A method and system for predicting microbial DNA methylation regulation of gene expression

Abstract

The application discloses a method and system for predicting gene expression regulated by microbial DNA methylation, comprising genome sequencing, obtaining genome assembly data and DNA methylation modification signals; genome assembly data annotation, obtaining transcription regulation regions and gene coding regions; selecting methylation sites from the DNA methylation modification signals, obtaining a first position overlapping region of the methylation sites and the transcription regulation regions and a second position overlapping region of the methylation sites and the gene coding regions; and predicting the relationship between the methylation sites and the genes according to the two position overlapping regions. The application establishes a one-to-one correspondence between genes and DNA methylation by using SMRT sequencing technology, can obtain genes in the whole genome that may be regulated by DNA methylation, thereby reducing the experimental time, narrowing the range of genes regulated by DNA methylation, and improving the interpretation of the regulation mode of microbial DNA methylation in a high-throughput manner.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioinformatics data processing, and in particular to a method and system for predicting microbial DNA methylation regulation of gene expression. BACKGROUND

[0002] At present, with the emergence of third-generation sequencing technologies such as single molecule real-time sequencing (SMRT-seq), DNA methylation modification on the genome can be directly obtained by sequencing, which accelerates the research on the biological function of DNA methylation.

[0003] DNA methylation, as a key genomic modification mode of epigenetic regulation of microorganisms (such as bacteria, archaea, fungi, and viruses), is an essential research content for exploring the adaptive and toxic regulation mechanisms of microorganisms. In order to study the influence and mechanism of DNA methylation modification on gene expression regulation, the traditional method is to use gene knockout to construct a mutant lacking DNA methylation modification, and compare it with the wild type, and compare the gene expression and microbial phenotype changes caused by the lack of DNA methylation through biological wet experiment means, but the experiment has the actual difficulties of long time and low throughput. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a method and system for predicting microbial DNA methylation regulation of gene expression, which can directly build a one-to-one correspondence between genes and DNA methylation on the basis of SMRT sequencing technology, can obtain genes in the whole genome that may be regulated by DNA methylation, thereby greatly reducing the experimental time and narrowing the range of DNA methylation regulated genes, and improving the interpretation of the regulation mode of microbial DNA methylation in a high-throughput manner.

[0005] The present application also provides a system, an electronic device and a computer readable storage medium for performing the method for predicting microbial DNA methylation regulation of gene expression.

[0006] The method for predicting microbial DNA methylation regulation of gene expression according to the first aspect of the present application comprises:

[0007] Obtaining the genome of the microorganism, and performing single molecule real-time sequencing on the genome to obtain genome assembly data and DNA methylation modification signals;

[0008] Performing genome annotation on the genome assembly data to obtain transcription regulation regions and gene coding regions;

[0009] select a methylation site from the DNA methylation modification signal, obtain a first position overlapping region where the methylation site and the transcription regulation region are positionally overlapped, and a second position overlapping region where the methylation site and the gene coding region are positionally overlapped;

[0010] predict the relationship between the methylation site and the gene of the microorganism according to the first position overlapping region or the second position overlapping region.

[0011] According to the control method provided in the embodiments of the present application, at least the following beneficial effects are achieved:

[0012] In order to improve the technical defects of the traditional research method, the present method can directly build a one-to-one correspondence between genes and DNA methylation on the basis of SMRT sequencing technology, and users can use the present method to obtain the genes in the whole genome that are regulated by DNA methylation, thereby greatly reducing the experimental time, narrowing the range of genes regulated by DNA methylation, and improving the interpretation of the regulation mode of microbial DNA methylation in a high-throughput manner. Especially for microbial species that have not been studied a lot previously, such as clinical pathogenic microorganisms and environmental microorganisms, the present method can quickly obtain the relationship between the methylation of the whole genome and the genes.

[0013] According to some embodiments of the present application, the selecting a methylation site from the DNA methylation modification signal, obtaining a first position overlapping region where the methylation site and the transcription regulation region are positionally overlapped, and a second position overlapping region where the methylation site and the gene coding region are positionally overlapped comprises:

[0014] obtaining all methylation sites from the DNA methylation modification signal;

[0015] dividing all the methylation sites into first methylation sites, second methylation sites and third methylation sites, wherein the first methylation sites are the methylation sites in the DNA methylation modification signal whose signal coverage is lower than a coverage threshold and / or whose signal recognition quality value is lower than a recognition quality value threshold; the second methylation sites and the third methylation sites are the methylation sites in the DNA methylation modification signal whose signal coverage is higher than the coverage threshold and whose signal recognition quality value is higher than the recognition quality value threshold, and the second methylation sites correspond to a methylation rate higher than a methylation rate threshold, and the third methylation sites correspond to a methylation rate lower than a methylation rate threshold;

[0016] The first position overlapping region where the first methylation site, the second methylation site and the third methylation site respectively overlap with the transcriptional regulatory region is obtained, and the second position overlapping region where the first methylation site, the second methylation site and the third methylation site respectively overlap with the gene coding region is obtained.

[0017] According to some embodiments of the present application, the method further comprises:

[0018] The DNA sequence corresponding to the first position overlapping region in the genome is extracted; wherein the DNA sequence is the transcriptional regulatory sequence where the first position overlapping region is located;

[0019] The potential feature sequence is predicted according to the DNA sequence;

[0020] The target gene is predicted according to the methylation site and the feature sequence; wherein the target gene refers to a gene potentially regulated by DNA methylation and the feature sequence.

[0021] According to some embodiments of the present application, the feature sequence comprises a DNA sequence represented in fasta format or matrix form.

[0022] According to some embodiments of the present application, the genome is sequenced by SMRT-seq.

[0023] According to some embodiments of the present application, the prediction of the relationship between the methylation site and the gene of the microorganism according to the first position overlapping region or the second position overlapping region comprises:

[0024] According to the transcriptional regulatory sequence position information of the DNA methylation event, the gene information is predicted, or according to the DNA methylation occurring in the gene coding region, the positional relationship of the DNA methylation site relative to the gene is predicted.

[0025] The system for predicting the DNA methylation regulation of the gene expression of the microorganism according to the second aspect of the embodiments of the present application comprises:

[0026] The genome acquisition unit is configured to acquire the genome of the microorganism.

[0027] The single molecule real-time sequencing unit is configured to perform single molecule real-time sequencing on the genome to obtain genome assembly data and DNA methylation modification signals.

[0028] The genome annotation unit is configured to perform genome annotation on the genome assembly data to obtain transcriptional regulatory regions and gene coding regions.

[0029] a methylation site selection unit configured to select a methylation site from the DNA methylation modification signal;

[0030] a position overlap detection unit configured to obtain a first position overlap region in which the methylation site and the transcription regulation region overlap in position, and a second position overlap region in which the methylation site and the gene coding region overlap in position;

[0031] a first regulated gene prediction unit configured to predict a relationship between the methylation site and a gene of the microorganism according to the first position overlap region or the second position overlap region.

[0032] Since the system for predicting microbial DNA methylation regulation gene expression adopts all the technical solutions of the method for predicting microbial DNA methylation regulation gene expression in the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments.

[0033] According to some embodiments of the present application, the system further comprises:

[0034] a DNA sequence extraction unit configured to extract a DNA sequence corresponding to the first position overlap region in the genome; wherein the DNA sequence refers to the transcription regulation sequence in which the first position overlap region is located;

[0035] a binding site prediction unit configured to predict a potential feature sequence according to the DNA sequence;

[0036] a second regulated gene prediction unit configured to predict a target gene according to the methylation site and the feature sequence; wherein the target gene refers to a gene regulated by DNA methylation and a binding site, and the feature sequence includes a DNA sequence represented in a fasta format or a matrix form.

[0037] An electronic device according to a third aspect embodiment of the present application comprises at least one control processor and a memory connected in communication with the at least one control processor; the memory stores instructions executable by the at least one control processor, and the instructions are executed by the at least one control processor to enable the at least one control processor to perform a method for predicting microbial DNA methylation regulation gene expression as described above.

[0038] Since the electronic device adopts all the technical solutions of the method for predicting microbial DNA methylation regulation gene expression in the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments.

[0039] A computer readable storage medium according to a fourth embodiment of the present application has computer executable instructions for causing a computer to perform a method of predicting microbial DNA methylation regulating gene expression as described above.

[0040] The computer readable storage medium has all the technical solutions of the method of predicting microbial DNA methylation regulating gene expression of the above embodiments, and thus has all the beneficial effects brought by the technical solutions of the above embodiments.

[0041] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0042] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which:

[0043] Figure 1 is a flowchart of a method of predicting microbial DNA methylation regulating gene expression provided by a first embodiment of the present application;

[0044] Figure 2 is a flowchart of a method of predicting microbial DNA methylation regulating gene expression provided by a second embodiment of the present application;

[0045] Figure 3 is a flowchart of a method of predicting microbial DNA methylation regulating gene expression provided by a third embodiment of the present application;

[0046] Figure 4 is a flowchart of a method of predicting microbial DNA methylation regulating gene expression provided by a fourth embodiment of the present application;

[0047] Figure 5 is a structural diagram of a system of predicting microbial DNA methylation regulating gene expression provided by a fifth embodiment of the present application;

[0048] Figure 6 is a structural diagram of an electronic device provided by a sixth embodiment of the present application. DETAILED DESCRIPTION

[0049] Embodiments of the present application are described in detail below with reference to the attached drawings, which show by way of example, embodiments in which the principles of the present application can be applied. The same or similar elements are denoted by the same or similar reference numerals throughout the drawings. The embodiments described below are examples only, and are not to be construed as limiting the present application.

[0050] In the description of the present application, if the first, second, etc. are described, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features or the sequence of indicated technical features.

[0051] In the description of the present application, it should be understood that the orientation description, such as the orientation or position relationship indicated by up, down, etc. is based on the orientation or position relationship shown in the drawings, only for the purpose of facilitating the description of the present application and simplifying the description, and is not intended to indicate or imply that the device or element indicated must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation of the present application.

[0052] In the description of the present application, it should be noted that, unless otherwise explicitly limited, the words such as setting, installing, connecting, etc. should be broadly understood, and those skilled in the art can reasonably determine the specific meaning of the above words in the present application in combination with the specific content of the technical solution.

[0053] The first embodiment is as follows:

[0054] Taking bacteria as an example, referring to Figure 1 The present application provides a method for predicting the expression of bacterial DNA methylation regulation genes, comprising the following steps:

[0055] Step S101, obtaining the genome of the target strain, and performing single molecule real-time sequencing on the genome to obtain genome assembly data and DNA methylation modification signals.

[0056] Single molecule real-time sequencing (SMRT-seq) is a third-generation sequencing technology, and DNA methylation modification on the genome can be directly obtained by sequencing, so in the sequencing process of the present step S101, the sequencing of the same strain of bacteria simultaneously obtains the genome assembly data and DNA methylation modification signals of the bacteria. Specifically, the data can be obtained by software SMRTLink. The target strain in step S101 refers to the bacteria specified by the user, which is not specifically limited here.

[0057] Step S102, performing genome annotation on the genome assembly data to obtain transcription regulation regions and gene coding regions.

[0058] In some embodiments, the genomic assembly data is subjected to genome annotation using PROKKA v3.0 to obtain the gene coding region, and the transcriptional regulatory region information of each gene in the target strain is obtained using the R computer programming language, and in some embodiments, the transcriptional regulatory region is by default 500 bp upstream of the transcription start site and 100 bp downstream of the transcription start site. In this step S102, the transcriptional regulatory region of a gene refers to the key region for the transcription factor and other DNA-binding proteins to regulate the initiation of gene transcription, which is of great significance for predicting the influence of DNA methylation modification on transcriptional regulation. For example, the transcription factor binds to the transcriptional regulatory region to attract RNA polymerase, thereby initiating transcription and starting gene expression. Therefore, predicting the transcriptional regulatory region can assist the user in obtaining the DNA methylation sites occurring in this region alone. It is worth noting that existing studies have shown that DNA methylation occurs in the transcriptional regulatory region, affecting the binding and regulation of transcription factors and other gene transcription initiation mechanisms, further affecting gene expression. For example, in Salmonella, Dam methyltransferase-induced methylation occurs in the regulatory region of the OxyR transcription factor bound to the gene opvAB, thereby controlling the expression of opvAB. The gene coding region refers to the gene position obtained in the genome annotation.

[0059] Step S103, selecting a methylation site from the DNA methylation modification signal, obtaining a first position overlapping region where the methylation site and the transcriptional regulatory region are positionally overlapped, and a second position overlapping region where the methylation site and the gene coding region are positionally overlapped. Through the single molecule real-time sequencing of step S101, the DNA methylation modification signal can be obtained, which includes information such as methylation sites, methylation rates, and methylation types.

[0060] Step S104, predicting the relationship between the methylation site and the gene of the target strain according to the first position overlapping region or the second position overlapping region.

[0061] The relationship between the methylation site and the gene of the target strain refers to the positional relationship of DNA methylation with respect to the gene. The step S103 and the step S104 are mainly to determine whether the methylation site and the transcription regulatory sequence or the methylation site and the gene coding region have positional overlap according to the site information of the methylation site and the transcription regulatory sequence or the methylation site and the gene coding region on the genome, so as to obtain the positional information of the transcription regulatory sequence of the DNA methylation event, and then use the positional information of the transcription regulatory sequence of the DNA methylation event to deduce the gene information (the transcription regulatory region is predicted by the gene position in the step S102, and therefore a one-to-one correspondence between the gene and the transcription regulatory region is established, so that the gene information can be deduced from the positional information of the transcription regulatory region), or directly obtain that the DNA methylation occurs in the gene coding region (the gene coding region refers to the gene position obtained from the genome annotation), so as to build the relationship between the DNA methylation site and the gene.

[0062] The method for predicting bacterial DNA methylation regulation of gene expression provided by the embodiment has the following beneficial effects:

[0063] The traditional research method starts from the perspective of molecular biology, relies on gene knockout, phenotypic analysis and multi-omics analysis to explore the mechanism, for example, a DNA methylation modification deficient strain is constructed by using gene knockout, and is compared with a wild strain, and the gene expression and bacterial phenotype changes caused by the DNA methylation deficiency are compared by biological wet experiment means. The experiment of the research method has the actual difficulties of long time and low throughput.

[0064] In order to improve the technical defects of the traditional research method, the method can directly build a one-to-one correspondence between the gene and the DNA methylation on the basis of the SMRT sequencing technology, and the user can use the method to obtain the genes in the whole genome that may be regulated by the DNA methylation, so as to greatly reduce the experimental time, reduce the range of the DNA methylation regulated genes, and improve the interpretation of the regulation mode of the bacterial DNA methylation in a high-throughput manner. Especially for strains that have not been studied by a large number of experiments before, such as clinical pathogenic bacteria, the method can quickly obtain the relationship between the methylation of the whole genome and the gene.

[0065] The second embodiment;

[0066] Taking a bacterium as an example, referring to Figure 2 The method for predicting bacterial DNA methylation regulation of gene expression provided by the embodiment comprises the following steps:

[0067] In step S201, the genome of a target strain is obtained, the genome is subjected to SMRT-seq genome sequencing, and the genome assembly data and the DNA methylation modification signal are obtained by SMRTLink.

[0068] Step S202, using PROKKA v3.0 to perform genome annotation on the genome assembly data to obtain transcription regulation regions and gene coding regions.

[0069] Step S203, obtaining all methylation sites from the DNA methylation modification signal.

[0070] Step S204, dividing all methylation sites into first methylation sites, second methylation sites and third methylation sites, wherein the first methylation sites refer to methylation sites with signal coverage lower than a coverage threshold and / or signal recognition quality value lower than a recognition quality value threshold in the DNA methylation modification signal; the second methylation sites and the third methylation sites refer to methylation sites with signal coverage higher than the coverage threshold and signal recognition quality value higher than the recognition quality value threshold in the DNA methylation modification signal, and the second methylation sites correspond to a methylation rate higher than a methylation rate threshold, and the third methylation sites correspond to a methylation rate lower than the methylation rate threshold.

[0071] According to existing research, different DNA methylation modification levels may play an important role in gene expression regulation, and changes in DNA methylation modification level can be used as a mechanism signal for epigenetic regulation. Therefore, dividing more specific DNA methylation modification levels is beneficial to obtain more specific mechanisms. Unlike the first embodiment, the step S204 of the present embodiment divides into different DNA methylation modification levels, specifically: first, by setting a coverage threshold and a recognition quality value threshold to distinguish the first methylation sites with abnormally low methylation modification signals from other methylation sites (including the second methylation sites and the third methylation sites) with normal methylation modification signals, it is worth noting that the coverage refers to the number of effective pulse signals detected by methylation at the current position; the recognition quality value refers to the quality reliability of the effective pulse signals detected by methylation at the current position. Then, set a methylation rate threshold to divide the other methylation sites into second methylation sites with a methylation rate higher than the methylation rate threshold and third methylation sites with a methylation rate lower than the methylation rate threshold.

[0072] Step S205, obtaining first position overlapping regions where the first methylation sites, the second methylation sites and the third methylation sites respectively overlap with the transcription regulation regions, and obtaining second position overlapping regions where the first methylation sites, the second methylation sites and the third methylation sites respectively overlap with the gene coding regions.

[0073] Step S206, predicting the relationship between the first methylation sites, the second methylation sites and the third methylation sites and the genes of the target strain according to the first position overlapping regions or the second position overlapping regions.

[0074] Different from the first embodiment, the method considers that different DNA methylation modification levels may play an important role in gene expression regulation, and in step S204, the methylation sites are divided into first methylation sites with abnormally low methylation modification signals and other methylation sites with normal methylation modification signals according to coverage and recognition quality values, and the other methylation sites are divided into second methylation sites with high methylation rates and third methylation sites with low methylation rates according to methylation rates, so as to divide three different DNA methylation modification levels; then, a first position overlapping region in which the first methylation sites, the second methylation sites and the third methylation sites are positionally overlapped with transcription regulatory regions, and a second position overlapping region in which the first methylation sites, the second methylation sites and the third methylation sites are positionally overlapped with gene coding regions are respectively obtained; finally, gene relationship prediction is respectively performed on the methylation sites with different methylation modification levels based on the first position overlapping region and the second position overlapping region. Compared with the method of the first embodiment, the method can predict the relationship between DNA methylation with different modification levels and gene positions, and capture the dynamic changes of methylation modification in the bacterial genome by dividing different methylation modification levels, that is, it is possible to reveal the regulation process of DNA methylation.

[0075] The third embodiment;

[0076] With reference to Figure 3 Based on the second embodiment, a method for predicting bacterial DNA methylation regulation of gene expression further includes the steps of:

[0077] In step S207, DNA sequences corresponding to the first position overlapping region in the genome are extracted. Here, the DNA sequence refers to the transcription regulatory sequence where the first position overlapping region is located.

[0078] In step S208, potential characteristic sequences are predicted according to the DNA sequence. The characteristic sequence (motif) includes DNA sequences represented in fasta format or matrix form, and the characteristic sequence includes but is not limited to transcription factor binding sequences, CRISPR sequences, -10 sequences or -35 sequences.

[0079] In step S209, target genes are predicted according to the methylation sites and the characteristic sequences; wherein the target gene refers to a gene that may be jointly regulated by DNA methylation and the characteristic sequence.

[0080] Taking the characteristic sequence as the transcription factor binding sequence as an example, the transcription factor (including sigma factor) realizes the regulation of gene expression by binding to the DNA sequence, and the DNA methylation may regulate the gene expression by affecting the binding of the transcription factor to the DNA sequence. Through steps S207 to S209, genes that may be jointly regulated by transcription factor binding and DNA methylation can be found.

[0081] This step first extracts the corresponding sequence information in the genome from the position information of the transcription regulatory sequence with DNA methylation (which is obtained in step S205), then scans the sequence for potential transcription factor binding sites, and finally predicts the genes co-regulated by DNA methylation and binding sites.

[0082] The method can divide methylation sites with different DNA methylation levels and adjust the default threshold using parameters. It can extract transcription regulatory sequences with DNA methylation and predict transcription factor binding to obtain genes that may be co-regulated by transcription factors and DNA methylation. Users can analyze pathways, functional enrichment, and other analyses of genes with different methylation levels or genes co-regulated by DNA methylation and transcription factors obtained by the method, which can further predict the physiological functions of bacteria that may be affected by DNA methylation.

[0083] The method directly builds a one-to-one correspondence between genes and DNA methylation based on SMRT sequencing technology, and users can use the method to obtain genes that may be regulated by DNA methylation and transcription factor binding in the whole genome, thereby greatly reducing experimental time, narrowing the range of genes regulated by DNA methylation, and improving the interpretation of the regulation mode of bacterial DNA methylation in a high-throughput manner. Especially for strains that have not been extensively experimentally studied before, such as clinical pathogenic bacteria, this tool can quickly obtain the relationship between the methylation of the whole genome and the genes. The method also supports multiple operating systems (such as MAC, Windows, LinuX), and users without computer background can quickly start analyzing data according to the user manual.

[0084] The fourth embodiment;

[0085] For ease of understanding, taking microorganisms as bacteria as an example, referring to Figure 4 The present application also provides a method for predicting the expression of genes regulated by bacterial DNA methylation, comprising the following steps:

[0086] Step S301, identifying the genomic assembly data and DNA methylation modification pattern and site from the target strain.

[0087] Extract the target strain genome for SMRT-seq genome sequencing. The raw sequencing data is analyzed by HGAP4 analysis process in SMRTLink for genome assembly; the "Base Modification Analysis and Motif Analysis" module in SMRTLink is used for whole genome DNA methylation identification to determine the position and type of DNA methylation, and the reference sequence is selected as the complete genome after assembly.

[0088] Step S302, genome annotation is performed on the genome assembly data to obtain gene coding region and transcription regulation region information.

[0089] The genome assembly data is subjected to genome annotation using PROKKA v3.0 to obtain information such as gene coding region, and the transcription regulation region information of each gene in the bacteria is obtained using R computer programming language. The transcription regulation region is by default 500 bp upstream of the transcription start site and 100 bp downstream of the transcription start site, but it should be noted that the user can modify the transcription regulation region. The transcription regulation region of a gene is a key region for transcription factors and other DNA binding proteins to regulate the initiation of gene transcription, and it is of great significance for predicting the influence of DNA methylation modification on transcription regulation. The gene annotation of the gene coding region and the transcription regulation region position information in this step will be used in steps S305 and S306 to infer the relationship between DNA methylation and transcription factor binding genes.

[0090] Step S303, the signal quality of DNA methylation is judged, and the first methylation site and other methylation sites are divided;

[0091] In single molecule real-time sequencing, DNA methylation modification recognition is determined by the duration of the detected pulse (hereinafter referred to as pulse signal) of the base during sequencing, but due to individual differences in the bacterial methylation group itself and other sequencing factors (such as sequencing depth and amplification times, etc.), the detection signals of different methylation sites in the same genome sequencing result are different. By setting the coverage threshold and the recognition quality value threshold, the first methylation site with abnormally low detection methylation modification signal and other methylation sites are distinguished. It should be noted that the low detection pulse signal of the first methylation site cannot be directly considered as an error caused by sequencing, but it may also be due to the methylation level exhibited by the epigenetic regulation of the bacteria itself.

[0092] Step S304, the DNA methylation modification level is judged, and the other methylation sites are divided into second methylation sites and third methylation sites.

[0093] DNA methylation modification is derived from DNA methyltransferase recognizing specific DNA motifs and methylating specified bases in the motifs, and different DNA methylation modification levels of different motifs in different positions of the genome enable DNA methylation to dynamically participate in epigenetic regulation of bacteria. Therefore, in order to capture the dynamically changing DNA methylation modification level, in single molecule real-time sequencing, the proportion of molecules that evaluate the methylation modification of the current position motif, i.e. the methylation rate, is selected, which is defined as the probability of methylation occurring in all raw sequencing sequences aligned to the position for the same motif position. Here, the methylation rate threshold is set to 0.75, and the sites with a methylation rate higher than 0.75 are the second methylation sites, and the sites with a methylation rate lower than 0.75 are the third methylation sites. It should be noted that the user can modify the methylation rate threshold.

[0094] Step S305, respectively determining the DNA methylation sites occurring in the transcription regulatory region and the gene coding region.

[0095] For three different DNA methylation modification levels (first methylation site, second methylation site and third methylation site), gene correlation analysis is respectively performed.

[0096] According to the DNA methylation site and the transcription regulatory region or the gene coding sequence in the genomic site information, it is judged whether there is an overlapping position, so as to obtain the transcription regulatory sequence position information of the DNA methylation event and further deduce the gene information, or directly obtain the DNA methylation occurring in the gene coding region to build the relationship between the DNA methylation site and the gene.

[0097] The shell script is used to integrate the methylation information (methylation site, methylation rate, methylation type) and the gene information (regulatory sequence position, distance between methylation and transcription start site, gene position, coding direction of gene, gene name, gene description) into the same file, wherein the methylation information and the gene information are obtained by step S101 genome sequencing.

[0098] Step S306, extracting the transcription regulatory sequence of DNA methylation to predict the binding of transcription factors.

[0099] Transcription factors (including sigma factors) regulate gene expression by binding to DNA sequences, and DNA methylation can regulate gene expression by affecting the binding of transcription factors to DNA sequences. The present method supports the user-provided transcription factor description file to find genes that may be simultaneously regulated by transcription factor binding and DNA methylation.

[0100] The step firstly extracts the corresponding sequence information in the genome with the transcription regulatory sequence position information of DNA methylation, then scans the sequence to find potential transcription factor binding sites (TFBS), and finally predicts the genes regulated by transcriptional factor and methylation (RTMG).

[0101] The following functions can be realized by the method:

[0102] 1) DNA methylation level statistics table and distribution graph. The user can modify the parameters according to the DNA methylation level distribution graph and then perform secondary analysis according to the user's research content.

[0103] 2) Predict the relationship between different levels of DNA methylation and gene positions, and provide specific DNA methylation modification information, gene position and function information. The user can infer the biological function regulated by DNA methylation according to the above information, especially in the gene family composed of adjacent genes, that is, the potential regulatory mechanism can be further inferred according to the function of the gene family;

[0104] 3) Different transcription factors and DNA methylation genes are combined to predict, on the basis of the results of 2), further supplement the genes and corresponding functions regulated by transcription factors and DNA methylation.

[0105] In addition, the user can also use the gene set obtained by the above analysis to do other personalized pathway and function analysis, and further predict the influence of DNA methylation on bacterial physiological function and virulence phenotype.

[0106] Compared with the prior art, the method can directly build a one-to-one correspondence between genes and DNA methylation based on SMRT sequencing technology, and the user can use the method to obtain the genes in the whole genome that may be regulated by DNA methylation and the transcription factor binding condition, thereby greatly reducing the experimental time, narrowing the range of DNA methylation regulated genes, and improving the interpretation of the regulation mode of bacterial DNA methylation in a high-throughput manner. Especially for strains that have not been studied much before, such as clinical pathogenic bacteria, the method can quickly obtain the relationship between the whole genome methylation and genes. The method also supports multiple operating systems (MAC, Windows, LinuX), and users without computer background can quickly start analyzing data according to the user manual.

[0107] Fifth embodiment;

[0108] Reference Figure 5 , a system for predicting the regulation of gene expression by bacterial DNA methylation is provided, the system comprising a genome acquisition unit 1001, a single molecule real-time sequencing unit 1002, a genome annotation unit 1003, a methylation site selection unit 1004, a position overlap detection unit 1005, a first regulated gene prediction unit 1006, a DNA sequence extraction unit 1007, a binding site prediction unit 1008, and a second regulated gene prediction unit 1009, wherein:

[0109] The genome acquisition unit 1001 is configured to acquire the genome of a target strain.

[0110] The single molecule real-time sequencing unit 1002 is configured to perform single molecule real-time sequencing on the genome to obtain genome assembly data and DNA methylation modification signals.

[0111] The genome annotation unit 1003 is configured to perform genome annotation on the genome assembly data to obtain transcription regulatory regions and gene coding regions.

[0112] The methylation site selection unit 1004 is configured to select methylation sites from the DNA methylation modification signals.

[0113] The position overlap detection unit 1005 is configured to obtain a first position overlap region where the methylation sites overlap with the transcription regulatory regions, and a second position overlap region where the methylation sites overlap with the gene coding regions.

[0114] The first regulated gene prediction unit 1006 is configured to predict the relationship between the methylation sites and the genes of the target strain based on the first position overlap region or the second position overlap region.

[0115] The DNA sequence extraction unit 1007 is configured to extract the DNA sequence corresponding to the first position overlap region in the genome.

[0116] The binding site prediction unit 1008 is configured to predict potential feature sequences based on the DNA sequence.

[0117] The second regulated gene prediction unit 1009 is configured to predict target genes based on the methylation sites and the feature sequences; wherein the target genes refer to genes that may be jointly regulated by DNA methylation and binding sites, and the feature sequences include DNA sequences represented in fasta format or matrix form, such as but not limited to transcription factor binding sequences, CRISPR sequences, -10 sequences, or -35 sequences.

[0118] In some embodiments, specifically, the methylation site selection unit 1004 is configured to divide all the methylation sites into first methylation sites, second methylation sites and third methylation sites, wherein the first methylation sites refer to the methylation sites with signal coverage lower than the coverage threshold and / or signal recognition quality value lower than the recognition quality value threshold in the DNA methylation modification signal; the second methylation sites and the third methylation sites refer to the methylation sites with signal coverage higher than the coverage threshold and signal recognition quality value higher than the recognition quality value threshold in the DNA methylation modification signal, and the second methylation sites correspond to the methylation rate higher than the methylation rate threshold, and the third methylation sites correspond to the methylation rate lower than the methylation rate threshold.

[0119] The position overlap detection unit 1005 is configured to obtain first position overlap regions in which the first methylation sites, the second methylation sites and the third methylation sites respectively overlap with the transcription regulation region, and obtain second position overlap regions in which the first methylation sites, the second methylation sites and the third methylation sites respectively overlap with the gene coding region.

[0120] It should be noted that the system embodiment and the above-mentioned method embodiment are based on the same inventive concept, and therefore the related content of the above-mentioned method embodiment is also applicable to the system embodiment.

[0121] The sixth embodiment;

[0122] With reference to Figure 6 The electronic device 6000 can be any type of smart terminal, such as a mobile phone, a tablet computer, a personal computer, etc.

[0123] Specifically, the electronic device 6000 includes one or more control processors 6001 and a memory 6002, Figure 6 For example, the control processor 6001 is taken as an example.

[0124] The control processor 6001 and the memory 6002 can be connected through a bus or other means, Figure 6 For example, the connection through the bus is taken as an example.

[0125] The memory 6002 is a non-transitory computer readable storage medium, which can be used to store non-transitory software programs, non-transitory computer executable programs and modules. The control processor 6001 executes various functional applications and data processing of the system for predicting the gene expression regulated by bacterial DNA methylation by running the non-transitory software programs, instructions and modules stored in the memory 6002, i.e., the method for predicting the gene expression regulated by bacterial DNA methylation of the above-mentioned method embodiment.

[0126] The memory 6002 can include a program storage area and a data storage area, where the program storage area can store an operating system, at least one application required by the at least one function, and the data storage area can store data created according to the use of the system for predicting the regulation of gene expression by bacterial DNA methylation, and the like. In addition, the memory 6002 can include a high-speed random access memory, and can also include a non-transitory memory, such as at least one magnetic disk storage device, a flash memory device, or other non-transitory solid-state memory device. In some embodiments, the memory 6002 can optionally include a memory disposed remotely with respect to the control processor 6001, which can be connected to the electronic device through a network. Examples of the above network include, but are not limited to, the Internet, an intranet, a local area network, a mobile communication network, and a combination thereof.

[0127] The one or more modules are stored in the memory 6002, and when executed by the one or more control processors 6001, perform the method of predicting the regulation of gene expression by bacterial DNA methylation in the above method embodiments, for example, perform the method steps S101 to S104 in the above description of Figure 1 , perform the method steps S201 to S206 in Figure 2 , perform the method steps S207 to S209 in Figure 3 , and achieve the functions of the units 1001 to 1009 in Figure 5 .

[0128] A seventh embodiment;

[0129] The embodiments of the present application also provide a computer readable storage medium, which stores computer executable instructions, and the computer executable instructions are executed by one or more control processors, for example, executed by the control processor 6001 in Figure 6 , so that the above one or more control processors 6001 perform the method of predicting the regulation of gene expression by bacterial DNA methylation in the above method embodiments, for example, perform the method steps S101 to S104 in the above description of Figure 1 , perform the method steps S201 to S206 in Figure 2 , perform the method steps S207 to S209 in Figure 3 , and achieve the functions of the units 1001 to 1009 in Figure 5 .

[0130] The device embodiments described above are only schematic, and the units described as separate components can or can not be physically separate, i.e., can be located in one place, or can be distributed on multiple network units. Part or all of the modules can be selected according to actual needs to achieve the purpose of the present embodiment scheme.

[0131] Through the above description of the embodiments, those skilled in the art can clearly understand that the embodiments can be implemented by means of software plus a general hardware platform. Those skilled in the art can understand that all or part of the processes in the above-mentioned embodiment methods can be completed by a computer program instructing related hardware, and the program can be stored in a computer readable storage medium. When the program is executed, the program can include the processes of the above-mentioned embodiment methods. The storage medium can be a magnetic disc, an optical disc, a read-only memory (ROM) or a random access memory (RAM), etc.

[0132] The above describes the embodiments of the present application in detail in combination with the drawings, but the present application is not limited to the above-mentioned embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the present application.

Claims

1. A method of predicting regulation of gene expression by microbial DNA methylation, characterized in that, The method comprises: obtaining a genome of a microorganism, and performing single molecule real-time sequencing on the genome to obtain genome assembly data and DNA methylation modification signals; performing genome annotation on the genome assembly data to obtain transcriptional regulatory regions and gene coding regions; selecting methylation sites from the DNA methylation modification signals, obtaining first position overlapping regions in which the methylation sites overlap with the transcriptional regulatory regions, and second position overlapping regions in which the methylation sites overlap with the gene coding regions; the selecting methylation sites from the DNA methylation modification signals, obtaining first position overlapping regions in which the methylation sites overlap with the transcriptional regulatory regions, and second position overlapping regions in which the methylation sites overlap with the gene coding regions comprises: obtaining all methylation sites from the DNA methylation modification signals; dividing all the methylation sites into first methylation sites, second methylation sites and third methylation sites, wherein the first methylation sites are the methylation sites in the DNA methylation modification signals whose signal coverage is lower than a coverage threshold and / or whose signal recognition quality value is lower than a recognition quality value threshold; the second methylation sites and the third methylation sites are the methylation sites in the DNA methylation modification signals whose signal coverage is higher than the coverage threshold and whose signal recognition quality value is higher than the recognition quality value threshold, and the second methylation sites correspond to a methylation rate higher than a methylation rate threshold, and the third methylation sites correspond to a methylation rate lower than the methylation rate threshold; respectively obtaining first position overlapping regions in which the first methylation sites, the second methylation sites and the third methylation sites overlap with the transcriptional regulatory regions, and respectively obtaining second position overlapping regions in which the first methylation sites, the second methylation sites and the third methylation sites overlap with the gene coding regions; predicting the relationship between the methylation sites and the genes of the microorganism according to the first position overlapping regions or the second position overlapping regions.

2. The method of predicting microbial DNA methylation regulation of gene expression according to claim 1, wherein, The method further comprises: extracting DNA sequences corresponding to the first position overlapping regions in the genome; wherein the DNA sequences are transcriptional regulatory sequences in which the first position overlapping regions are located; predicting potential feature sequences according to the DNA sequences; predicting target genes according to the methylation sites and the feature sequences; wherein the target genes refer to genes potentially jointly regulated by DNA methylation and feature sequences.

3. The method of predicting microbial DNA methylation regulation of gene expression according to claim 2, wherein, The feature sequences comprise DNA sequences represented in fasta format or matrix form.

4. The method of predicting microbial DNA methylation regulation of gene expression according to claim 3, wherein, The genome is sequenced by SMRT-seq.

5. The method of predicting the regulation of gene expression by microbial DNA methylation according to claim 1, wherein, The predicting the relationship between the methylation sites and the genes of the microorganism according to the first position overlapping regions or the second position overlapping regions comprises: According to the transcription regulatory sequence position information of the DNA methylation event, gene information is predicted, or according to the DNA methylation occurring in the coding region of the gene, the position relationship of the DNA methylation site relative to the gene is predicted.

6. A system for predicting regulation of gene expression by microbial DNA methylation, comprising, The system comprises: a genome acquisition unit configured to acquire a genome of a microorganism; a single molecule real-time sequencing unit configured to perform single molecule real-time sequencing on the genome to obtain genome assembly data and DNA methylation modification signals; a genome annotation unit configured to perform genome annotation on the genome assembly data to obtain transcription regulatory regions and gene coding regions; a methylation site selection unit configured to select methylation sites from the DNA methylation modification signals; a position overlap detection unit configured to acquire a first position overlap region in which the methylation sites overlap the transcription regulatory regions, and a second position overlap region in which the methylation sites overlap the gene coding regions; the acquisition of the first position overlap region in which the methylation sites overlap the transcription regulatory regions, and the second position overlap region in which the methylation sites overlap the gene coding regions comprises: acquiring all methylation sites from the DNA methylation modification signals; dividing all the methylation sites into first methylation sites, second methylation sites and third methylation sites, wherein the first methylation sites are the methylation sites in the DNA methylation modification signals with a signal coverage lower than a coverage threshold and / or a signal recognition quality value lower than a recognition quality value threshold; the second methylation sites and the third methylation sites are the methylation sites in the DNA methylation modification signals with a signal coverage higher than the coverage threshold and a signal recognition quality value higher than the recognition quality value threshold, and the second methylation sites correspond to a methylation rate higher than a methylation rate threshold, and the third methylation sites correspond to a methylation rate lower than the methylation rate threshold; respectively acquiring the first position overlap region in which the first methylation sites, the second methylation sites and the third methylation sites overlap the transcription regulatory regions, and respectively acquiring the second position overlap region in which the first methylation sites, the second methylation sites and the third methylation sites overlap the gene coding regions; a first regulated gene prediction unit configured to predict the relationship between the methylation sites and the genes of the microorganism according to the first position overlap region or the second position overlap region.

7. The system for predicting the regulation of gene expression by microbial DNA methylation according to claim 6, wherein, The system further comprises: a DNA sequence extraction unit configured to extract a DNA sequence corresponding to the first position overlap region in the genome; wherein the DNA sequence refers to a transcription regulatory sequence in which the first position overlap region is located; a binding site prediction unit configured to predict potential feature sequences according to the DNA sequence; and a second regulated gene prediction unit configured to predict the relationship between the methylation sites and the genes of the microorganism according to the potential feature sequences. A second regulated gene prediction unit is configured to predict a target gene according to the methylation site and the feature sequence; wherein the target gene refers to a gene regulated by DNA methylation and a binding site, and the feature sequence includes a DNA sequence represented in a fasta format or a matrix form.

8. An electronic device, comprising: The method comprises: at least one control processor and a memory connected in communication with the at least one control processor; the memory stores instructions executable by the at least one control processor, and the instructions are executed by the at least one control processor to enable the at least one control processor to perform the method for predicting microbial DNA methylation regulated gene expression according to any one of claims 1 to 5.

9. A computer-readable storage medium, characterized in that, The computer readable storage medium stores computer executable instructions for causing a computer to perform the method for predicting microbial DNA methylation regulated gene expression according to any one of claims 1 to 5.

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