Design FISH Probes
Use DECIPHER's DesignProbes function to design the optimal FISH probes for targeting one group of RNA sequences in the presence of many non-target groups, as described in:ES Wright et al. (2014) "Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification." Applied and Environmental Microbiology, doi:10.1128/AEM.01685-14.
How do I design FISH probes?
First it is necessary to install DECIPHER and load the library in R. Next, set the "fas" variable to the path to the FASTA file of aligned sequences (e.g., "~/mySeqs.fas").Hide output
library(DECIPHER)
> library(RSQLite)
>
> # load sequences into a database
> fas <- "~/mySeqs.fas"
> dbConn <- dbConnect(SQLite(),
+ ":memory:")
> Seqs2DB(fas,
+ type="FASTA",
+ dbFile=dbConn,
+ identifier="")
Reading FASTA file chunk 1
118 total sequences in table Seqs.
Time difference of 0.05 secs
>
> # identify the sequences based on their description
> x <- dbGetQuery(dbConn,
+ "select description from Seqs")$description
> ns <- unlist(lapply(strsplit(x,
+ split=" "),
+ FUN=`[`,
+ 1L))
> Add2DB(myData=data.frame(identifier=ns),
+ dbFile=dbConn)
Expression:
update `Seqs` set `identifier` = (select
`temp`.`identifier` from `temp` where
`temp`.`row_names` = `Seqs`.`row_names`) where
exists (select `temp`.`identifier` from `temp`
where `temp`.`row_names` = `Seqs`.`row_names`)
Added to table Seqs: "identifier".
Time difference of 0.01 secs
>
> tiles <- TileSeqs(dbConn)
|========================================| 100%
Time difference of 13.73 secs
> head(tiles)
row_names start end start_aligned end_aligned
1 1 1 27 1 27
2 2 2 28 2 28
3 3 3 29 3 29
4 4 4 30 4 30
5 5 5 31 5 31
6 6 6 32 6 32
misprime width id coverage
1 FALSE 1594 Acinetobacter 1
2 FALSE 1594 Acinetobacter 1
3 FALSE 1594 Acinetobacter 1
4 FALSE 1594 Acinetobacter 1
5 FALSE 1594 Acinetobacter 1
6 FALSE 1594 Acinetobacter 1
groupCoverage target_site
1 1 AGAGTTTGATCATGGCTCAGATTGAAC
2 1 GAGTTTGATCATGGCTCAGATTGAACG
3 1 AGTTTGATCATGGCTCAGATTGAACGC
4 1 GTTTGATCATGGCTCAGATTGAACGCT
5 1 TTTGATCATGGCTCAGATTGAACGCTG
6 1 TTGATCATGGCTCAGATTGAACGCTGG
>
> probes <- DesignProbes(tiles,
+ identifier="Streptococcus", # target group
+ target="SSU") # target molecule
Streptococcus (751 candidate probes):
|========================================| 100%
Time difference of 79 secs
> head(probes)
identifier start start_aligned permutations
1 Streptococcus 1 1 1
2 Streptococcus 2 2 1
3 Streptococcus 3 3 1
4 Streptococcus 4 4 1
5 Streptococcus 5 5 1
6 Streptococcus 6 6 1
score probe.1 probe.2
1 -15.2240.... TCCTGAGCCAGGATCAAACTCT
2 -11.1516.... GTCCTGAGCCAGGATCAAACTC
3 -11.0664.... GTCCTGAGCCAGGATCAAACT
4 -9.45344.... TCGTCCTGAGCCAGGATCAAAC
5 -9.70312.... TTCGTCCTGAGCCAGGATCAAA
6 -9.60355.... TCGTCCTGAGCCAGGATCAA
probe.3 probe.4 efficiency.1 efficiency.2
1 0.5451167 NA
2 0.5822476 NA
3 0.5014876 NA
4 0.5493680 NA
5 0.5397903 NA
6 0.5470627 NA
efficiency.3 efficiency.4 FAm.1 FAm.2
1 NA NA 36.45083 NA
2 NA NA 37.66186 NA
3 NA NA 35.04946 NA
4 NA NA 36.58840 NA
5 NA NA 36.27878 NA
6 NA NA 36.62911 NA
FAm.3 FAm.4 coverage.1 coverage.2
1 NA NA 1 NA
2 NA NA 1 NA
3 NA NA 1 NA
4 NA NA 1 NA
5 NA NA 1 NA
6 NA NA 1 NA
coverage.3 coverage.4
1 NA NA
2 NA NA
3 NA NA
4 NA NA
5 NA NA
6 NA NA
mismatches
1 Acinetobacter (10.7%,5.70kcal/mol,-18.4%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCATGGCTCAGAT) Actinomyces (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA) Bacillus (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA) Bacteroides (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA) Bifidobacterium (9.7%,5.98kcal/mol,-19.3%;TCCTGAGCCAGGATCAAACTCT/AGGGTTCGATTCTGGCTCAGGA) Clostridium (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA) Deinococcus (51.9%,0.26kcal/mol,-0.9%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGG) Enterococcus (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA) Escherichia (10.7%,5.70kcal/mol,-18.4%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCATGGCTCAGAT) Helicobacter (29.3%,2.63kcal/mol,-8.5%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGAG) Lactobacillus (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA) Listeria (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA) Neisseria (29.3%,2.63kcal/mol,-8.5%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGAT) Porphyromonas (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA) Propionibacterium (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA) Pseudomonas (10.7%,5.70kcal/mol,-18.4%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCATGGCTCAGAT) Rhodobacter (29.3%,2.63kcal/mol,-8.5%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGAA) Staphylococcus (54.5%,0.00kcal/mol,0.0%;TCCTGAGCCAGGATCAAACTCT/AGAGTTTGATCCTGGCTCAGGA)
2 Actinomyces (58.2%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAC) Bacillus (44.4%,1.38kcal/mol,-4.5%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAT) Bacteroides (44.4%,1.38kcal/mol,-4.5%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAT) Clostridium (58.2%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAC) Deinococcus (41.1%,1.72kcal/mol,-5.6%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGGT) Enterococcus (58.2%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAC) Helicobacter (18.7%,4.47kcal/mol,-14.5%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGAGT) Lactobacillus (58.2%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAC) Listeria (58.2%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAC) Neisseria (18.7%,4.47kcal/mol,-14.5%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGATT) Porphyromonas (44.4%,1.38kcal/mol,-4.5%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAT) Propionibacterium (58.2%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAC) Rhodobacter (18.7%,4.47kcal/mol,-14.5%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGAAT) Staphylococcus (44.4%,1.38kcal/mol,-4.5%;GTCCTGAGCCAGGATCAAACTC/GAGTTTGATCCTGGCTCAGGAT)
3 Actinomyces (50.1%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAC) Bacillus (36.6%,1.38kcal/mol,-4.6%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAT) Bacteroides (36.6%,1.38kcal/mol,-4.6%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAT) Clostridium (50.1%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAC) Deinococcus (33.5%,1.72kcal/mol,-5.8%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGGT) Enterococcus (50.1%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAC) Helicobacter (14.2%,4.47kcal/mol,-15.0%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGAGT) Lactobacillus (50.1%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAC) Listeria (50.1%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAC) Neisseria (14.2%,4.47kcal/mol,-15.0%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGATT) Porphyromonas (36.6%,1.38kcal/mol,-4.6%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAT) Propionibacterium (50.1%,0.00kcal/mol,0.0%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAC) Rhodobacter (14.2%,4.47kcal/mol,-15.0%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGAAT) Staphylococcus (36.6%,1.38kcal/mol,-4.6%;GTCCTGAGCCAGGATCAAACT/AGTTTGATCCTGGCTCAGGAT)
4 Actinomyces (54.9%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGACGA) Bacillus (34.5%,2.08kcal/mol,-6.7%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGATGA) Bacteroides (34.5%,2.08kcal/mol,-6.7%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGATGA) Clostridium (54.9%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGACGA) Deinococcus (17.9%,4.27kcal/mol,-13.8%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGGTGA) Enterococcus (54.9%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGACGA) Lactobacillus (54.9%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGACGA) Listeria (54.9%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGACGA) Porphyromonas (34.5%,2.08kcal/mol,-6.7%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGATGA) Propionibacterium (54.9%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGACGA) Staphylococcus (34.5%,2.08kcal/mol,-6.7%;TCGTCCTGAGCCAGGATCAAAC/GTTTGATCCTGGCTCAGGATGA)
5 Actinomyces (54.0%,0.00kcal/mol,0.0%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGACGAA) Bacillus (33.6%,2.08kcal/mol,-6.7%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGATGAA) Bacteroides (33.6%,2.08kcal/mol,-6.7%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGATGAA) Bifidobacterium (13.6%,4.98kcal/mol,-16.1%;TTCGTCCTGAGCCAGGATCAAA/TTCGATTCTGGCTCAGGATGAA) Clostridium (54.0%,0.00kcal/mol,0.0%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGACGAA) Deinococcus (16.9%,4.34kcal/mol,-14.0%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGGTGAA) Enterococcus (54.0%,0.00kcal/mol,0.0%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGACGAA) Lactobacillus (54.0%,0.00kcal/mol,0.0%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGACGAA) Listeria (54.0%,0.00kcal/mol,0.0%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGACGAA) Porphyromonas (33.6%,2.08kcal/mol,-6.7%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGATGAA) Propionibacterium (54.0%,0.00kcal/mol,0.0%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGACGAA) Staphylococcus (33.6%,2.08kcal/mol,-6.7%;TTCGTCCTGAGCCAGGATCAAA/TTTGATCCTGGCTCAGGATGAA)
6 Actinomyces (54.7%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGACGA) Bacillus (34.3%,2.08kcal/mol,-7.2%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGATGA) Bacteroides (34.3%,2.08kcal/mol,-7.2%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGATGA) Bifidobacterium (18.1%,4.22kcal/mol,-14.7%;TCGTCCTGAGCCAGGATCAA/TCGATTCTGGCTCAGGATGA) Clostridium (54.7%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGACGA) Deinococcus (17.7%,4.27kcal/mol,-14.9%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGGTGA) Enterococcus (54.7%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGACGA) Lactobacillus (54.7%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGACGA) Listeria (54.7%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGACGA) Porphyromonas (34.3%,2.08kcal/mol,-7.2%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGATGA) Propionibacterium (54.7%,0.00kcal/mol,0.0%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGACGA) Staphylococcus (34.3%,2.08kcal/mol,-7.2%;TCGTCCTGAGCCAGGATCAA/TTGATCCTGGCTCAGGATGA)
>
> dbDisconnect(dbConn)