how to design probes using decipher
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hoonhuiyi • 0
@hoonhuiyi-23342
Last seen 14 months ago

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?

R decipher • 205 views
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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 each identifier. If the groups are ill-defined then these warning messages are expected.

Please provide a reproducible example if you need more assistance.

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An aligned Fasta file with sequence descriptions:

KU221427.1.1442 Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae;Pseudomonas;bacterium L54 HE591468.1.1765 Eukaryota;Amorphea;Obazoa;Opisthokonta;Holozoa;Choanozoa;Metazoa;Porifera;Demospongiae;Poecilosclerida; Desmapsamma anchorata KY623367.1.1439 Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae;Pseudomonas;Pseudomonas sp. AB000392.1.1478 Bacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae;Vibrio;Vibrio halioticoli

I have defined the groups exactly the same as when I was designing primer for genus level:

x <- dbGetQuery(dbConn, "select description from Seqs")$description
x <- strsplit(x, ";", fixed=TRUE)
x <- sapply(x, tail, n=1)
x <- strsplit(x, " ", fixed=TRUE)
x <- sapply(x, head, n=1)
Add2DB(data.frame(identifier=x, stringsAsFactors=FALSE), dbConn)
> tiles <- TileSeqs(dbConn, add2tbl="Tiles")

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:

probes <- DesignProbes(tiles, identifier="Pseudomonas",start=120, end=1450) PseudomonasWarning message: In DesignProbes(tiles, identifier = "Pseudomonas", start = 120, : All target sites have too many permutations: Pseudomonas

probes <- DesignProbes(tiles, identifier="Pseudomonas") Pseudomonas (1 candidate probe) identifier start start_aligned permutations score 150 Pseudomonas 517 3694 2 -10.4613.... probe.1 probe.2 probe.3 probe.4 efficiency.1 150 CCCGTAGGACGTATGCGGT CCCTTAGGGCGTATGCGGT <na> <na> 0.6183057 efficiency.2 efficiency.3 efficiency.4 FAm.1 FAm.2 FAm.3 FAm.4 150 0.7408706 NA NA 39.32682 44.42303 NA NA coverage.1 coverage.2 coverage.3 coverage.4 150 0.89743590 0.02564103 NA NA

Although 1 probe was found, this probe sequence could not be found in the fasta file of pseudomonas sequences that were used as input.

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