Model organism circRNA database system and application thereof

Abstract

The invention relates to a model organism circRNA database system and application thereof.The database system is composed of a database layer, a persistent layer, a business logic layer and a presentation layer and comprises a species meta-information mining module, a sequencing data downloading module, a reference gene information Index module, a circRNA identification module, a circRNA spatiotemporal expression module, an experimental verification module, a scientific naming module and a circRNA genome structure amp; the kit comprises a DNA sequence module, a mature sequence module, an ORF prediction and coding potential module, an IRES region mining module, a miRNA regulation and control site module, an m6A modification site module and a species conservative module. According to the method, multiple advanced technologies are integrated, front and back end separation and micro-servitization are achieved, the database comprises circRNA characteristics of different model organisms, a professional analysis method and a data basis are provided for circRNA research workers, and important data support is provided for research of human diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioinformatics, and in particular relates to a model organism circRNA database system and applications thereof. Background Art

[0002] Circular RNA (circRNA) is a unique class of non-coding RNA, which is distinguished from traditional linear RNA by its unique circular structure. Due to its high stability and tissue specificity, circRNA is involved in a variety of biological processes such as gene expression regulation and disease occurrence. In recent years, the in vitro synthesis and in vivo delivery technology of circRNA has gradually matured, laying a solid foundation for its development into a new generation of RNA drugs. The study of circRNA in model organisms such as mice, rats, naked mole rats, and zebrafish is of particular importance. These model organisms have become ideal tools for studying the mechanisms of human diseases due to their clear genetic background, physiological characteristics, and disease models. At present, many research teams have invested in the functional analysis and database construction of circRNA in model organisms. However, existing databases are usually limited to a single species or a specific category, with relatively single functions, and it is difficult to fully meet the needs of researchers for comprehensive analysis and comparison of multi-species circRNA data.

[0003] Although patent publication number CN109285587A provides a circRNA database system and its application, the system has certain limitations in design. First, the amount of data in its database is limited, and the needs of big data processing were not fully considered during the development process. In addition, the application scope of the system is relatively narrow, mainly limited to humans, and fails to cover circRNA information of other model organisms. More importantly, the scalability of the database architecture is poor, and future expansion needs were not taken into account during the design.

[0004] Therefore, in the current circRNA research field, there is an urgent need to develop a circRNA comprehensive information processing and modular construction method that can be applied to a variety of model organisms to effectively support scientific research and provide researchers with more flexible and convenient tools. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention constructs a database system that can more comprehensively characterize the characteristics of circRNA in different model organisms, providing professional analysis methods and data basis for circRNA researchers and providing important data support for the study of human diseases.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A database system for comprehensive information of circRNA of model organisms, the database system includes thirteen modules, namely: species meta-information mining module, sequencing data download module, reference gene information Index module, circRNA identification module, circRNA spatiotemporal expression module, experimental verification module, scientific naming module, circRNA genome structure & mature sequence module, ORF prediction and coding potential module, IRES region mining module, miRNA regulatory site module, m6A modification site module, species conservation module.

[0008] Furthermore, the database system operation architecture consists of a database layer, a persistence layer, a business logic layer and a presentation layer; the database layer is responsible for data storage and management, and is used to store data processed by thirteen modules; the persistence layer is responsible for mapping data objects in the business logic layer to the table structure in the database layer to realize data persistence operations; the business logic layer receives requests from the presentation layer, calls the persistence layer to perform data operations, and returns the results to the presentation layer; the presentation layer is responsible for presenting the user interface.

[0009] Furthermore, the circRNA identification module includes fastq data preprocessing, reference genome alignment, circRNA identification and data integration.

[0010] Furthermore, the fastq data preprocessing includes downloading the fastq data using a multi-threaded aspera download analysis method, and preprocessing the fastq data using a multi-threaded analysis framework of a high-performance computing system.

[0011] Furthermore, the reference genome alignment includes downloading the reference genome sequence of the species and the corresponding GTF annotation file from the Ensembl database, and using STAR and BWA to perform reference genome alignment respectively.

[0012] Furthermore, the circRNA identification is performed using two algorithms, DCC and CIRI2; the DCC identifies possible circRNAs by detecting reverse splicing events from exon to exon, and has high sensitivity and specificity, especially in complex genomic regions; the CIRI2 utilizes a local alignment method based on the Burrows-Wheeler Transform (BWT) and Smith-Waterman algorithms to identify reverse splicing events and filter out false positive results, and can better handle genomic regions containing repetitive sequences.

[0013] Furthermore, the data integration includes integrating the coordinates and expression information of circRNAs identified by the two algorithms.

[0014] Furthermore, the basic naming format of the scientific naming module is: species scientific naming code abbreviation_gene name or chromosome name_7 digits.

[0015] Furthermore, the species scientific naming code abbreviations are collected from KEGG; the gene name is used to indicate whether the circRNA is transcribed from the gene, otherwise it indicates that the circRNA is transcribed from the intergenic region; the size of the 7-digit number is sorted according to the position of the circRNA in the gene or chromosome.

[0016] Furthermore, the circRNA genome structure & mature sequence module infers the optimal circRNA genome structure by analyzing the positional relationship between the circRNA genome coordinates and the known gene exons, and calculates the mature sequence of the circRNA based on the exon information in the structure.

[0017] Furthermore, the ORF prediction and coding potential module includes using the EMBOSS::getORF tool to identify ORFs in circRNA sequences, and using CPC2 to calculate the coding potential of ORF sequences.

[0018] Furthermore, the IRES region mining module performs IRES resolution on the circRNA intermediate sequence and the circularization region BSJ through IRESfinder.

[0019] Furthermore, the miRNA regulatory site module identifies the miRNA binding site of each circRNA by designing a multiple miRanda prediction method, setting the threshold alignment score: 120 to 160 and free energy: -10 to -20 kcal / mol to ensure its sensitivity and specific binding.

[0020] Furthermore, the miRanda prediction method predicts potential binding sites by aligning miRNA sequences with target RNA sequences based on sequence complementarity; the threshold is alignment score: 140, free energy: -15.

[0021] Furthermore, the m6A modification site module includes using the SRAMP algorithm to predict m6A modification sites and integrating genome structure information to obtain high-throughput sequencing m6A modification sites.

[0022] Furthermore, the genome structure information includes the circRNA genome structure in the circRNA genome structure & mature sequence module and the information collected and collated by m6A-Atlas2.0; the SRAMP algorithm can clearly present the methylation results of the predicted RNA sequence modification sites, and display the position and score of the bases, as well as the credibility of the predicted sites through charts.

[0023] Furthermore, the species conservation module evaluates its conservation through multiple methods such as sequence comparison analysis, genome coordinate Liftover conversion and multi-species PhastCons Score.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The present invention adopts text mining technology to systematically collect, download and analyze high-quality, high-throughput whole transcriptome sequencing data of model organisms and their species physiological characteristics information. According to the characteristics of the data, a suitable combination of high-performance computing strategies is selected to conduct modular analysis and recording for circRNA identification, spatiotemporal expression, experimental verification, scientific naming, genome linear structure and mature sequence analysis, ORF prediction and coding potential, IRES region, miRNA regulatory sites, m6A modification sites and species conservation. The integrated data module serves as the background support of the human-computer interaction database network system. Users can query, analyze and download the circRNA characteristics of various model organisms through a friendly database portal website, providing professional analysis methods and data foundations for circRNA researchers, and providing important data support for the study of human diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a flow chart of the construction of the circbank2.0 model organism circRNA database system of the present invention;

[0027] Figure 2 This is a diagram of the architecture of the development and application of the circbank2.0 database of comprehensive information on circRNAs of model organisms of the present invention;

[0028] Figure 3 This is a schematic diagram of the circbank2.0 database website backend management system;

[0029] Figure 4 This is the schematic diagram of the homepage of the circbank2.0 database website;

[0030] Figure 5 This is a schematic diagram of the browsing and screening system of the circbank2.0 database website. DETAILED DESCRIPTION

[0031] The following will be combined with the accompanying drawings of the specification to provide a detailed and complete description of the technical solutions of the embodiments of the present invention. It should be noted that the described embodiments are only examples of some of the present invention and do not represent all possible implementation methods of the present invention. Based on the embodiments of the present invention, any other implementation methods obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

[0032] Example 1 A database system for comprehensive information of circRNA in model organisms

[0033] The database system of model organism circRNA comprehensive information includes thirteen modules: species meta-information mining module, sequencing data download module, reference gene information Index module, circRNA identification module, circRNA spatiotemporal expression module, experimental verification module, scientific naming module, circRNA genome structure & mature sequence module, ORF prediction and coding potential module, IRES region mining module, miRNA regulatory site module, m6A modification site module, and species conservation module. The flowchart of the model organism circRNA database system construction is as follows: Figure 1 shown.

[0034] Figure 1 In the above, the species metadata mining module first retrieves and downloads the high-throughput transcriptome sequencing metadata of the species through the NCBI Gene Expression Omnibus (GEO) and Sequence Read Archive (SRA) databases. Then, the metadata existing in both databases is screened out, and preliminary candidate circRNA metadata is obtained according to the following parallel screening criteria: LibraryStrategy: rna-seq / ncrna-seq / other, LibrarySelection: PCR / random / cDNA / sizefractionation, LibrarySource: transcriptomic / other. Subsequently, metadata containing the following keywords are further filtered out through text mining: ffpe, oligo(dt), poly-dt, poly-a, poly(a), 10X, 3', smart, chromium, single-cell, single cell, scrna, cel-seq2. Finally, the remaining metadata is used as the candidate circRNA dataset of the species, and the sample, tissue and differentiation stage information in the metadata is recorded as key information.

[0035] In order to solve the problem of downloading large quantities of data, the sequencing data download module utilizes the parallel computing advantages of multi-core servers. The sequencing data download module adopts a multi-threaded Aspera download analysis tool, which is specifically used for downloading large quantities of fastq data of species in the species metadata mining module of the present invention. In the present invention, it is mainly used to download high-throughput transcriptome sequencing metadata.

[0036] The reference gene information Index module downloads the reference genome sequence of the species and the corresponding GTF annotation file from the Ensembl database. Then, these data are used to construct indexes for STAR and BWA software and construct structural information of known genes.

[0037] The circRNA identification module integrates four parts: fastq data preprocessing, reference genome alignment, circRNA identification, and data integration. First, the high-throughput transcriptome sequencing metadata downloaded from the sequencing data download module is preprocessed using fastp using the multi-threaded analysis framework of the high-performance computing system. Then, STAR and BWA are used to align the reference genome in the reference gene information Index module, respectively. Subsequently, circRNAs are identified using DCC and CIRI2 tools. Finally, the circRNA coordinates obtained by the two algorithms are integrated with the expression information to generate circRNA information.

[0038] The circRNA spatiotemporal expression module integrates the circRNA information identified in the circRNA identification module with the species metadata screened in the species metadata mining module.

[0039] The experimental verification information module obtains data related to circRNA experimental verification by mining texts collected from PubMed.

[0040] The scientific naming module adopts the following rules for naming: species scientific naming code abbreviation_gene name or chromosome name_7-digit number. Among them, the species scientific naming code abbreviation comes from the KEGG database; the gene name indicates that the circRNA is derived from the transcription of the gene. If the circRNA is derived from the intergenic region, the chromosome name is used; the 7-digit number is arranged in order according to the position of the circRNA in the gene or chromosome, and the larger the number, the later the position.

[0041] The circRNA genome structure & mature sequence module combines the gene annotation GTF file obtained in the reference gene information Index module and the circRNA information generated in the circRNA identification module. First, the genome coordinates of the circRNA reverse sequence in the circRNA identification module are compared with the exon distance of the known gene to infer the optimal genome structure of the circRNA. Then, based on the exon information in the inferred circRNA genome structure, the mature sequence of the circRNA is further calculated.

[0042] The ORF prediction and coding potential module is divided into two parts: the first part uses the EMBOSS::getORF method to identify ORFs that can cross cyclization sites; the second part uses CPC2 to calculate the coding potential of the identified ORF sequences.

[0043] The IRES region mining module uses the IRESfinder tool to perform IRES identification on the middle sequence and circularization region (BSJ) of circRNA based on the circRNA mature sequence obtained in the circRNA genome structure & mature sequence module.

[0044] The miRNA regulatory site module extracts miRNA information from the miRBase database and NCBIPubMed text mining according to species information, combines the extracted miRNA information with the circRNA mature sequence obtained in the circRNA genome structure & mature sequence module, and establishes the miRNA regulatory site module. By designing multiple miRanda prediction methods, the miRNA binding site of each circRNA in the species is identified, and the threshold alignment score: 140, free energy: -15 is set to ensure the sensitivity and specificity of its identification.

[0045] The m6A modification site module is divided into two parts: the first part predicts the m6A modification site based on the circRNA mature sequence obtained in the circRNA genome structure & mature sequence module through the SRAMP algorithm; the second part integrates the high-throughput sequencing data collected and collated in the m6A-Atlas2.0 based on the circRNA genome structure obtained in the circRNA genome structure & mature sequence module to obtain the m6A modification site information.

[0046] The species conservation module includes three parts: first, the coordinates of the circRNA in the circRNA identification module are transformed using the liftoff and liftover tools; second, the middle sequence of the circRNA in the circRNA identification module and the sequence of 100nt near the cyclization site are compared by blast (the similarity score must be greater than 0.5); finally, the UCSC PhastCons score of the circRNA exon is calculated.

[0047] The database system operation architecture consists of a database layer, a persistence layer, a business logic layer and a presentation layer. The bottom layer is the database layer, which uses the MySQL relational database management system to store application data, uses technical means such as indexing and query optimization to improve database query efficiency, reduce data redundancy and storage costs, and is used to store data processed by thirteen modules. The database layer is connected to the persistence layer through a persistence framework to provide underlying data support for the business logic layer. The middle layer is the persistence layer and the business logic layer. The persistence layer maps Java objects to database tables (the database layer consists of multiple tables) through the XML mapping file or annotation method of MyBatis to achieve data persistence operations, and at the same time uses the dynamic SQL function of MyBatis to dynamically construct query conditions according to business logic to improve query efficiency. The persistence layer is responsible for data transmission and conversion between the database layer and the business logic layer, so that data can flow efficiently and consistently between the two. The business logic layer builds microservice applications through Spring Boot, and combines Spring Cloud components to implement core functions such as service registration and discovery, load balancing and configuration management; on this basis, write business logic code to process requests from the presentation layer, perform data operations by calling the persistence layer, and finally return the processing results. The business logic layer is responsible for processing core business and is the core of the system operation. It is responsible for converting user requests from the front-end presentation layer into specific business operations and relies on the persistence layer for data interaction. To ensure the stable operation of the application, MavenActuator is integrated to implement monitoring functions such as health checks and collection of measurement information; in addition, LogBack is used as a log framework to record key events during the application operation, which facilitates problem troubleshooting and performance analysis. The bottom layer is the presentation layer, which uses Vue3 as the front-end framework and builds a user-friendly interface based on its responsive system and component-based development features. As the interactive entrance of the system, the presentation layer passes the user's request to the business logic layer and finally displays the processing results. In order to improve development efficiency, Vite is used as a front-end construction tool, and its fast cold start and hot module replacement (HMR) functions are used to speed up the development process. In order to enhance the code quality and maintainability, TypeScript was used to perform static type checking on JavaScript code. Finally, the ElementPlus UI component library was integrated to further accelerate the development and implementation of the interface using its rich UI components. Figure 2 shown.

[0048] Example 2 Model organism circRNA database circbank2.0 website

[0049] The website of the model organism circRNA database circbank2.0 system constructed by the present invention is as follows Figure 3-5 As shown in the figure, the backend management system of circbank2.0 website includes advertising management, information management and download management, etc. The homepage of circbank2.0 website includes the global search bar of the database, circRNA feature analysis tools, data download and content construction instructions, etc. The browsing and filtering system of circbank2.0 website can filter, view and download data through different options.

[0050] The model organism circRNA database circbank2.0 website, taking fish as an example, the web browsing and screening system is as follows Figure 5 The circbank2.0 website displays relevant information of circRNAs in fish species, including circRNA ID, location, chain, splicing length, circRNA type, gene symbol, tissue information, miRNA binding site, m6A modification and verification status.

Claims

1. A database system for comprehensive information of circRNA of model organisms, characterized in that: The database system includes thirteen modules, namely: species meta-information mining module, sequencing data download module, reference gene information Index module, circRNA identification module, circRNA spatiotemporal expression module, experimental verification module, scientific naming module, circRNA genome structure & mature sequence module, ORF prediction and coding potential module, IRES region mining module, miRNA regulatory site module, m6A modification site module, and species conservation module.

2. The database system as claimed in claim 1, characterized in that: The operation architecture of the database system includes a database layer, a persistence layer, a business logic layer and a presentation layer that store the processed thirteen modules.

3. The database system as claimed in claim 1, characterized in that: The circRNA identification module includes fastq data preprocessing, reference genome alignment, circRNA identification and data integration processes.

4. The database system as claimed in claim 1, characterized in that: The circRNA genome structure & mature sequence module infers the optimal circRNA genome structure by analyzing the positional relationship between the circRNA genome coordinates and the known gene exons, and calculates the mature sequence of the circRNA based on the exon information in the structure.

5. The database system as claimed in claim 1, characterized in that: The ORF prediction and coding potential module includes using the EMBOSS::getORF tool to identify ORFs in circRNA sequences and using CPC2 to calculate the coding potential of ORF sequences.

6. The database system as claimed in claim 1, characterized in that: The IRES region mining module uses IRESfinder to perform IRES resolution on the circRNA intermediate sequence and the circularization region BSJ.

7. The database system as claimed in claim 1, characterized in that: The miRNA regulatory site module identifies the miRNA binding site of each circRNA by designing a multiple miRanda prediction method. During the prediction process, the threshold alignment score is set to 120 to 160 and the free energy is set to -10 to -20 kcal / mol.

8. The database system as claimed in claim 1, characterized in that: The m6A modification site module includes using the SRAMP algorithm to predict the m6A modification sites, and then integrating the genome structure information to obtain the modification sites of m6A for high-throughput sequencing.

9. An application of the database system as claimed in claim 1 in evaluating the characteristics of circRNAs in different model organisms.

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