The probes are sorted by quality. The quality tries to reflect how good each probe separates between target- and non-target group.
Therefore it detects the column COL in the decrease-temparature-list, where more than twice as much non-target species would get hitten, than mishits (i.e. non-group hits) were encountered by matching the probe.
Using that column the quality is calculated using the formula
quality = groupsize * COL + ------------------
1000 + hits[COL]
COL is in range 0 .. 19
mishits = probehits - groupsize
probehits = number of hits reported by probe match (zero mismatches)
COL is lowest column where: hits[COL] > 2*mishits
if no column matches the criterion above, COL=19 is used
Probes with equal qualities are sorted by absolute position and then alphabetically. It does not take G+C content, temperature or ECOLI position into account.
It is up to the user to study the list carefully and choose his candidates. In our lab we often found the 20th probe was the best.