Frequently Asked Questions for ExonSurfer
Which organisms are supported by ExonSurfer?
Currently, ExonSurfer supports Homo sapiens, Mus musculus, Rattus norvegicus, Danio rerio, and Drosophila melanogaster from Ensembl’s release 108, and Arabidopsis thaliana and Oryza sativa from release 57. These correspond to GenBank Assembly IDs:
- Homo sapiens (GRCh38)
- Mus musculus (GRCm39)
- Rattus norvegicus (mRatBN7.2)
- Danio rerio (GRCz11)
- Drosophila melanogaster (BDGP6.32)
- Arabidopsis thaliana (TAIR10)
- Oryza sativa (IRGSP-1.0)
Can ExonSurfer design primers for transcripts not present in Ensembl’s database?
Yes, ExonSurfer can design primers for transcripts not found in Ensembl. The sequences must be uploaded in GenBank format or FASTA format (including the junction placement in the FASTA header). ExonSurfer will check the specificity against Ensembl’s database, treating every match as a possible off-target.
Can ExonSurfer design primers for novel transcripts?
Yes, ExonSurfer allows primer design for novel transcripts. This should be uploaded in FASTA format (including the junction placement in the FASTA header). ExonSurfer will check the specificity against Ensembl’s database, treating every match as a possible off-target.
Why do I get off-targets in my target gene?
ExonSurfer allows transcript-specific primer design. If you only select some transcripts from a gene, or if you upload a sequence in a file, ExonSurfer treats every other match in the database as an off-target.
How does ExonSurfer avoid common polymorphisms?
By default, ExonSurfer uses masked sequences for the Homo sapiens genome. We filtered NCBI’s dbSNP database and kept all polymorphisms that had a frequency larger than 1% in any population. Polymorphisms without frequency information were discarded. These positions have been changed in the genome to an “N” and primers cannot contain any “N” in their sequence.
How does ExonSurfer design primers for transcript-specific detection?
ExonSurfer searches for unique exon junctions that all the targeted transcripts have in common. To do that, we compare the sorted exon identifiers from all the transcripts of a gene. If this is not feasible because the junctions present in the targeted transcripts are also present in untargeted isoforms, ExonSurfer reports these other isoforms as off-targets.
Can ExonSurfer design primers for multiple targets simultaneously?
Yes, as long as they belong to the same gene. ExonSurfer allows primer design for some or all the transcripts of a gene. This feature is not currently supported for file input.
What types of PCR experiments is ExonSurfer suitable for?
ExonSurfer is suitable for dye-based quantitative PCR experiments.
Is ExonSurfer free to use?
Yes, ExonSurfer is a free online tool available to anyone with an internet connection. We ask you to cite our software:
Monfort-Lanzas, P., Rusu, E.C., Parrakova, L. et al. ExonSurfer: a web-tool to design primers at exon–exon junctions. BMC Genomics 25, 594 (2024). https://doi.org/10.1186/s12864-024-10456-2
How long does it take for ExonSurfer to design primers?
You can choose the "Number of primer pairs to design" in the Primer Parameters section to best suit your time requirements. The mean time for designing the different number of primer pairs are:
- 100 primers: 17 seconds
- 1000 primers: 73 seconds
- 3000 primers: 119 seconds
These values are constantly updated with the time execution information from new designs.