This contains one or more sequences that are used by RNAhybrid to hybridize the miRNA(s) on. RNAhybrid uses all this sequences to find minimal free energy hybridisations between miRNA(s) and target sequence(s). Sequences should be in RNA.fasta format but RNAhybrid can also use DNA.fasta files. A single Sequences one can use can contain up to 50000 basepairs.

contains one or more micro RNA(s) that RNAhybrid uses to hybridize with the RNA sequences and to find the minimal free energy hybridization. A single micro RNA sequence can contain up to 2000 basepairs.

RNAhybrid computes the minimal free energy hybridization's the results are shown by increasing energy. By using the parameters given by bibiserv2 you can specialize your results the way you want them to be.

Usage

Sequences data

The first step of using RNAhybrid is to choose which Sequences you want to use. There are two windows that you use for your submission. The first window is for the DNA/RNA sequence(s) and the second for the miRNA(s) you want to hybridize. RNAhybrid will map the miRNA(s) on the DNA/RNA sequence(s). Its your choice whether you use a file or you want to copy and paste the input. The server also provides an example, this example uses a bacterial sequence and a miRNA of the same bacteria. and can be used to learn how this tool is working. After submitting your sequence data you go on to step 2.

Parameters

In the second step you choose the parameter set you want to use for your analyses. RNAhybrid provides you with a lot of useful parameters. The first parameter is named "hits per target" it is used to calibrate how many hybridisations you get to see as output. This is competing to the "energy threshold" parameter, here you choose the maximal MFE (minimum free energy) hybridisations that RNAhybrid displays, of course if you have only displays the output that has less hybridisations. For example if you choose a "energy threshold" of -20 and 30 "hits per target" RNAhybrid will not display 30 hits if their are only 10 hits with less or equal then the energy threshold.

You can decide if you want an approximate estimate of p-values for the target sequence therefore RNAhybrid lets you choose between three sequence groups (3utf_fly, 3utf_human, 3utf_worm). RNAhybrid can calculate better results with this p-values if you search in sequences of the given groups. Otherwise if you don't want to use these sequences you have the opportunity to use the "helix constraint" parameter. This parameter has a "from" and a "to" field here you can say that your structure should have a helix between those two parameters. This starts counting by one on your target sequence. When using this parameter you are also allowed to use the "no G:U in seed" parameter. Using this RNAhybrid tries not to calibrate structures that have G:U bindings in the seed.

Now its possible to confine special loop structures. The first possibility is given with the "max internal loop size" parameter. Depending on how you choose the length of the loop RNAhybrid displays only structures with this as the maximal internal loop length. For example use 0 as "max internal loop size" and you get structures with no internal loops at all. Internal loops are loops with both strands having no binding side, whereas bulge loops are loops where only one strand has no binding. As said before the other opportunity is using the "max bulge loop size". Basically this parameter is used in the same way as the "max internal loop size" parameter only that it delimits the size of bulge loops instead of internal loops. So if you for example set this parameter to 0 you get structures with only internal loops.

Last but not least there is the "compact output" parameter using this parameter the displayed output is only one line of output for each hit. I would suggest to use this parameter if you only want to know if your results are the same as RNAhybrid would calibrate. By submitting the combination of parameters you want to use RNAhybrid starts calibrating the results.

Get the result(s)

The results are shown in a very specific way. First of all you see the Header which includes the version of RNAhybrid and the Dataset which is shown. Right under the separation line the first result is shown. First of all the target sequence length and the miRNA length are pointed out. Furthermore the minimal-free-energy is shown for the displayed structure, right below this information you can see whether a p-value is used or which p-value is used. Then the position is marked in which this structure could be found on the target sequence, below that position mark RNAhybrid shows how the structure looks like. If you have less or equal to ten results you get a picture of the structure in addition. This picture can be downloaded from the results by clicking on the links. RNAhybrid separates all different Results with a line, so this line is the indicator for going on to the next Result.