of 0-15 bp from the ends in a gradient manner, so as to eliminate the mutations caused by oxidative damage;

[0080] For the molecules satisfying the above conditions, the number of single nucleotide mutation bases is calculated as the mutation base data of the sample.

[0081] Specifically, the number n of filtered bases of the mutation bases located at the ends is filtered according to whether the soft truncation is processed or not, and satisfies the following formula:

[0082]

[0083] Wherein, C is 1 if the soft truncation is processed, and C is 0 if the soft truncation is not processed.

[0084] Based on different cluster size thresholds, whether the soft truncation is processed or not, and the length of the mutation bases located at the 5'-end and 3'-end, different combinations are used for verification, and the combination modes are shown in Table 2.

[0085] Table 2: Detection results of different base model combination conditions

[0086]

[0087] Note: a The soft truncation is processed, 0 indicates that the soft truncation is not processed, that is, the molecules containing the soft truncation are retained, and 1 indicates that the soft truncation is processed, that is, the molecules containing the soft truncation are filtered out;

[0088] b End filtering length (bp), indicating the length of the mutation bases located at the 5'-end and 3'-end of the molecules filtered out;

[0089] c CV(mean), indicating the average value of the coefficient of variation of the mutation frequency of the whole system (control group and each concentration treatment group); the value is less than 10%, the result is relatively stable, and the smaller the value is, the more stable it is, and the value is greater than or equal to 10%, the stability is relatively poor.

[0090] In the v201-v203 version, we evaluated the influence of different cluster size thresholds on the results. As shown in Table 2 and the attached Figure 1As shown, the sample mutation frequency gradually decreases as the cluster size threshold increases.v201 version: Mann-Kendall trend test (MK p-value 1<0.05) indicates that there is a significant dose-dependent effect, and the minimum mutagenic concentration is 10 μg / mL;v202 version: the results meet the three conditions of the evaluation module. However, as the dose increases, the difference fold of mutation frequency of different dose samples is small, especially the difference fold of 10 and 100 μg / mL samples is 3.2 and 3.3 respectively, which may be due to the fact that the cluster threshold of v202 version is set to 2, and the real mutations are filtered out, resulting in a decrease in the discrimination of the treatment group, and the CV value is 11.242%, showing poor stability;v203 version: Mann-Kendall trend test results show that MK p-value 1 is 0.2207 (greater than 0.05), indicating that there is no significant dose-dependent effect, and the condition combination of v203 version cannot be used for mutagenicity evaluation. Therefore, the best cluster size threshold of the base model is 1.

[0091] When the cluster size threshold is 2, the processing soft cutoff and the end filtering length are different in the version of different conditions, v208 and v220: MK p-value 1 is 0.0864, indicating that these two condition combinations have no dose-dependent effect.v202, v205, v211, v214, v217 and v224: These condition combinations meet the three conditions of the evaluation module, but the CV value is greater than 10%, indicating that the result stability is poor.

[0092] When the cluster size threshold is 3, the processing soft cutoff and the end filtering length are different in the version of different conditions, the MK p-value 1 of the following versions is greater than 0.05, v206 and v221: MK p-value 1 is 0.0864;v203, v209, v212, v215, v218 and v224: MK p-value 1 is 0.2207. This indicates that eight condition combinations have no dose-dependent effect, so mutagenicity cannot be evaluated when the cluster size threshold is 3.

[0093] Comprehensive analysis shows that no matter whether the processing soft cutoff or the end filtering length, the cluster size threshold of 1 is the best choice. When the cluster size threshold is 2, the result stability is poor; and when the cluster size threshold is 3, mutagenicity cannot be evaluated. Therefore, it is recommended to set the cluster size threshold of the base model to 1.

[0094] Based on the optimal cluster size threshold 1, we evaluated the impact of different end filtering lengths on mutagenicity assessment without handling soft clipping (parameter 0). The condition combination versions used include: v201 (end filtering length 0bp), v207 (end filtering length 5bp), v213 (end filtering length 10bp), v219 (end filtering length 15bp). The results are shown in Table 2 and the accompanying Figure 2 Figures, all of which meet the three conditions of mutagenicity assessment and can successfully assess mutagenicity. In particular, the coefficients of variation (CV) of v207 and v213 are 7.164% and 7.664% respectively, indicating that the assessment results under these two conditions are relatively stable.

[0095] Based on the optimal cluster size threshold 1, we also evaluated the impact of different end filtering lengths on mutagenicity assessment with handling soft clipping (parameter 1). The condition combination versions used include: v204 (end filtering length 0bp), v210 (end filtering length 5bp), v216 (end filtering length 10bp), v222 (end filtering length 15bp). As shown in Table 2 and the accompanying Figure 3 Figures, all of which meet the three conditions of mutagenicity assessment and can successfully assess mutagenicity. The coefficients of variation (CV) of v204 and v222 are 8.091% and 8.29% respectively, indicating that the results under these two conditions are relatively stable.

[0096] Therefore, whether handling soft clipping or not, the number of filtered bases n that filter the mutated bases located at the end satisfies the following formula:

[0097]

[0098] Where C is 1 if soft clipping is handled, and 0 if soft clipping is not handled. Stable detection and assessment results can be obtained.

[0099] The accompanying Figure 4The average relative proportion change of six types of base substitution (C>A, C>G, C>T, T>A, T>C, T>G) after treatment of different concentrations of compounds (0, 10, 100, 120, 150 μg / mL) based on v207 version statistics is shown. The abscissa is different types of mutation base substitution, including its complementary pairing. The ordinate is the average value of the proportion of different types of mutation bases (the average value under the same concentration). The analysis results show that C>T substitution occupies an absolute dominant position in all concentration groups, and its proportion increases significantly from 66.7% in the 10 μg / mL group to 85.5% in the 150 μg / mL group, showing an obvious dose-dependent growth trend. Relatively speaking, the proportions of the other five types of base substitution (C>A, C>G, T>A, T>C, T>G) in each concentration group remain at a low level (less than 15% in total), and fluctuate less with the concentration change. This specific mutation spectrum characteristic shows that the genotoxic effect of EMS is mainly realized by inducing C>T mutation, and its mechanism of action may be related to specific DNA damage pathways such as cytosine deamination or oxidative damage (such as 8-oxoG generation). This data not only confirms the concentration-dependent genotoxic effect of EMS, but also provides important experimental evidence for further studying its molecular mechanism of action.

[0100] Embodiment 2

[0101] The embodiment of the present application also provides a compound mutagenicity evaluation system, comprising: a human cell culture and treatment module for culturing required human cells and then treating the human cells with different concentrations of the compound;

[0102] a sequencing module for extracting genomic DNA from the human cells and performing sequencing;

[0103] a calculation module for obtaining total base data and mutation base data based on the sequencing data and calculating a mutation frequency;

[0104] an evaluation module, which evaluates the mutagenicity of the compound and determines the minimum mutagenic concentration by using the following conditional evaluation system,

[0105] (1) using the Mann-Kendall method to perform trend test on the sample mutation frequency and the compound concentration of each concentration treatment group and the control group, and satisfying p-value 1<0.05, that is, the sample mutation frequency has a dose-dependent effect on the compound;

[0106] (2) using ANOVA one-way analysis of variance to test the sample mutation frequency of each concentration treatment group and the sample mutation frequency of the control group, and satisfying p-value 2<0.05, that is, the sample mutation frequency of the treatment group and the sample mutation frequency of the control group have a significant difference;

[0107] (3) The mutation frequency of each concentration treatment group sample is compared with the mutation frequency of the control group to obtain the fold change of the sample mutation frequency of each concentration treatment group relative to the control group, denoted as FC; FC≥α is met,

[0108] The lowest concentration that meets the above three conditions is the minimum mutagenic concentration of the compound system.

[0109] The sequencing includes: the concentration A260 / A280 ratio of the human-derived cell genomic DNA is 1.8-2.0, and the DNA fragment size is controlled to be 180-220 bp; then the DNA fragments are filled, and molecular identifiers are introduced during the ligation process; the DNA library is hybridized in a liquid phase environment using a biotin probe and a blocking solution, after eluting the captured library, the target fragments are enriched by PCR amplification to obtain a final library; the final library is sequenced to obtain raw data, and the raw data is quality controlled, aligned, and consistency analyzed to finally obtain sequencing data.

[0110] For the sequencing data, first, the total base data of the sample is obtained, and then the mutant bases common to different concentration samples and control samples are removed; next, the sample mutant base data is obtained, and the sample mutation frequency is calculated.

[0111] The total base data is obtained in the following manner: the sequencing data is screened, and the following conditions are met:

[0112] (1) The number of clusters of each sequence during consistency analysis is ≥1, and unclustered sequences are excluded; (2) the MAQ of each sequence after consistency analysis is ≥60; based on the above screened sequences, the following rules are followed for molecular selection: (1) if either strand of a DNA molecule double helix is covered by a sequencing molecule (single strand coverage), the molecule is retained; (2) if both strands of a DNA molecule double helix are covered by a sequencing molecule (double strand coverage), only one molecule is retained; finally, the number of bases contained in the molecules that meet the above rules is counted as the total base data of the sample.

[0113] The mutant base data is obtained in the following manner: molecules that simultaneously meet the following conditions are screened: (1) contain at least one single nucleotide mutation, without considering short insertion and deletion base mutations; (2) the MAQ of each sequence after consistency analysis is ≥60; (3) both strands of the double helix are covered by a sequencing molecule (double strand coverage); (4) the number of clusters of each sequence during consistency analysis is ≥1; (5) soft truncation is processed; (6) mutant bases located at the 5'-end and 3'-end are filtered; for the molecules that meet the above conditions, the number of single nucleotide mutation bases is calculated as the mutant base data of the sample.

[0114] Whether or not to process soft truncation, the number of filtered bases n located at the end of the mutant base satisfies the following formula:

[0115]

[0116] Wherein, if the soft clipping is processed, C is 1, and if the soft clipping is not processed, C is 0.

[0117] Computer system

[0118] The embodiment of the present application provides a computer system, including one or more processors; a memory having one or more programs stored thereon, when the one or more programs are executed by the one or more processors, the one or more processors implement the above-mentioned evaluation method.

[0119] The memory can include a random access memory (RAM) and can also include a read-only memory (ROM). The memory can be used to store instructions, programs, codes, code sets or instruction sets. The memory can include a program storage area and a data storage area, wherein the program storage area can store instructions for implementing an operating system, instructions for implementing at least one function (such as a histogram equalization function, etc.), instructions for implementing each method embodiment described below, etc. The data storage area can also store data created by the electronic device during use (such as image matrix data, etc.).

[0120] The processor can include one or more processing cores. The processor connects various parts in the entire electronic device through various interfaces and lines, executes various functions of the electronic device and processes data by running or executing instructions, programs, code sets or instruction sets stored in the memory, and calling data stored in the memory. Optionally, the processor can be implemented in at least one of a hardware form of a digital signal processing (DSP), a field-programmable gate array (FPGA), and a programmable logic array (PLA). The processor can be integrated with a combination of one or several of a central processing unit (CPU) and a modem. Among them, the CPU mainly processes operating systems and application programs, etc.; the modem is used to process wireless communication. It can be understood that the above-mentioned modem can also not be integrated into the processor, but be implemented separately through a communication chip.

[0121] Computer-readable storage medium

[0122] The embodiment of the present application also provides a computer readable storage medium, wherein instructions are stored in the computer readable storage medium, and the instructions perform the above evaluation method when executed by one or more processors, to evaluate the mutagenicity of the system and the minimum mutagenic concentration.

[0123] Comparative Example 1

[0124] Detection by flow cytometry

[0125] PIG-A gene mutation rate was detected on the 12th day of the expression period after TK6 cells were treated with different concentrations of ethyl methanesulfonate (EMS). All dosing concentration groups were centrifuged at room temperature for 5 min, 2x10 6 cells were taken per portion in parallel in two portions, washed once with PBS solution, 180 μL of whole antibody staining solution was added, and incubated at 4°C for 30 min in the dark. After incubation, PBS buffer was washed once, 500 μL of nucleic acid staining solution was added, and incubated at 4°C for 10 min in the dark. PBS buffer was washed once, 800-1000 μL of fixing solution was added to resuspend the cells, and transferred to a flow tube for flow analysis. The sample was stored on ice during the analysis process, and the flow cytometer stopped detecting when the total number of cells reached one million. The PIG-A gene frequency was the frequency of GPI(-) cells in one million GPI(+) cells (i.e., the frequency of the CD55-CD59- / CD19+ cell population in the CD19+ cell population), and the data was expressed as Mean±SD. ANOVA single factor variance analysis was performed, and P<0.05 was considered statistically significant. When there was a dose-effect correlation, it was determined to be positive.

[0126] The results are shown in the accompanying Figure 5 The abscissa is the EMS concentration, and the ordinate is the frequency of GPI(-) cells. As can be seen from the results, as the EMS concentration increases, the frequency of mutant cells also increases, and there is a dose-dependent effect. When the EMS concentration reaches 100 μg / mL, the frequency of mutant cells has a significant difference, and is determined to be positive. In contrast, the results of the base model evaluation show that when the EMS concentration is only 10 μg / mL, the mutation frequency of the sample has a significant difference, and the sensitivity is significantly higher than the minimum mutagenic concentration (100 μg / mL) detected by flow cytometry. This shows that the base model has higher sensitivity and can effectively detect mutant cells at a lower concentration, showing its advantage in mutation detection.

[0127] Comparative Example 2

[0128] Use of molecular models

[0129] Establishment of molecular model calculation method:

[0130] S1, count the total number of molecules in the sample.

[0131] The molecular sequences obtained through molecular tag consistency analysis are filtered as follows, and the molecular sequences meeting the conditions are subjected to the next step of statistics:

[0132] (1) The number of clusters of each sequence of a sequencing molecule is ≥1 when consensus is analyzed, and the sequences without clusters are excluded to reduce the noise generated by sequencing errors;

[0133] (2) The Mapping Quality (MAQ) of each sequence of a sequencing molecule is ≥60 after consistency analysis, to ensure that high-confidence aligned sequences are obtained;

[0134] The molecules meeting the above conditions are counted according to the following rules:

[0135] (1) If either strand of a double helix of a DNA molecule is covered by a sequencing molecule (single-stranded coverage), the total number of sample molecules is increased by 1;

[0136] (2) If both strands of a double helix of a DNA molecule are covered by a sequencing molecule (double-stranded coverage), the total number of sample molecules is still increased by 1, and no duplicate counting is performed.

[0137] The total number of molecules meeting the above conditions (including single-stranded and double-stranded coverage) is finally counted as the total number of molecules of the sample.

[0138] S2, Obtain common mutation sites

[0139] After the above molecular tag consistency analysis, the mutant VCF files of the control group and the cell lines of different concentrations of EMS treatment group are obtained. By comparing these VCF files, the common mutation sites of the control sample (0 μg / mL EMS treatment) and each EMS concentration treatment sample are identified. These mutation sites include both genetic mutations and random mutations generated during the culture of the cell line. Finally, each sample will obtain a list of common mutation sites.

[0140] S3, Statistic the total number of mutant molecules of the sample

[0141] The molecular sequences obtained through molecular tag consistency analysis are filtered as follows, and the molecular sequences meeting the conditions are subjected to the next step of statistics:

[0142] (1) The minimum cluster support threshold of each sequence of a mutant molecule is set to 1 when consensus is analyzed for a sequencing molecule;

[0143] (2) one sequence of a sequencing molecule, after consistency analysis, the Mapping Quality (MAQ) of each sequence is ≥60, ensuring that the sequence with high confidence alignment is obtained;

[0144] (3) one sequencing molecule contains at least one mutant base, and the mutant base is not included in the common mutation list obtained in S2;

[0145] (4) both strands of a double-stranded molecule are covered by a sequencing molecule (double-stranded coverage);

[0146] (5) processing soft cutoff;

[0147] (6) filtering the mutant base located at the 5'-end and the 3'-end;

[0148] For condition (5), whether to retain soft cutoff or not, the number of bases n of the mutant base located at the end of condition (6) is satisfied by the following formula, and the selection of this part of the condition follows the best evaluation condition of Example 1:

[0149]

[0150] Wherein, if processing soft cutoff, C is 1, and if not processing soft cutoff, C is 0.

[0151] Finally, the total number of molecules satisfying the above conditions is counted as the total number of mutant molecules of the sample;

[0152] S4, calculating the mutation frequency of the sample

[0153] The mutation frequency of the sample is calculated according to the total number of mutant molecules of the sample obtained in S3 and the total number of molecules of the sample obtained in S2. The mutation frequency of the sample = the total number of mutant molecules / the total number of molecules of the sample

[0154] S5, evaluating the mutagenicity of the molecular model

[0155] The following condition evaluation system is used to evaluate the mutagenicity and determine the minimum mutagenic concentration,

[0156] (1) using Mann-Kendall method to perform trend test on the sample mutation frequency and compound concentration of each concentration treatment group and control group, and satisfying p-value 1<0.05, that is, the mutation frequency has a dose-dependent effect on the compound;

[0157] (2) using ANOVA one-way analysis of variance to test the sample mutation frequency of each concentration treatment group and the sample mutation frequency of the control group, and satisfying p-value 2<0.05, that is, the sample mutation frequency of the treatment group and the sample mutation frequency of the control group have significant difference;

[0158] (3) The mutation frequency of each concentration treatment group sample is compared with the mutation frequency of the control group to obtain the change multiple of the sample mutation frequency of each concentration treatment group relative to the control group, denoted as FC; FC≥a is met;

[0159] The lowest concentration that meets the above three conditions is the minimum mutagenic concentration of the compound system.

[0160] The optimal evaluation condition combination obtained according to the base model includes the cluster size threshold, the processing soft cutoff, and the end filtering length. When applied to the molecular model, the final condition combination and its detection results are shown in Table 3.

[0161] Table 3 Detection results of different condition combinations of the molecular model

[0162]

[0163] Note: a The processing soft cutoff, 0 represents no processing soft cutoff, i.e., retaining the molecules containing the soft cutoff, and 1 represents processing soft cutoff, i.e., filtering out the molecules containing the soft cutoff;

[0164] b The end filtering length (bp) represents the length of the filtered mutation bases located at the 5'-end and 3'-end of the molecule;

[0165] c CV(mean) represents the average value of the coefficient of variation of the sample mutation frequency of the entire system (the control group and each concentration treatment group); the value less than 10% is relatively stable, and the smaller the value, the more stable; the value ≥10% is relatively poor in stability.

[0166] In the v101-v104 versions, the optimal condition combination optimized by the base model is applied to the molecular model for evaluation. The results (Table 3) show that the p-value of the MK test of the four versions is 0.0275 (p-value 1<0.05), confirming the existence of the concentration-dependent effect. Through ANOVA single factor variance analysis, it is found that the difference between the EMS lowest concentration treatment (10 μg / mL) and the control group has statistical significance (p-value 2 is 0.0005, 0.0002, 0.0018, and 0.0002, respectively), and the change multiple (FC) is all ≥a, indicating that the molecular model can effectively evaluate the mutagenicity of the system under these four conditions, and the minimum mutagenic concentration is determined to be 10 μg / mL.

[0167] The mutation frequency analysis shows (Appendix Figure 6 ), the mutation frequency of the control group is 10 -6 orders of magnitude, while the EMS treatment group is 10 -5 orders of magnitude. Although this resolution is better than the error rate of the conventional NGS technology (10 -4 ~10-2 ), but there are the following defects: 1) the mutation frequency of the control group is significantly higher than the background mutation frequency of human body 10 -9 ~ 10 -7 ; 2) the mutation frequency detection sensitivity of the base model (10 -8 , EMS treatment group 10 -8 ~ 10 -7 ) is 1-2 orders of magnitude lower. This shows that, although using the number of molecules to calculate the mutation frequency has wide application in detecting mutation events such as SNV, InDel and SV, due to its insufficient sensitivity (the minimum detection limit is only 10 -5 ), there are obvious limitations in evaluating the mutagenicity of compounds.

[0168] It should be noted that, Figure 1 , Figure 2 , Figure 3 and Figure 6 Each subgraph in the above, the horizontal axis represents the treatment condition, including the control group (0 μg / mL) and four EMS concentration treatment groups (10 μg / mL, 100 μg / mL, 120 μg / mL, 150 μg / mL), the vertical axis represents the sample mutation frequency, the significant difference of ANOVA one-way variance analysis marked on the column chart in the figure is represented using (*P<0.05, **P<0.01, ****P<0.0001), and the number above the column chart represents the fold change (FC) of the sample of different concentration treatment group and the sample of the control group.

[0169] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for the assessment of the mutagenic potential of a compound, characterized in that, The method comprises the following steps: culturing qualified human cells, and then treating the human cells with different concentrations of the compound; extracting genomic DNA from the human cells and sequencing; obtaining total base data and mutation base data based on the sequencing data, and calculating the mutation frequency; the mutation frequency meets the following three conditions: (1) using the Mann-Kendall method to perform trend test on different compound concentration treatment groups, and meeting p-value 1<0.05; (2) using ANOVA single factor variance analysis to test the mutation frequencies of the sample in each concentration treatment group and the control group, and meeting p-value 2<0.05; (3) obtaining the change fold FC of the mutation frequency of the sample in each concentration treatment group relative to the control group, and meeting FC≥α, wherein the value of α is related to whether the processing soft cutoff is used and the number of terminal filtered bases; the lowest concentration meeting the above three conditions is the minimum mutagenic concentration of the compound system.

2. The evaluation method according to claim 1, wherein the sequencing comprises: the concentration A260 / A280 ratio of the genomic DNA of the human cells is 1.8-2.0, and the DNA fragment size is controlled to be 180-220 bp; then the DNA fragments are filled, and molecular identifiers are introduced in the linking process; the DNA library is hybridized in a liquid phase environment using a biotin probe and a blocking solution, the captured library is eluted, the target fragments are enriched through PCR amplification, and the final library is obtained; the final library is sequenced to obtain raw data, and the raw data is quality controlled, aligned and consistency analyzed to finally obtain sequencing data.

3. The evaluation method according to claim 2, wherein for the sequencing data, first, the total base data of the sample is obtained; then, the mutation bases common to the samples treated with different concentrations of the compound and the control sample are removed, next, the sample mutation base data is obtained, and the sample mutation frequency is calculated.

4. The evaluation method according to claim 3, wherein the total base data is obtained in the following manner: the sequencing data is screened, and the following conditions are met for statistics: (1) the number of clusters of each sequence in the consistency analysis is ≥1, and the unclustered sequences are excluded; (2) the MAQ of each sequence after the consistency analysis is ≥60; based on the screened sequences, the molecules are selected according to the following rules: (1) if either strand of a DNA molecule double helix is covered by a sequencing molecule, the molecule is retained; (2) if both strands of a DNA molecule double helix are covered by a sequencing molecule, only one molecule is retained; finally, the number of bases contained in the molecules meeting the above rules is counted as the total base data of the sample.

5. The evaluation method according to claim 3, wherein the mutation base data is obtained in the following manner: molecules meeting the following conditions are screened out: ​ ​ ​ ​ 1) at least one single nucleotide mutation, regardless of short insertion and deletion base mutations; (2) MAQ of each sequence after consistency analysis ≥60; (3) both strands of the double helix are covered by sequencing molecules; (4) the number of clusters of each sequence during consistency analysis ≥1; (5) soft truncation is processed; (6) filtering of mutation bases located at the 5'-end and 3'-end; For molecules meeting the above conditions, the number of single nucleotide mutation bases is calculated as the mutation base data of the sample.

6. The evaluation method according to claim 5, characterized in that, The number of filtered bases n, which are filtered mutation bases located at the end, satisfies the following formula whether or not the soft truncation is processed: Wherein, C is 1 if the soft truncation is processed, and C is 0 if the soft truncation is not processed.

7. A system for assessing the mutagenicity of a compound, characterized by It comprises: A human cell culture and processing module for culturing qualified human cells, and then treating the human cells with different concentrations of the compound; A sequencing module for extracting genomic DNA from the human cells and sequencing; A calculation module for obtaining total base data and mutation base data based on the sequencing data, and calculating mutation frequency; An evaluation module, which uses the following condition evaluation system to evaluate mutagenicity and determine the minimum mutagenic concentration: (1) Trend test is performed on different compound concentration treatment groups using the Mann-Kendall method, and p-value 1<0.05 is met; (2) ANOVA single factor variance analysis is used to test the mutation frequency of each concentration treatment group and the control group sample, and p-value 2<0.05 is met; (3) The mutation frequency of each concentration treatment group sample and the mutation frequency of the control group sample are compared to obtain the fold change FC of each concentration treatment group relative to the control group, and FC≥α is met, wherein the value of α is related to whether the soft truncation is processed and the number of end filtering bases; The minimum concentration that meets the above three conditions is the minimum mutagenic concentration of the compound system.

8. The evaluation system according to claim 7, characterized in that, The sequencing comprises: The concentration ratio A260 / A280 of the human cell genomic DNA is 1.8-2.0, and the DNA fragment size is controlled at 180-220 bp; Then the DNA fragments are filled, and molecular identifiers are introduced during the ligation process; The DNA library is hybridized in a liquid environment using a biotin probe and a blocking solution, and after eluting the captured library, the target fragments are enriched by PCR amplification to obtain the final library; The final library is sequenced to obtain raw data, and the raw data is quality controlled, aligned and consistency analyzed to finally obtain sequencing data.

9. The evaluation system according to claim 7, characterized in that, For the sequencing data, first, the total base data of the sample is obtained; Then, the mutation bases common to the samples treated with different concentrations of the compound and the control samples are removed; Next, the sample mutation base data is obtained, and the sample mutation frequency is calculated.

10. The evaluation system according to claim 9, characterized in that, The total base data of the sample is obtained in the following manner: The sequencing data is screened, and the following conditions are met: (1) The number of clusters of each sequence in the consistency analysis is ≥1, and the unclustered sequences are excluded; (2) The MAQ of each sequence after the consistency analysis is ≥60; Based on the screened sequences, the following rules are used for molecular selection: (1) If either strand of a DNA molecule double helix is covered by a sequencing molecule, the molecule is retained; (2) If both strands of a DNA molecule double helix are covered by a sequencing molecule, only one molecule is retained; and finally, the number of bases contained in the molecules meeting the above rules is counted as the total base data of the sample.

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