Usage instructions CIRCprimerXL
Input variables
| parameter | default | options |
|---|---|---|
| species | human | human, mouse, rat, zebrafish, Xenopus tropicalis, or Caenorhabditis elegans Of note, the SNP filter is only activated when designing primers for human circRNAs. |
| splicing | yes | yes: the template sequence is spliced no: the template sequence is not spliced |
| template length | 150 (which results in a total template length of 300 nucleotides) | any integer between 50 and 500 |
| amplicon length | calculated based on template length | any integer between 50 and 500 |
| primer GC settings | 30-50-80 (minimum – optimum – maximum) | any integer between 15 and 85 |
| primer Tm settings | 58-59-60 (minimum – optimum – maximum) | any integer between 50 and 70 |
| SNP filter | strict | strict: avoids common SNPs in primer design loose: avoids common SNPs in the 3' end half of primer |
| off-targets filter | strict | strict or loose (see Table 1 in manuscript) |
Output variables
| column name | description |
|---|---|
| circ_ID | circ id assigned to each circRNA (unique within one run) |
| chr | circRNA chromome |
| start | circRNA start position |
| end | circRNA end position |
| primer_ID | primer ID generated by primer3 |
| FWD_primer | forward primer |
| REV_primer | reverse primer |
| FWD_pos | relative position of forward primer |
| FWD_length | length of forward primer |
| REV_pos | relative position of reverse primer |
| REV_length | length of reverse primer |
| FWD_Tm | melt temperature of forward primer |
| REV_Tm | melt temperature of reverse primer |
| FWD_GC | GC content of forward primer |
| REV_GC | GC content of reverse primer |
| amplicon | amplicon sequence amplified by the primer pair |
| PASS | result of filtering (PASS if the primer pair passed all filters, FAIL if the primer pair failed one or more filters) |
| start_annotation | exons used for the template sequence on the right side of the BSJ |
| end_annotation | exons used for the template sequence on the left side of the BSJ |
| splicing | spliced or unspliced template sequence was used |