It is possible to select a fasta file from your computer and upload it into the application. The uploaded file will appear in the "FASTA sequence" text-box below, where you can check for formatting errors before lunching the analysis. Also in this case you must submit a "FASTA file name" to be used (see "FASTA file name" help box below for more details).

A custom name referring to your FASTA-formatted sequence is required. This name identifies the unique probe-set derived from the submitted FASTA-formatted file and it will replace its header. Please remind that setting the same "FASTA file name" for multiple input sequences when using "Input - Multi" module, will prevent you to distinguish between the resulting probes set. The following characters are not allowed: ":", ".", "/", ".", "<", ">" and the "underscore".

A FASTA-formatted sequence is required to let OligoMiner workflow mines for probes. It is possible to write or cut & paste a single, up to 100 Kbs long, sequence. Please remember that any information contained into the header of your FASTA-formatted sequence will be lost replaced by the "FASTA file name" of your choice.

The minimum allowed probe length. Default is 36 base pairs.

The maximum allowed probe length. Default is 41 base pairs.

The minimum allowed percent G + C inside each probes. Default is 20%.

The maximum allowed percent G + C inside each probes. Default is 80%.

The minimum allowed adjusted Tm, default is 42° C, calculated using nearest-neighbor thermodynamics. 'Nearest neighbor' is a model where oligonucleotides are treated like sequences of dinucleotides with experimentally derived thermodynamic parameters for each nucleotide pair.

The maximum allowed adjsuted Tm, default is 47° C, calculated using nearest-neighbor thermodynamics. 'Nearest neighbor' is a model where oligonucleotides are treated like sequences of dinucleotides with experimentally derived thermodynamic parameters for each nucleotide pair.

A list of sequences separated by commas and with no spaces that will be avoided, if present in the FASTA-formatted input, during the designing of probes. Default list is: 'AAAAA,TTTTT,CCCCC,GGGGG'.

The minimum spacing between adjacent probes, default is 0 bases. This does not necessary mean that the probes will be separated exactly by the set value but that they will never be more closer than it.

The mM Na+ (salt) concentration that will be used experimentally for the hybridization step of FISH, default is 390 mM.

The percent formamide concentration that will be used experimentally for the hybridization step of FISH, default is 50%.

The nearest neighbor table of thermodynamic parameters to be used for melting temperature calculation. Default is DNANN3 that is suitable for DNA FISH experiments. Switch to RNANN3 if you plan to perform RNA FISH. Please remind that the Tm displayed on the resulting probes set is calculated again during the "Linear Discriminant Analysis (LDA)" step using DNANN3 thermodynamic table, thus it will systematically differ from the Tm ranges previosly set if using RNANN3 thermodynamic table. It is suggested to always use DNANN3 thermodynamic table also for RNA/RNA FISH or DNA/RNA FISH experiments.

The reference genome index to be used for the alignement of the probes using NGS aligner "bowtie2". Take into account that using FASTA-formatted sequences coming from species too far from the selected reference genome index could not produce any suitable results.

Specify the temperature-specific linear discrimination model to use in LDA. Default value is 42° C. Select the temperature model closer to your experimental FISH condition (hybridization temperature).

The probability threshold for classifying a candidate sequence as likely to have off-target binding using the LDA model. Default value is 0.5 meaning that the LDA will filter those probes with a probability of duplexing non-target sites higher than 50%. Selecting a smaller values will improve precision (fewer false positives), but at the expense of recall (more false negatives). Selecting larger values will improve recall at the expense of precision.

The length, in base pairs, of the kmers to be searched inside the probes sequence. Choose the correct kmer length basing also on the probes mean length. Default value is 18 bp.

Filter probes with off-target kmers occurring at least the specified number of time. In default mode if a probes has 5 or more kmer that binds an off target sequence then the entire probe will be discarded.

This is the temperature at which the probes will be hybridized. The default value is 47 °C.

The probability threshold for a probe having a linear structure to use for discarding. Default value is 0.1. With default setting all the probes that have a 10% probability or more to have a linear structure will be saved.

The mM Na+ concentration at which probes will be hybridized, default value is 390 mM. Take into accounts that NUPACK's allowable range of Na+ concentration is 50-1100 mM

The percent formamide at which probes will be hybridized, default value is 50%.

The mM Na+ concentration of the new experimental condition, default value is 390 mM.

The percent formamide of the new experimental condition, default value is 50%.

The final probes orientation will be identical to the selected genomic strand. Choosing plus(+) will give you probes with sequence identical to the plus(+) strand of your submitted FASTA-formatted sequence. On the contrary, selecting minus(-), will give you probes with sequence identical to the minus(-) strand of your submitted FASTA-formatted sequence.