Circular RNAs (circRNAs) are a type of single-stranded covalently closed endogenous RNA molecules that generated by back-splicing. CircRNAs have attracted great attention in recent years and have been reported to regulate various physiological and pathological processes via sponging miRNAs, acting as RNA binding protein (RBP) decoys, and encoding functional peptides. However, there is still lacks a graphical user interface-based web tool for circRNA interactive analysis and visualization.
circPlot provides an user-friendly interface and a suite of plotting and analysis functions for rapid and intuitive exploration, visualization and analysis of pre-analyzed or user's own data:
1) comprehensive annotations of circRNA such as basic information, genomic position, sequence conservation, somatic mutation, secondary structure, epigenetic modification and divergent primer;
2) expression landscape of circRNA across multiple caner types, non-tumor tissues, cancer cell lines and non-tumor cells;
3) putative mechanisms of circRNA including miRNA sponge, RBP decoy and translating into peptide;
4) expression correlation analysis between circRNA and gene or miRNA in various tumor and non-tumor tissues, cell lines and cells.
Overall, circPlot is an user-friendly and powerful web application for visualizing, exploring and analyzing circRNAs without any programming skills.
Circular RNAs (circRNAs) are a class of endogenous RNA molecules with single-stranded covalently closed structure, which generate via the back-splicing event where the downstream splice donor is linked to the upstream splice acceptor. CircRNAs have attracted great attention in recent years and have merged as versatile regulator that extensively implicated in various physiological and pathological processes including cancer. CircRNAs have been demonstrated to exert their functions through multiple aspects of mechanisms such as regulating gene transcription, sponging miRNAs, acting as RNA binding protein (RBP) decoys, and encoding functional peptides (Figure 1).
Figure 1. Mechanisms of circRNA function
circPlot is an user-friendly interactive web server for comprehensively annotating circRNA and providing publication-quality plots to assist in circRNA research. It has integrated the sequencing data of 3180 samples covering 26 types of cancer, 107 types of normal tissue, 111 types of cancer cell line and 254 types of normal cell from our previous studies and public datasets. Besides, other publicly available datasets such as RBP CLIP-seq, somatic mutation sites, epigenetic modification sites are also included.
The circPlot web application contains 5 menus, namely Home, Search, Exploration, Tutorial and Contact (Figure 2). The Home page provides a brief introduction to the circPlot, the Search and Exploration pages are dedicated to query or analyze circRNA respectively, the Tutorial page contains detailed instructions on how to easily use the circPlot, the Contact page is designed for the feedback of website questions and comments to the developer.
Figure 2. The graphical interface of the circPlot web application
A suite of plotting and analysis functions is provided to facilitate the interactive analysis and visualization of pre-analyzed or user provided circRNA (Figure 3).
Figure 3. Schematic overview of the circPlot web application
Herein we provide the detailed instructions on how to easily use the circPlot web application.
Currently, circPlot has integrated the basic information, expression profile and putative mechanism of 815624 circRNAs. The Search page provides 5 search options, namely circRNA ID, circRNA sequence, gene, miRNA and RBP for users to query a circRNA of interest. The search results are shown upon clicking the “Submit” button. Besides, users can try the provided examples in each section directly.
As shown in Figure 1, users can choose the “circRNA ID” search option and then enter the circRNA ID or circRNA coordinate in the search box to query circRNA. The circRNA ID in 12 databases (circBase, circBank, circAtlas, DeepBase3, CIRCpedia2, circRNADb, exoRBase2, TSCD, CSCD2, MiOncoCirc, TransCirc, riboCIRC) and circRNA coordinate in hg19 or hg38 version are supported in the circPlot.
Figure 1. Query a circRNA of interest by searching circRNA ID
Users can choose the “circRNA sequence” search option and then paste the circRNA sequence in the box to query circRNA (Figure 2).
Figure 2. Query a circRNA of interest by searching circRNA sequence
Users can choose the “gene” search option and then paste the gene name, gene aliases or gene ID in the box to query circRNA (Figure 3). Currently, gene ID in the NCBI, Ensembl, geneAliases, MIM, HGNC, AllianceGenome or IMGT are supported in the circPlot.
Figure 3. Query a circRNA of interest by searching gene
Users can choose the “miRNA” search option and then paste the miRNA name, miRNA accession or miRNA aliases in the box and set the number of miRNA binding site to query circRNA (Figure 4). Currently, only miRNA name, miRNA accession or miRNA aliases in the miRBase (Release 22.1) database are supported in the circPlot.
Figure 4. Query a circRNA of interest by searching miRNA
Users can choose the “RBP” search option and then paste the gene name, gene aliases or gene ID in the box and set the number of RBP binding site to query circRNA (Figure 5). Currently, gene ID in the NCBI, Ensembl, geneAliases, MIM, HGNC, AllianceGenome or IMGT are supported in the circPlot.
Figure 5. Query a circRNA of interest by searching RBP
If the circRNA is not include in our pre-analyzed results, users can analyze this circRNA by simply selecting the Exploration page and typing circRNA coordinate or circRNA sequence in the corresponding input box. Subsequently, users should click the “Submit” button to trigger the analysis and the results will display upon the server has finished the analysis. In addition, users can use the predefined examples for a try.
Users can choose the “circRNA coordinate” option and then paste the circRNA name and circRNA coordinate in the box to analyze circRNA (Figure 6). It is worth noting that the circRNA name and coordinate must be in proper format.
Figure 6. Analyze a circRNA by circRNA coordinate
Besides, users can analyze a circRNA by circRNA sequence. Users should firstly choose the “circRNA sequence” option and then select to provide circRNA sequence by typing (Figure 7) or uploading (Figure 8) the circRNA sequence. It is worth noting that the circRNA name and sequence must be in proper format.
Figure 7. Users can type circRNA sequence and then analyze circRNA
Figure 8. Users can upload circRNA sequence and then analyze circRNA
To show the analysis and visualization results of a circRNA, users should firstly select a circRNA of interest via clicking the row of result table. As the circRNA is selected, a suite of analyses and visualization result will be displayed for this circRNA (Figure 9), including:
Figure 9. Overview of results of selected circRNA
The circPlot provides the interactivity between users and analysis visualization results, which is summary blow:
The “General information” section provides basic information of circRNA, and has 8 menus, namely circRNA overview, host gene overview, genomic position, circularization diagram, base modification, secondary structure, somatic mutation and divergent primer.
This section provides the basic information of circRNA, including host gene, linear transcript, genomic coordinates, aliases of circRNA in circRNA databases and microarray platforms, spliced sequence, and the conserved mouse circRNA reported in circBase database. Besides, users can click the hyperlink to view detailed information recorded in the corresponding database (Figure 10).
Figure 10. Basic information of circRNA
The basic information of circRNA host gene such as linear transcript, aliases in other databases, and gene summary are shown in this section. Users can click the hyperlink to view detailed information recorded in the corresponding database (Figure 11).
Figure 11. Basic information of circRNA host gene
This module is designed to visualize the genomic position and sequence conservation of circRNA using pre-analyzed result (Figure 12) or user's own data (Figure 13).
Figure 12. Visualization of circRNA genomic position and sequence conservation of pre-analyzed results
Figure 13. Visualization of circRNA genomic position of user provided data
In this section, users can select the “Pre-analyzed result” (Figure 14) or “Visualize your data?” (Figure 15) option to display the schematic diagram of circRNA back-splicing. Besides, users can set the relative position of circRNA divergent and convergent primer sets to show the schematic diagram of primer sets.
Figure 14. Visualization of schematic diagram of circRNA back-splicing of pre-analyzed results
Figure 15. Visualization of schematic diagram of circRNA back-splicing of user provided data
In this section, users can select the “Pre-analyzed result” (Figure 16) or “Visualize your data?” (Figure 17) option to visualize the base modification sites on circRNA.
Figure 16. Visualization of base modification sites on circRNA of pre-analyzed results
Figure 17. Visualization of base modification sites on circRNA of user provided data
This module is designed to visualize the secondary structure of circRNA using pre-analyzed result (Figure 18) or user's own data (Figure 19). The statistics on circRNA secondary structure illustrates the number and relative position of 3 kinds of structure (namely hairpin, internal-loop and multi-branched loop). Besides, users can download the sequence of circRNA secondary structure, which can be analyzed or visualized by other web tools, such as RNAfold and forna.
Figure 18. Visualization of secondary structure of circRNA of pre-analyzed results
Figure 19. Visualization of secondary structure of circRNA of user provided data
In this section, users can select the “Pre-analyzed result” (Figure 20) or “Visualize your data?” (Figure 21) option to visualize the somatic mutation sites on circRNA.
Figure 20. Visualization of somatic mutation sites on circRNA of pre-analyzed results
Figure 21. Visualization of somatic mutation sites on circRNA of user provided data
This module is designed to design and visualize the divergent primer of circRNA using pre-analyzed result (Figure 22) or user's own data (Figure 23). Users can click the row of result table to view the details and visualization results of selected primer sets.
Figure 22. Visualization of divergent primer of circRNA of pre-analyzed results
Figure 23. Visualization of divergent primer of circRNA of user provided data
Besides, users can design the divergent primer using their own circRNA sequence by selecting “Design circRNA divergent primer?” option. The circRNA sequence can be provided via typing (Figure 24) or uploading (Figure 25).
Figure 24. Users can type circRNA sequence and then design and visualize circRNA divergent primer
Figure 25. Users can upload circRNA sequence and then design and visualize circRNA divergent primer
The “Expression profile” section provides circRNA expression profiles in cancers, normal tissues, cancer cell lines and normal cells. Currently, 3 plot types, namely “Box plot”, “Dot plot” and “Bar plot” (Figure 26) are provided for the visualization results this section, which users can choose according to their needs. Collectively, this module contains valuable resources to benefit the circRNA research community.
Figure 26. Three plot types are provided for the visualization results
The “Tumor tissue” module provides the details and visualization result of circRNA expression profiles in tumor and their adjacent normal tissues (Figure 27). Besides, the differential expression results are also provided, helping users to screen out the cancer-specific or ubiquitously altered circRNAs in cancer. Alternatively, ssers can also select to upload and visualize their own plotting data (Figure 28).
Figure 27. The visualization, details and differential expression results of circRNA in tumor and its adjacent normal tissues
Figure 28. Users can upload and visualize circRNA expression profiles in tumor and its adjacent normal tissues
The “Non-tumor tissue” section is designed to display circRNA expression profiles in normal tissues of the pre-analyzed result (Figure 29) or users provided data (Figure 30).
Figure 29. The visualization results and details of circRNA in normal tissues
Figure 30. Users can upload and visualize circRNA expression profiles in normal tissues
In “Cancer cell line” module, herein we provide the visualization results and details of circRNA in cancer cell lines (Figure 31), helping users to select the appropriate cancer cell lines and facilitating circRNA researches in cancer. This module also allows the users to visualize their own plotting data (Figure 32).
Figure 31. The visualization results and details of circRNA in cancer cell lines
Figure 32. Users can upload and visualize circRNA expression profiles in cancer cell lines
The expression profiles of circRNA in normal cells is provided in the “Non-tumor cell” module (Figure 33). Besides, users are permited to upload and visualize their own data as followed the instructions (Figure 34).
Figure 33. The visualization results and details of circRNA in normal cells
Figure 34. Users can upload and visualize circRNA expression profiles in normal cells
In the “Putative mechanism” section, we provide the visualization and analysis results of circRNA-miRNA interaction, circRNA-RBP interaction and circRNA coding potential. This section suggests valuable clues to circRNA mechanism research and will significantly contribute to research on circRNAs.
The “circRNA-miRNA interaction” module shows miRNA binding sites on circRNA and sequence alignments between circRNA and miRNA. Users should click the rows of result table to select miRNAs of interest and set the color for each miRNA (Figure 35). Upon the rows have selected, the visualization results is shown and will update when the selected rows changed. The sequence alignments of circRNA-miRNA interaction can be exported in plain text format. Alternatively, users can upload and visualize their own data (Figure 36).
Figure 35. The visualization results of circRNA-miRNA interaction
Figure 36. Users can upload and visualize circRNA-miRNA interaction
The “circRNA-RBP interaction” module shows RBP binding sites on circRNA. Users should click the rows of result table to select RBP of interest and set the color for each RBP (Figure 37). Upon the rows have selected, the visualization results is shown and will update when the selected rows changed. The sequence of RBP binding site can be exported in plain text format. Users can upload and visualize their own data (Figure 38).
Figure 37. The visualization result of RBP binding sites on circRNA
Figure 38. Users can upload and visualize circRNA-RBP interaction
The “circRNA coding potential” module is designed to display ORF, IRES element and m6A modification sites on circRNA. Users should click the rows of result table to select ORF and then the visualization results will show (Figure 39). Users can download the sequence of ORF and IRES element. The basic properties such as amino acid composition and molecular weight of selected ORF can be downloaded. Besides, the details of N-Glycosylation, mucin type O-Glycosylation, and S, T and Y phosphorylation modification sites are also provided and can be exported in plain text format. As the selected rows changed, the analysis and visualization results will update immediately. Users can upload and visualize their own data (Figure 40).
Figure 39. The visualization result of circRNA coding potential
Figure 40. Users can upload and visualize circRNA coding potential
The circRNA candidates that downloaded from circBase database are filtered to obtain exonic circRNAs. The annotations such as genomic coordinates, host gene and exon index of exonic circRNAs are retrieved from circBase database.
# download circRNA candidates and annotations
wget http://www.circbase.org/download/hsa_hg19_circRNA.bed
The putative spliced sequences of retained circRNAs are obtained from circBase database. The mature sequences of miRNAs (miRBase 22 release) are downloaded from miRBase database.
# download putative spliced sequences of circRNAs
wget http://www.circbase.org/download/human_hg19_circRNAs_putative_spliced_sequence.fa.gz
# unpack
gunzip human_hg19_circRNAs_putative_spliced_sequence.fa.gz
# download mature sequences of miRNAs
wget ftp://mirbase.org/pub/mirbase/CURRENT/mature.fa.gz
# unpack
gunzip mature.fa.gz
The miRNA binding sites on exonic circRNA are predicted by miRanda (v3.3a) with following parameters:
# prediction of miRNA binding sites on circRNA
miRanda miRNA.fa circRNA.fa -go -8 -ge -2 -sc 120 > circRNA_miRanda.result
The RBP binding sites on circRNA that inferred by RBP CLIP-seq experiments are retrieved from starBase database using the provided Web API.
# example command line
# get data of all RBPs for TP53 (in human)
curl 'http://starbase.sysu.edu.cn/api/RBPTarget/?assembly=hg19&geneType=circRNA&RBP=TP53&clipExpNum=1&pancancerNum=0&target=all&cellType=all' > hg19_TP53_circRNA_interaction
The relative positions of RBP binding sites on circRNA are determined by bedtools (v2.28.0) using following command line:
bedtools intersect -a RBP_binding_sites.bed -b circRNA_exon_coordinate.bed -wb -s > RBP_relative_position_circRNA
The putative spliced sequences of circRNAs are submitted to ORFfinder software to search the putative open reading frames (ORFs) in circRNA sequence. Only ORFs with minimal 25 aa in length are kept.
The Internal Ribosome Entry Site (IRES) sequence has been extensively demonstrated to initiate the cap-independent translation of circRNA. The IRESfinder (v1.1.0) software is used to predict the IRES element on circRNAs that harbor putative ORFs.
python IRESfinder.py -f circRNA.fa -o circRNA.result -m 2 -w 174 -s 50
The circPlot package is a wrapper of the ggplot2 and ggforce packages to facilitate the visualization of circRNA. The circPlot_circularization function is used to view the back-splicing event of circRNA. The circPlot_miRNA_binding function is utilized to visualize the miRNA binding sites on circRNA, while circPlot_miRNA_alignment function is designed to show the details of sequence complementary between circRNA and miRNA. The circPlot_RBP function is employed to display the RBP binding sites on circRNA. The circPlot_coding_potential function is applied to visualize the relative position of predicted ORF and IRES elements on circRNA. All R scripts of the circPlot package are freely available at https://github.com/zimuliving/circPlot.
The circPlot interactive web server is built using shiny and shinydashboard packages. The data visualization is done with circPlot package. The interactive user interface is implemented with shiny framework and DT package. The source codes and data that used to develop the circPlot interactive web portal are freely available at https://github.com/zimuliving/circPlot.
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