Welcome to circPlot, an interactive web portal for circRNA exploration, analysis and visualization


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.

Loading...

Exploration

Notes: Users can enter the circRNA ID of 14 circRNA databases in the box
Examples: circBase circAtlas3 circBank CSCD2 circRNADb TransCirc Reset: reset
Notes: The running time ranging from two to five minutes
Notes: circRNA name cannot contain space, slash, backslash, special character and symbol
Notes: The coordinate system must be 0-based, and the coordinate must be in the following
format: chromosome:start-end|strand
Example: example Reset: reset
Notes: The running time depends on size of coordinate range and ranging from two to five minutes
Notes: circRNA name cannot contain space, slash, backslash, special character and symbol
Example: example Reset: reset
Notes: circRNA name cannot contain space, slash, backslash, special character and symbol


Notes: The running time depends on length of inputed circRNA sequence and ranging from two to ten minutes
Notes: The job ID cannot contain space, slash, backslash, special character and symbol
Example: example Reset: reset
Notes: The running time ranging from one to five minutes

Analysis result


Notes: Click the row to display the results of selected circRNA

Result visualization



Host gene:

Transcript:

Location (0-based):

Length (bp):

IDs in database:

IDs in microarray platform:

Sequence:


                                

Conserved mouse circRNA:


                                

Gene name:

Description:

Gene type:

Aliases:

Other ID:

Summary:


   
Notes: Prepare your upload file based on example data.

Customize plot

Download plot and data

Customize plot

Download plot

Schematic diagram of circRNA genomic position

Loading...

Schematic diagram of circRNA genomic position

Loading...

Notes: Prepare your upload file based on example data.

Customize plot

Download plot and data

Customize plot

Download plot

circRNA block color

Schematic diagram of circRNA back-splicing

Loading...

circRNA block color

Schematic diagram of circRNA back-splicing

Loading...

Notes: Prepare your upload file based on example data.


Customize plot

Download plot and data


Customize plot

Download plot

circRNA block color

Schematic diagram of base modification site on circRNA

Loading...

Details of base modification site on circRNA

circRNA block color

Schematic diagram of base modification site on circRNA

Loading...

Notes: Prepare your upload file based on example data.


Customize plot

Download plot and data


Customize plot

Download plot

circRNA block color

Schematic diagram of minimum free energy secondary structure of circRNA

Loading...

Details of circRNA MFE secondary structure

Notes: The structure is presented in dot-bracket notation, where the characters "(" and ")" correspond to the 5' base and 3' base in the base-pair, respectively, while "." denotes an unpaired base.


circRNA block color

Schematic diagram of minimum free energy secondary structure of circRNA

Loading...

Notes: Prepare your upload file based on example data.


Customize plot

Download plot and data


Customize plot

Download plot

circRNA block color

Schematic diagram of somatic mutation on circRNA

Loading...

Details of somatic mutation on circRNA

circRNA block color

Schematic diagram of somatic mutation on circRNA

Loading...

Notes: Prepare your upload file based on example data.

circRNA divergent primer sets

Loading...
Notes: Click the row to display the schematic diagram of circRNA divergent primer sets. BSJ: back-spliced junction site

Customize plot

Download plot and data

Customize plot

Download plot

circRNA block color

Schematic diagram of circRNA divergent primer sets

Loading...

Sequence of amplification product

circRNA block color

Schematic diagram of circRNA divergent primer sets

Loading...


Select all dataset: all dataset Reset: reset
Notes: Using all datasets by default when no dataset selected
Notes: Prepare your upload file based on example data.

Customize plot

Plot type

Download plot

Customize plot

Plot type

Download plot

Visualization of circRNA expression profile in tumors

Loading...

Visualization of circRNA expression profile in tumors

Loading...

Differential expression result


Expression profile



Select all dataset: all dataset Reset: reset
Notes: Using all datasets by default when no dataset selected
Notes: Prepare your upload file based on example data.

Customize plot

Plot type

Download plot

Customize plot

Plot type

Download plot

Visualization of circRNA expression profile in non-tumor tissues

Loading...

Visualization of circRNA expression profile in non-tumor tissues

Loading...

Expression profile



Select all dataset: all dataset Reset: reset
Notes: Using all datasets by default when no dataset selected
Notes: Prepare your upload file based on example data.

Customize plot

Plot type

Download plot

Customize plot

Plot type

Download plot

Visualization of circRNA expression profile in cancer cell lines

Loading...

Visualization of circRNA expression profile in cancer cell lines

Loading...

Expression profile



Select all dataset: all dataset Reset: reset
Notes: Using all datasets by default when no dataset selected
Notes: Prepare your upload file based on example data.

Customize plot

Plot type

Download plot

Customize plot

Plot type

Download plot

Visualization of circRNA expression profile in non-tumor cells

Loading...

Visualization of circRNA expression profile in non-tumor cells

Loading...

Expression profile




Notes: Prepare your upload file based on example data.

Notes: Click the rows to display the schematic diagram of circRNA-miRNA interaction

Customize plot

Download plot and data


Customize plot

Download plot

circRNA and miRNA color

Schematic diagram of miRNA binding site on circRNA

Loading...

circRNA and miRNA color

Schematic diagram of miRNA binding site on circRNA

Loading...

Customize plot

Download plot


Customize plot

Download plot

Schematic diagram of alignment between circRNA and miRNA

Loading...

Alignment between selected circRNA and miRNA


                                

Schematic diagram of alignment between circRNA and miRNA

Loading...

Alignment between selected circRNA and miRNA


                                


Notes: Prepare your upload file based on example data.

Notes: Click the row to display the schematic diagram of circRNA-RBP interaction

Customize plot

Download plot and data


Customize plot

Download plot

circRNA and RBP color

Schematic diagram of RBP binding site on circRNA

Loading...

Details of RBP binding site on circRNA


circRNA and RBP color

Schematic diagram of RBP binding site on circRNA

Loading...

Notes: Prepare your upload file based on example data.

Notes: Click the row to display the schematic diagram of circRNA coding potential

Customize plot

Download plot and data


Customize plot

Download plot

circRNA block color

Schematic diagram of circRNA coding potential

Loading...

circRNA block color

Schematic diagram of circRNA coding potential

Loading...

Property of circRNA-encoded peptide

Number of amino acids:

Molecular weight:

Number of negatively charged residues (Asp + Glu):

Number of positively charged residues (Arg + Lys):

Amino acid composition of circRNA-encoded peptide

ORF, m6A and IRES sequences of circRNA-encoded peptide


                                

Predicted modification on circRNA-encoded peptide



Notes: Prepare your upload file based on example data.
Loading...
Notes: Click the row to visualize the expression correlation result between circRNA and selected gene

Select all dataset: all dataset Reset: reset
Notes: Using all datasets by default when no dataset selected

Customize plot

Download plot


Customize plot

Download plot

Visualization of expression correlation between circRNA and gene

Loading...

Expression profile of circRNA and gene



Results of expression correlation analysis


Gene overview

Gene name:

Description:

Gene type:

Aliases:

Other ID:

Summary:

Visualization of expression correlation between circRNA and gene

Loading...


Notes: Prepare your upload file based on example data.
Loading...
Notes: Click the row to visualize the expression correlation result between circRNA and selected miRNA

Select all dataset: all dataset Reset: reset
Notes: Using all datasets by default when no dataset selected

Customize plot

Download plot


Customize plot

Download plot

Visualization of expression correlation between circRNA and miRNA

Loading...

Expression profile of circRNA and miRNA



Results of expression correlation analysis


miRNA overview

miRNA name:

Accession:

Sequence:

Aliases:

Precursor:

Predicted targets:

Visualization of expression correlation between circRNA and miRNA

Loading...

ID converter

Notes: Users can enter the circRNA ID of 14 circRNA databases in the box
Examples: hsa_circ_0072309 hsa-LIFR_0005 hsa_circLIFR_002 Reset: reset
Notes: The coordinate system must be 0-based, and the coordinate must be in the following
format: chromosome:start-end|strand
Example: chr5:38523520-38530768|- Reset: reset
Notes: The coordinate system must be 0-based, and the coordinate must be in the following
format: chromosome:start-end|strand
Example: chr5:38523418-38530666|- Reset: reset

ID convert result

Loading...
Notes: Click the row to the datails of circRNA ID converter result

Details

Location (0-based):

circRNA database:

circRNA microarray platform:

Differential expression analysis

Reset: reset

Details of dataset of selected tumor

Loading...
Notes: Click the rows to select datasets to perform circRNA differential expression analysis

Results of differential expression analysis

Customize plot

Download plot

Volcano plot displays the differentially expressed circRNA

Loading...

Customize plot

Download plot

Heatmap plot displays the differentially expressed circRNA

Loading...

Differential expression result


Customize plot

Plot type

Download plot

Visualization of miRNA expression profile in cell_lines

Loading...

Expression profile


Customize plot

Plot type

Download plot

Visualization of miRNA expression profile in cells

Loading...

Expression profile


Primer design

Notes: circRNA name cannot contain space, slash, backslash, special character and symbol
Example: example Reset: reset
Notes: circRNA name cannot contain space, slash, backslash, special character and symbol


Primer design result

Loading...
Notes: Click the row to display the schematic diagram of circRNA divergent primer sets. BSJ: back-spliced junction site

Customize plot

Download plot

Schematic diagram of circRNA divergent primer sets

Loading...

Sequence of amplification product

Gene list

Loading...
Notes: Click the row to visualize the expression profile of selected gene

Gene expression profile

Customize plot

Plot type

Download plot

Visualization of gene expression profile in tumors

Loading...

Differential expression result


Expression profile


Customize plot

Plot type

Download plot

Visualization of gene expression profile in tissues

Loading...

Expression profile


Customize plot

Plot type

Download plot

Visualization of gene expression profile in cell_lines

Loading...

Expression profile


Customize plot

Plot type

Download plot

Visualization of gene expression profile in cells

Loading...

Expression profile


Customize plot

Plot type

Download plot

Visualization of gene expression profile in TCGA

Loading...

Expression profile


Customize plot

Plot type

Download plot

Visualization of gene expression profile in CCLE

Loading...

Expression profile


Gene overview

Gene name:

Description:

Gene type:

Aliases:

Other ID:

Summary:

miRNA list

Loading...
Notes: Click the row to visualize the expression profile of selected miRNA

miRNA expression profile

Customize plot

Plot type

Download plot

Visualization of miRNA expression profile in tumors

Loading...

Differential expression result


Expression profile


Customize plot

Plot type

Download plot

Visualization of miRNA expression profile in tissues

Loading...

Expression profile


Customize plot

Plot type

Download plot

Visualization of miRNA expression profile in cell_lines

Loading...

Expression profile


Customize plot

Plot type

Download plot

Visualization of miRNA expression profile in cells

Loading...

Expression profile


Customize plot

Plot type

Download plot

Visualization of miRNA expression profile in TCGA

Loading...

Expression profile


Customize plot

Plot type

Download plot

Visualization of miRNA expression profile in CCLE

Loading...

Expression profile


miRNA overview

miRNA name:

Accession:

Sequence:

Aliases:

Precursor:

Predicted targets:

What are circRNAs?

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: a web server for circRNA exploration, analysis and visualization

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).

  • general information module
    • detailed annotations of circRNA and its host gene, including genomic coordinate, aliases, sequence and description
    • analysis and visualization of circRNA back-splicing schematic diagram, genomic position and sequence conservation, base modification and somatic mutation sites, secondary structure, and divergent primer
  • expression profile module
    • visualization of expression landscape of circRNA in cancers, normal tissues, cancer cell lines and normal cells
  • putative mechanism module
    • analysis and visualization of circRNA-miRNA, circRNA-RBP interactions, and circRNA coding potential



Figure 3. Schematic overview of the circPlot web application

Herein we provide the detailed instructions on how to easily use the circPlot web application.

Query a circRNA

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.

  • query a circRNA by searching circRNA ID

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

  • query a circRNA by searching circRNA sequence

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

  • query a circRNA by searching gene

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

  • query a circRNA by searching miRNA

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

  • query a circRNA by searching RBP

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

Analyze a circRNA

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.

  • analyze a circRNA by circRNA coordinate

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

  • query a circRNA by circRNA sequence

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

Overview of results of selected 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:

  • General information of circRNA
    • Basic information of circRNA and its host gene
    • Visualization of circRNA back-splicing schematic diagram, genomic position, sequence conservation and secondary structure
    • Visualization of somatic mutation and base modification sites on circRNA
    • Design and visualization of circRNA divergent primer sets
  • Expression profiles of circRNA in cancers, normal tissues, cancer cell lines and normal cells
  • Putative mechanisms of circRNA
    • Visualization of miRNA binding sites on circRNA and alignment between circRNA-miRNA interaction
    • Visualization of RBP binding sites on circRNA
    • Visualization of circRNA coding potential



Figure 9. Overview of results of selected circRNA

The circPlot provides the interactivity between users and analysis visualization results, which is summary blow:

  1. Users can select to display the analysis and visualization results through clicking the row of result table
  2. The tooltip or detailed information emerged when the mouse hover over the input box or elements within the plot
  3. Users can utilize the modebar and figure legend to manipulate the plot, such as show or hide elements, zoom in or zoom out the plot
  4. Users can customize the plot by clicking the “Visualization parameters” dropdown menu and then setting the visualization parameters. As any parameter changed, the plot will update immediately
  5. The plot is available for download in various formats by clicking the “Download parameters” dropdown menu in corresponding section. Besides, all plotting data and analysis results are publicly accessible and can be downloaded
  6. Users can prepare their own data and upload to the circPlot for customized visualization by selecting “Visualize your data?” option

General information of circRNA

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.

  • Basic information of circRNA

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

  • Basic information of circRNA host gene

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

  • Genomic position and sequence conservation of circRNA

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

  • Schematic diagram of circRNA back-splicing

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

  • Epigenetic modification sites on circRNA

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

  • Secondary structure of circRNA

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

  • Somatic mutation sites on circRNA

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

  • Divergent primer of circRNA

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

Expression landscape of circRNA

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

  • Expression profile of circRNA in cancers

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

  • Expression profile of circRNA in 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

  • Expression profile of circRNA in cancer cell lines

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

  • Expression profile of circRNA in normal cells

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

Putative mechanism of circRNA

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.

  • miRNA binding sites on circRNA

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

  • RBP binding sites on circRNA

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

  • circRNA coding potential

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

Data analysis

Obtain circRNA candidates

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

Prediction of miRNA binding sites on circRNA

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

Analysis of RNA binding proteins (RBPs) binding sites on circRNA

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

Prediction of coding potential of 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

Visualization of circRNA

The circPlot package

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 web portal

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.

Data analysis

Obtain circRNA candidates

The