First posting here!
I am having a problem running primer3 (actually using EMBOSS eprimer32) with the option to exclude regions where primer should not overlap. I used the option -excludedregion. It seems to work for a single region but not a multiple region. For example:
eprimer32 -sequence input.fasta -prange 170-250 -excludedregion 345,360 -targetregion 180,335 -maxdifftm 1 -gcclamp 1 -opttm 58 -maxtm 60 -mintm 54 -optsize 20 -minsize 17 -maxsize 24 -outfile output.primer -numreturn 1 -mingc 30 -maxgc 70
does seem to work with primer designed at position
BUT the following with 2 regions to avoid:
eprimer32 -sequence input.fasta -prange 170-250 -excludedregion 20,25 345,360 -targetregion 180,335 -maxdifftm 1 -gcclamp 1 -opttm 58 -maxtm 60 -mintm 54 -optsize 20 -minsize 17 -maxsize 24 -outfile output.primer -numreturn 1 -mingc 30 -maxgc 70
is not working and primers are designed at position
340-360 , the second primer overlapping with my excluded region.
Any idea what I'm doing wrong?
Many thanks for any help or pointers!
PS: Help file for the option is saying:
-excludedregion range [(full sequence)] Primer oligos may not overlap any region specified in this tag. The associated value must be a space-separated list of (start),(end) pairs where (start) is the index of the first base of the excluded region, and and (end) is the last. This tag is useful for tasks such as excluding regions of low sequence quality or for excluding regions containing repetitive elements such as ALUs or LINEs. E.g. 401,407 68,70 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68.