Maintained by Shanliang Zhong
CircPrimer: a software for annotating circRNAs and determining specificity of circRNA primers
CircPrimer is a user-friendly tool for exploring circRNAs that does not require special user skills. CircPrimer enables users to extract the spliced sequences and genomic sequences of any circRNA, including novel circRNAs. Furthermore, circPrimer help users to design primers for circRNAs and to determine the specificity of the circRNA primers.
- Data Input
The CircRNA ID (e.g., hsa_circ_0000007), chromosome location (e.g., chr1:1735857-1737977-), gene symbol, or a file path of the text file (one chromosome location per line) can be input into the circRNA field according to your need. To input a file path, you press the Ctrl key and later double click the circRNA field to show the open file dialog. The chromosome, the start and end coordinates, and the strand orientation can be separated with any non-numeric character, except ‘,’.
- Searching circRNA in circBase
The CircRNA ID, chromosome location, and gene symbol are the accepted data for searching circRNA in the circBase. Input the data to circRNA field, click the “circBase” button, the circRNAs are listed at the right of the main form, if one or more circRNAs are obtained. Click one of the listed circRNAs, the spliced sequence of this circRNA is shown in the field “circRNA SEQ.” If you have checked “Annotate circRNA when click”, a form is present to show the annotated result of the circRNA (Figure 1). You can save the search results in fasta format by using the right-click list menu.
Note: RefGene “GRCh37.75” or “hg19” should be chosen before clicking a circRNA to annotate the circRNA.
Figure 1 The annotation result for hsa_circ_0002232. The data showed on the top-left is the data from the circBase. The data on the circle is annotated based on refGene. CDS, Coding sequence; UTR, Untranslated Region; e1, partial sequence of exon 1 (“e” represents partial sequence of the exon, while “i” represents partial sequence of the intron).
- Annotating a circRNA
The CircRNA ID and chromosome location are the accepted data for annotating a circRNA. Input the data to the circRNA field, click “Annotate” button, a form is present to show the annotated result of the circRNA.
Note: If inputting the circRNA ID, refGene “GRCh37.75” or “hg19” should be chosen. If inputting chromosome location, the right refGene version should be chosen before annotating.
- Extracting a spliced sequence
The CircRNA ID, chromosome location, and a file path are the accepted data. To input a file path, you can press Ctrl key and later double click the circRNA field to show the open file dialog. After inputting circRNA ID or chromosome location and clicking “UCSC RNA” button, the spliced sequence of the circRNA is shown in the “circRNA SEQ” field . When inputting a file path for a file with multiple chromosome locations, a circRNA list is present at the right of the main form. You can annotate a circRNA or export the circRNAs in fasta format using the method mentioned above.
Note: No matter which data is input, the sequence is not obtained from the circBase, but is extracted from the UCSC web site according to the annotation result.
- Extracting the genomic sequence
The CircRNA ID, chromosome location, and a file path are the accepted data. After inputting the circRNA ID or chromosome location and subsequently clicking the “UCSC DNA” button, the genomic sequence of the circRNA is shown in the “circRNA SEQ” field. If inputting a file path of a file with multiple chromosome locations, a text file in fasta format will be saved in the root directory of the application.
Note: No matter which data is input, the sequence is not obtained from the circBase, but is extracted from UCSC web site according to chromosome location.
- Designing divergent primer
Use the above mentioned method to get the spliced sequence for a circRNA, check “Divergent primer”, click “Template” button, and then click the link of “Primer3 (V4.1)” and paste the template into Primer3, and finally click “Pick Primers” in Primer3 to obtain divergent primers.
- Designing primers with one primer spanning the spliced junction
Use same method mentioned in section 6, but check “One primer spanning junction”.
- Checking specificity of primers
Check “Check Primer”, a panel is shown at the right of the main form. Input the primers and click the “check” button, the application will search circBase to show the circRNAs which could be amplified by the primers. The “length” in the grid is the product size; and “F0R” is a field to indicate the primer characteristics (1, convergent primer; 0, divergent primer; F+No., forward primer spanning the spliced junction [No. represents the base count spanned by the primer]; and R+No., reverse primer spanning the spliced junction). You can click the circRNA in the list to show the primer location visually (Figure 2).
Figure 2 Primer location is shown visually.
How to export the figure?
After showing the annotated result (e.g. Figure 1) using circPrimer, a file named “circRNA.bmp” will be generated at root directory of circPrimer.
How to cite？
Shanliang Zhong, Jinyan Wang, Qian Zhang, Hanzi Xu and Jifeng Feng. CircPrimer: a software for annotating circRNAs and determining the specificity of circRNA primers. BMC Bioinformatics. 2018, 19(1):292. DOI: 10.1186/s12859-018-2304-1. PMID: 30075703