Hi Erik,
I have the following issues with the design probe function:
probes <- DesignProbes(tiles, identifier="Streptococcus",start=120, end=1450, batchSize=100,numProbeSets=5)
StreptococcusWarning message: In DesignProbes(tiles, identifier = "Streptococcus", start = 120, : No target sites met the specified constraints: Streptococcus
probes <- DesignProbes(tiles, identifier="Pseudomonas",start=120, end=1450, batchSize=100,numProbeSets=5)
PseudomonasWarning message: In DesignProbes(tiles, identifier = "Streptococcus", start = 120, : All target sites have too many permutations: Pseudomonas
what does it mean and how can I solve it?
It means that the sequences corresponding to these identifiers are too diverse. In other words, there isn't a target site that meets your design constraints. I recommend looking at the
tiles
corresponding to each of these groups, and the original sequences corresponding to eachidentifier
. If the groups are ill-defined then these warning messages are expected.Please provide a reproducible example if you need more assistance.
An aligned Fasta file with sequence descriptions:
I have defined the groups exactly the same as when I was designing primer for genus level:
According to
sort(table(x))
, there are 79 sequences for Pseudomonas.After defining the groups and creating tiles, Using the design primer function, I have managed to obtained 2 PCR primers for Pseudomonas. however was unable to obtain probes for it using design probes function:
Although 1 probe was found, this probe sequence could not be found in the fasta file of pseudomonas sequences that were used as input.