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Introduction

CopomuS evaluates and ranks possible compensatory point mutations within IntaRNA mfe predictions to guide respective validation experiments.

When using CopomuS please cite :

Results are computed with CopomuS version part of IntaRNA package (check IntaRNA version information)

Overview

The following parameters are used to control the execution of CopomuS

Furthermore, additional information is available

Sequence Parameters

?  RNA-1

An RNA sequence where ambiguous nucleotides ('N') are not considered for base pairing.
The parameter constraints are: RNA/DNA sequence composed of 'ACGTUN' base encoding. String length has to be in range (7,2000) for each line. Maximally 1 line is allowed.
Defaults to ()

?  Index first position of RNA-1

Index of first sequence position in RNA-1. If not present, it defaults to 1. Setting to a negative number X will result in an indexing 'X,..,-1,+1,..(length+X)' omitting '0', as eg used for position information relative to an mRNA start codon.
The parameter constraints are: Input value has to be parsable as Integer. The value must be greater than or equal to -9999999 and must be smaller than or equal to 9999999.
Defaults to (1)

?  RNA-2

An RNA sequence where ambiguous nucleotides ('N') are not considered for base pairing.
The parameter constraints are: RNA/DNA sequence composed of 'ACGTUN' base encoding. String length has to be in range (7,2000) for each line. Maximally 1 line is allowed.
Defaults to ()

?  Index first position of RNA-2

Index of first sequence position in RNA-2. If not present, it defaults to 1. Setting to a negative number X will result in an indexing 'X,..,-1,+1,..(length+X)' omitting '0', as eg used for position information relative to an mRNA start codon.
The parameter constraints are: Input value has to be parsable as Integer. The value must be greater than or equal to -9999999 and must be smaller than or equal to 9999999.
Defaults to (1)

Mutation Parameters

?  Mutation candidates

Defines how candidate base pairs are identified. (mfe) only base pairs from the predicted mfe interaction are considered. (mfeSO) base pairs from the mfe and from suboptimal interactions within the mfe boundaries are taken into account.

?  Ignore AU base pairs

Do not consider mutation of AU base pairs
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (true)

?  Ignore GU base pairs

Do not consider mutation of GU base pairs
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (true)

?  Ignore GC base pairs

Do not consider mutation of GC base pairs
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (false)

?  Ignore lonely base pairs

Do not consider lonely base pairs, i.e. base pairs that can not stack to either side. Note: base pairs not filtered are not necessarily stacked in mfe interaction!
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (true)

?  Ignore helix ends

Do not consider base pairs for which no stacking is possible on both sides. Note: base pairs not filtered are not necessarily stacked on both sides in mfe interaction!
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (true)

?  Ignore helix ends of mfe

Do not consider base pairs for which no stacking is reported on both sides within mfe interaction!
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (false)

?  Mutations to generate

What bases are to be considered for mutations. (flip) only the reverse base pair is considered. (any) any non-complementary base pair is evaluated.

Evaluation Parameters

?  Classification combination

The ranking of considered mutations are identified based on a hierachical classification and evaluation using various measures. (mfeCover) since we are assuming IntaRNA predictions are correct, we enforce as a first filter that the mutation is also within the mfe boundaries for the mutated sequences. (E) enforces a destabilization of wildtype-mutant combinations compared to both mfe(wt) and mfe(mutants) (ie. both have to be exceeded by some internal energy threshold x)

?  Sorting within final classes

The final classes are each sorted by one of the following measures. (minDeltaE) defines enables sorting per class and is always done as the final step.

IntaRNA Parameters

?  Max. interaction length

Output only interactions that involve subsequences of lengths up to the given threshold. Set 0 or leave free to disable the constraint.
The parameter constraints are: Input value has to be parsable as Integer. The value must be greater than or equal to 0.
Defaults to ()

?  Max. absolute energy of an interaction

Output only interactions with an energy below or equal to this energy in kcal/mol. Note, if the minimum free energy (mfe) of any interaction is above this threshold, no interaction will be reported.
The parameter constraints are: Input value has to be parsable as Double. The value must be smaller than or equal to 0.
Defaults to (0)

?  No lonely base pairs

If enabled, no unstacked (lonely) base pairs are considered for prediction.
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (true)

?  No GU at helix ends

If enabled, no GU base pair is allowed at helix ends (ie. interaction ends or within loops).
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (true)

?  Min. number of basepairs in seed

Minimal number of intermolecular base pairs in the seed region. Note, for webserver use this value is restricted.
The parameter constraints are: Input value has to be parsable as Integer. The value must be greater than or equal to 2 and must be smaller than or equal to 12. Has to be greater than the number of unpaired bases within the seed (for webserver use).
Defaults to (7)

?  Max. Number of mismatches in seed

Maximal number of unpaired bases in the seed region in both sequences. Note, for webserver use this value is restricted.
The parameter constraints are: Input value has to be parsable as Integer. The value must be greater than or equal to 0 and must be smaller than or equal to 20.
Defaults to (0)

?  Maximal energy

Maximal overall interaction energy of the seed interaction to be considered for further interaction prediction. Note, a value too low will discard all seeds and thus result in no predicted interaction. A value too high will cause weaken the seed constraint.
The parameter constraints are: Input value has to be parsable as Double. The value must be greater than or equal to -999 and must be smaller than or equal to 999.
Defaults to (0)

?  Minimal unpaired probability (per RNA)

The minimal unpaired probabilitiy of the seed interaction sites (checked independently for query and target seed site) to be considered for further interaction prediction. Note, a value too high will discard all seeds and thus result in no predicted interaction.
The parameter constraints are: Input value has to be parsable as Double. The value must be greater than or equal to 0 and must be smaller than or equal to 1.
Defaults to (0)

?  Seed position (RNA-1)

Seed search and position is constrained to this region of RNA-1. The start and the end position of the region have to be given in 5' to 3' direction of the RNA starting from position 1 (or the user defined first index).
The parameter constraints are: Has to be in the format 'FROM-TO' to give the coordinates FROM where TO where the seed is to be found (index counting starts with 1).
Defaults to ()

?  Seed position (RNA-2)

Seed search and position is constrained to this region of RNA-2. The start and the end position of the region have to be given in 5' to 3' direction of the RNA starting from position 1 (or the user defined first index).
The parameter constraints are: Has to be in the format 'FROM-TO' to give the coordinates FROM where TO where the seed is to be found (index counting starts with 1).
Defaults to ()

?  Ignore seeds with GU base pairs

If enabled, no seeds containing GU base pairs are considered for prediction.
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (false)

?  Ignore seeds with GU ends

If enabled, no seeds with GU base pairs at their ends are considered for prediction.
The parameter constraints are: Input value has to be parsable as Boolean.
Defaults to (true)

?  Temperature for energy computation

Temperature in degrees Celsius used to rescale energy parameters.
The parameter constraints are: Input value has to be parsable as Double. The value must be greater than 0.
Defaults to (37.0)

?  Access. RNA-1: folding window size

Size of the averaging window in the local query ncRNA folding (RNAplfold -W) for the computation of accessibilities.
Local folding is key to reasonable folding results when facing larger RNA molecules, since it minimizes effects of incorrect long-range predictions (see local folding article).
Note, the folding window size should be about 50nt higher than the max. basepair distance.
If set to 0, no sliding window is used and the full sequence length is considered. The same holds, if the value is larger than the sequence length.
The parameter constraints are: Input value has to be parsable as Integer. The value must be greater than or equal to 0. The window size has to be at least as large as the maximal basepair distance.
Defaults to (150)

?  Access. RNA-1: max. basepair distance

Maximal distance of two paired bases in the local query ncRNA folding (RNAplfold -L) for computation of accessibilities.
Local folding is key to reasonable folding results when facing larger RNA molecules, since it minimizes effects of incorrect long-range predictions (see local folding article).
Note, max. basepair distance should be about 50nt less than the the folding window size.
If set to 0, the sliding window size value is also used for base pair span restrictions. The same holds, if the value is larger than the sequence length.
The parameter constraints are: Input value has to be parsable as Integer. The value must be greater than or equal to 0.
Defaults to (100)

?  Access. RNA-2: folding window size

Size of the averaging window in the local target RNA folding (RNAplfold -W) for the computation of accessibilities.
Local folding is key to reasonable folding results when facing larger RNA molecules, since it minimizes effects of incorrect long-range predictions (see local folding article).
Note, the folding window size should be about 50nt higher than the max. basepair distance.
If set to 0, no sliding window is used and the full sequence length is considered.
The parameter constraints are: Input value has to be parsable as Integer. The value must be greater than or equal to 0. The window size has to be at least as large as the maximal basepair distance.
Defaults to (150)

?  Access. RNA-2: max. basepair distance

Maximal distance of two paired bases in the local target RNA folding (RNAplfold -L) for computation of accessibilities.
Local folding is key to reasonable folding results when facing larger RNA molecules, since it minimizes effects of incorrect long-range predictions (see local folding article).
Note, max. basepair distance should be about 50nt less than the the folding window size.
If set to 0, the sliding window size value is also used for base pair span restrictions.
The parameter constraints are: Input value has to be parsable as Integer. The value must be greater than or equal to 0.
Defaults to (100)

?  Energy parameter set (Vienna package)

Defines what energy parameter set to be used within the Vienna RNA package to compute base pair probabilities, i.e. dot plots, for each input sequence. The parameter sets are provided by the Vienna RNA package (see version information) and are named according to the first author and year of the corresponding publication.

Output Description

Result table

CopomuS provides for all tested mutations the respective mutation encoding along with the mutation details. For each enabled classification measure, the resulting two-class assignment is given, i.e. '1' if the the mutation fulfills criteria of the classifier or '0' otherwise. The overall rank is generated by the selected final sorting measure while considering the classification. For detailed investigations, we also provide the minimum free energy values for the wildtyp (w) and mutated (m) sequences provided by IntaRNA. The full result table is available for download.

Interaction visualization

The ASCII visualization shows the mfe interaction of the wildtype sequences as reported by IntaRNA. It allows to localize the suggested mutations and to study their sequence and interaction context. Base pairs of potential seed interactions are marked by '+', while GU base pairs are highlighted with ':'.

Input Examples

?  RybB-tsx (Papenfort et al., 2010)

Verification of RybB-tsx interaction in Salmonella by Papenfort et al. (2010) was originally using C2G&G-8C. (note, here we use tsx sequence = genomic region [-200,+100] around start codon)
The example's result can be directly accessed here

List of Changes

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