Design FISH Probes - Inputs

1. Probes:

   Set constraints on the designed probes by limiting their lengths and maximum number of different permutations in order to cover the target group.

2. Hybridization Conditions:

   These experimental parameters serve as inputs for thermodynamic models that optimize probe sensitivity and specificity to the target group. Standard FISH conditions are provided as defaults. Hybridization efficiency specifies the minimum percentage of target hybridized to probe at equilibrium, where the melt point is defined as 50%. A higher value of hybridization efficiency will result in a greater target brightness at the expense of also increasing non-target signal.

3. Target group:

   Set the name of sequences belonging to the target group and the minimum fraction of sequences that must be covered by the probes. For example, specifying a coverage of 90% will ensure that the designed set of probe(s) perfectly match at least 90% of sequences in the target group. The target group should match the FASTA identifier of one or more sequences in the FASTA file (e.g., "XYZ" in the example below).

4. Target molecule:

   Choose the target RNA molecule, either the small-subunit ribosomal RNA (SSU rRNA), large subunit rRNA, or any other RNA (e.g., mRNA). This will set the method of determining target site accessiblity.

5. Comprehensive sequence database:

   Specify whether to search a comprehensive reference database of non-targets. Only applicable if the selected target molecule is "LSU" or "SSU". Selecting "Yes" will initiate a search for non-target genera through a large database containing sequences of the target molecule. Potential non-targets are recorded and used to adjust each probe's score, which will serve as an additional criteria to help in choosing the best probe(s). Potential non-target genera in the reference database are added to the results with "ref" followed by the genus name (e.g., "ref_Bacillus"). If the target group is present in the non-target database then it will have minimal effect on the ranking of results because all candidate probes will be penalized equally. However, if you wish to attempt to exclude genera similar to the target sequences then select "Yes, and exclude potential target genera". This option will first classify all sequences in the specified target group using the RDP classifier. Then potential target genera will be removed before searching the comprehensive reference database.

6. Target molecule:

   Choose the target RNA molecule, either the small-subunit ribosomal RNA (SSU rRNA), large subunit rRNA, or any other RNA (e.g., mRNA). This will set the method of determining target site accessiblity.

7. FASTA File:

   Choose a text file containing the sequence records to use for probe design. Here are some remarks on the input file:

   • Sequences must be in FASTA format where each new sequence record begins with a ">" symbol on a single line containing the name of the group where it belongs, and subsequent lines contain the actual sequence. There are no restrictions on the number of nucleotides per line, however a maximum of 10,000 nucleotides are permitted per sequence.
   • Sequences must be aligned (with gaps; "-") such that all input sequences are the same total length. Standard IUPAC ambiguity codes are permitted.
   • The size of the uploaded file is restricted to be less than 50 MB. This limit corresponds to roughly 35,000 sequences of length 1,000 nucleotides plus gaps. To analyze more than 50MB of sequences, please download DECIPHER for use on your computer.
   • Each FASTA record must have an identifier listing only the group to which it belongs. One or more sequences must be named the same as the target group. Up to 500 non-target groups are permitted. Each non-target group is weighed equally when scoring specificity.
     
     A correctly formatted input file for the target group "XYZ" might look like:
     >XYZ
     ACTG-ACTG...
     >XYZ
     ACTGGACTG...
     >ABC
     AGGG-ACTG...
     >ABC
     -GGC-ACTG...
     >Etc.
     ...