BLASTcirc is a tool for finding whether or not a query sequence is a circular RNA (circRNA) by searching against a genome or transcriptome. BLASTcirc can determine whether query sequences contain back-splicing sites based on basic local alignment, provides the statistical significance of the potential circRNAs using a global alignment algorithm, and optimizes a composite score (score A) for each alignment by taking into account all potential genomic locations of back-splicing sites.


Users can paste query nucleotide sequences in the main input box. Query sequences saved in a plain text file can also be uploaded using the "Choose File" button.




The database for BLASTcirc can be selected from the standard list using the pull-down menu. Five organisms (humans, rice, Arabidopsis, mouse and fruit fly) are included in the current version. The default setting of the Max distance between the start and end of a circRNA will be changed automatically according to the organism selected.




BLASTcirc implements the following selectable parameters for searching and filtering the results. Users can change the parameters based on their requirements.


Word sizeSame as the parameter used in BLASTn. It also defines the shortest length of the junction fragments for output (default 15nt).
Mismatch penaltySame as the parameter used in BLASTn (default -5).
Gap costsGap costs here mean gap existence and gap extension (default gap existence 15 and gap extension 3).
Max number of resultsThe maximum number of alignment results for output.
E-valueThe threshold of the expectation value for saving a candidate circRNA.
Output typeThere are two types of output: circle (default) or both circle and linear.
FilterWhether or not to filter out highly complex regions using DUST algorithm.
Mismatch in junction sitesWhether or not to allow mismatches in the back-splicing junction sites.
Splicing signal & exon boundaryThere are two options: (1) only output potential circRNAs with GT-AG splicing signal or back-splicing site locating at exon boundary; (2) output all circRNAs.
Max distance between start and end (bp)The maximum distance between the start and end locations of a potential circRNA on genome (default 50Kb for human and animals and 10Kb for plants).

Press the submit button. The results and descriptions will appear in a new page.


Result and description


The result page shows the hits and descriptions of each output item, and contains four components, including Graphic Summary, Descriptions, Alignments and Downloads.


The results of different queries can be selected to be shown in the page using the pull-down menu if there is more than one query sequence.





Its purpose is to provide a brief summary of the searching results. Colors represent scores of two parts of a back-splicing junction. Annotations of the hit will display when moving mouse onto the colored bars.





The Descriptions table provides a list of circRNA candidates found by BLASTcirc.





The headings of each column are described in the following table.


No.The order of all hits according to their reliability level.
HitThe circRNA reported in the format of chromosome (gene).
TypeType of hits: circRNA, linear transcript or uncertain.
ScoreThe raw score of the global alignment between the query sequence and the pseudo-subject sequence calculated based on the regular BLASTn scoring rules (for details see the Supplementary file of the BLASTcirc paper by Zhang et al., 2017).
Score AScore A consists of two components: raw score of global alignment of the query sequence based on the regular BLASTn scoring rules and a value representing different type of canonical splicing site (for details see the Supplementary file of the BLASTcirc paper by Zhang et al., 2017).
E-valueThe expectation value for the candidate circRNA.
SignalSplicing signal.
PositionThe exact genomic position of the circRNA, including chromosome, start and end positions (1-based).
SubjectIf a fragment of the back splicing junction crosses exon-intron boundary, it is the name of the matched transcript or the name of the matched chromosome.
VisualizationClick the eye icon to visualize the circRNA in a new page.

The detailed alignments of the L and R segments from a query sequence with their subject sequences in the database.





Three formats of the search result files generated by BLASTcirc and BLASTn are provided for download.




Visualization


CircRNAs can be visualized based on the gene annotation of genomic sequences.


Users can customize the output picture by changing parameters and selecting the information to be showed in the picture, such as mismatches, gaps, UTR regions and gene direction.





The left panel figure shows the genomic position of the circRNA. The colored rectangles represent transcripts with their names showing above and the corresponding gene names showing below. The grey area represents the exact position of the circRNA within its parent gene. The L and R fragments are shown on the boundary of the grey bar above the coordinate axis.

The right panel figure shows the putative structure of the circRNA. The line with arrow represents the direction of transcripts. The vertical line with two numbers represents the back-splicing site of the circRNA. The query sequence is aligned around the circRNA with its two (L and R) fragments shown in different colors to indicate the junction event. Different genomic components (CDS, intron, exon and UTR) are color-coded.

The picture can be downloaded in PDF or PNG format.



Provide the detailed alignment information of the L and R segments from a query sequence in the figures.


References


  1. Altschul, S.F, et al. (1990) Basic local alignment search tool, Journal of molecular biology, 25, 403-410.
  2. Chen, L., et al. (2016) PcircRNA_finder: a software for circRNA prediction in plants, Bioinformatics, 32, 3528-3529.
  3. Chu, Q., et al. (2017) PlantcircBase: a database for plant circular RNAs, Molecular Plant. DOI: http://dx.doi.org/10.1016/j.molp.2017.03.003
  4. Gao, Y., et al. (2015) CIRI: an efficient and unbiased algorithm for de novo circular RNA identification, Genome biology, 16, 4.
  5. Glazar, P., Papavasileiou, P. and Rajewsky, N. (2014) circBase: a database for circular RNAs, RNA, 20, 1666-1670.
  6. Guo, J.U., et al. (2014) Expanded identification and characterization of mammalian circular RNAs, Genome Biol, 15, 409.
  7. Hansen, T.B., et al. (2013) Natural RNA circles function as efficient microRNA sponges, Nature, 495, 384-388.
  8. Hoffmann, S., et al. (2014) A multi-split mapping algorithm for circular RNA, splicing, trans-splicing, and fusion detection, Genome Biology, 15, 34.
  9. Jeck, W.R., et al. (2013) Circular RNAs are abundant, conserved, and associated with ALU repeats, RNA, 19, 141-157.
  10. Memczak, S., et al. (2013) Circular RNAs are a large class of animal RNAs with regulatory potency, Nature, 495, 333-338.
  11. Salzman, J., et al. (2012) Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types, PLoS One, 7, e30733.
  12. Ye, C.Y., et al. (2015) Widespread noncoding circular RNAs in plants, New Phytol, 208, 88-95.
  13. Ye, C.Y., et al. (2016) Full-length sequence assembly reveals circular RNAs with diverse non-GT/AG splicing signals in rice, RNA biology, 1-9.
  14. Zhang, X.O., et al. (2016) Diverse alternative back-splicing and alternative splicing landscape of circular RNAs, Genome research, 26, 1277-1287.