GenoFrag: Primer Selection

Primer selection

Given a sequence, all the substrings of length sizePrimer (default value = 25) are considered as potential primers and are processed through a serie of filters. The primers which pass sucessfully all the filters are selected.

GC filter

Select primers with a minimum of nbGCMin (default value = 11) and a maximum of nbGCMax (default value = 13) G or C nucleotides.

Repeat filter

Dismiss primers with nbRepeat (default value = 4) successive identical nucleotides.

Hairpin filter

Dismiss primers having hairpins with the following features:

a duplex length equal to or greater than maxHpDup (default value = 3)

a loop equal to or greater than maxHpLoop (default value = 4)

           C A G G A T G T T G
                       | | |   C
                       | | |   C   loop
           A A G A C C C A A T
                      duplex
                      length

Stability filter

Check the 5' and 3' extremities (4 nucleotides) and select primers satisfying:

5' extremity: dG smaller than deltaG5 cal/mol (default value = -10000)
3' extremity: dG between deltaG3min (default value = -9000) and deltaG3max cal/mol (default value = -5500)

Nearest-Neighbor Sequence Depedence

1er nt / 2nd nt	dA	dC	dG	dT
dA	-1940	-1340	-1600	-1470
dC	-1950	-3070	-3600	-1600
dG	-1570	-3140	-3070	-1340
dT	-960	-1570	-1950	-1940

START / END Primers

If criteria stability in 5' and 3' is achieved for the primer, it is tagged as START.
If criterai stability in 5' and 3' is achieved for its inverse complemnt, the primer is tagged as END.

Auto-complementarity filter

Dismiss :

Primers having a number of matches with their reverse strand equal to or greater than maxAutoMatch (default value = 15)
Primers having matching words greater than 3 with their reverse strand and a dG smaller than deltaGAuto cal/mol (default value = -5000)
in the 3' extremity, primers having matching words greater than 2 with their reverse strand and a dG smaller than deltaGAuto3 cal/mol (default value = -3500)

Similarity filter

Dismiss primers having a potential secondary fixation site on its neighboring. The length of the scaning zone is equal to sizeScan (default value = 20000) upstream and downstream the primer site. A secondary site exists if the number of matches between the primer and this site is equal to or greater than maxSimMatch matches (default value = 15), with a maximum of maxSimGap gaps (default value = 0).