Good day,

my goal is to design fish probes for an uncultured bacterial species that is part of a microbiome. I do have the 16S sequence of the species and a dataset of V1-V2 16S amplicon sequences of the microbiome without taxonomic assignment. I want to use the DECIPHER package in R following the current documentation for FISH-probe design. I have to work around the fact that I don't have taxonomic assignment for my sequences. This is done by clustering the sequences using Clusterize() and identifying the cluster with my target sequences (cluster 45), which worked.

My problem is that when running DesignProbes() I get the following warning:

Warning message: In DesignProbes(tiles, identifier = "45", start = 10, end = 230) : No probes met the specified constraints: 45

This persists even when setting the constraints for probe design extremely low. It seems unlikely to me that there can be actually no probes designed, so I'm wondering if anyone has an idea of what the problem could be or if I just have to accept that no probes can be designed. Btw: oligoarrayaux is installed and works.

The following is my current code:

library(DECIPHER)
library(Biostrings)

seqs <-"/home/user/Desktop/FISH_probe_design/16S_V1_V2_.fasta"

seqs_raw <- readDNAStringSet(seqs)

seqs_raw <- OrientNucleotides(seqs_raw)

seqs_trim <- subseq(seqs_raw, start = 1, end = 246)

clus <- Clusterize(seqs_raw, cutoff = 0.05, processor=NULL)

dbConn <- dbConnect(SQLite(), "/home/user/R/FISH/seqs_1.sqlite")

Seqs2DB(seqs_trim, "DNAStringSet", dbConn, "")

Add2DB(data.frame(identifier=clus[[1]]), dbConn)

tiles <- TileSeqs(dbConn,
                  tblName = "Seqs",
                  identifier = "",
                  minLength = 26,
                  maxLength = 27,
                  maxTilePermutations = 10,
                  add2tbl = "Tiles")

probes <- DesignProbes(tiles, identifier="45", start=10, end=230)

Thank you for your time!