| -f | is a fasta formated input file |
| -p | is a comma separated value (.csv) file containing four or six primer sequences per line (first six columns; inner forward, inner reverse, middle forward, middle reverse, outer forward, outer reverse) |
| -A | maximum amplicon size (bp; default = 280) |
| -a | maximum interloop spacing (bp; default = 51) |
| -s | minimum space between inner primers (bp; default = 1) |
| -l | minimum space between inner and middle primers (bp; default = 25) |
| -I | the number of exact matches at the 3' ends of the inner primer pair (1–3; default = 3) |
| -M | the number of exact matches at the 3' ends of the middle primer pair (1–3; default = 3) |
| -O | the number of exact matches at the 3' ends of the outer primer pair (1–3; default = 3) |
| -i | percent of matching bases for the inner primer pair (excluding the 3' bases set with -I; default = 100) |
| -m | percent of matching bases for the middle primer pair (excluding the 3' bases set with -M; default = 100) |
| -o | percent of matching bases for the outer primer pair (excluding the 3' bases set with -O; default = 100) |
| -r | activate “relaxed” mode, primer GC content is not checked |
| -c | evaluate template sequences in both possible orientations |