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RNASUBOPT(1) User Commands RNASUBOPT(1) NAME RNAsubopt - manual page for RNAsubopt 2.7.2 SYNOPSIS RNAsubopt [OPTION]... DESCRIPTION RNAsubopt 2.7.2 calculate suboptimal secondary structures of RNAs Reads RNA sequences from stdin and (in the default -e mode) calculates all suboptimal secondary structures within a user defined energy range above the minimum free energy (mfe). It prints the suboptimal structures in dot-bracket notation followed by the energy in kcal/mol to stdout. Be care- ful, the number of structures returned grows exponentially with both se- quence length and energy range. Alternatively, when used with the -p option, RNAsubopt produces Boltzmann weighted samples of secondary structures. -h, --help Print help and exit --detailed-help Print help, including all details and hidden options, and exit --full-help Print help, including hidden options, and exit -V, --version Print version and exit -v, --verbose Be verbose. (default=off) Lower the log level setting such that even INFO messages are passed through. I/O Options: Command line options for input and output (pre-)processing -i, --infile=filename Read a file instead of reading from stdin. The default behavior of RNAsubopt is to read input from stdin. Using this parameter the user can specify an input file name where data is read from. -o, --outfile[=filename] Print output to file instead of stdout. This option may be used to write all output to output files rather than printing to stdout. The default filename is "RNAsubopt_out- put.sub" if no FASTA header precedes the input sequences and the --auto-id feature is inactive. Otherwise, output files with the scheme "prefix.sub" are generated, where the "prefix" is taken from the sequence id. The user may specify a single output file name for all data generated from the input by supplying an optional string as argument to this parameter. In case a file with the same filename already exists, any output of the program will be appended to it. Note: Any special characters in the filename will be replaced by the filename delimiter, hence there is no way to pass an entire direc- tory path through this option yet. (See also the "--filename-delim" parameter) --noconv Do not automatically substitute nucleotide "T" with "U". (default=off) --auto-id Automatically generate an ID for each sequence. (default=off) The default mode of RNAsubopt is to automatically determine an ID from the input sequence data if the input file format allows to do that. Sequence IDs are usually given in the FASTA header of input sequences. If this flag is active, RNAsubopt ignores any IDs re- trieved from the input and automatically generates an ID for each sequence. This ID consists of a prefix and an increasing number. This flag can also be used to add a FASTA header to the output even if the input has none. --id-prefix=STRING Prefix for automatically generated IDs (as used in output file names). (default=`sequence') If this parameter is set, each sequences' FASTA id will be prefixed with the provided string. FASTA ids then take the form ">pre- fix_xxxx" where xxxx is the sequence number. Note: Setting this pa- rameter implies --auto-id. --id-delim=CHAR Change the delimiter between prefix and increasing number for auto- matically generated IDs (as used in output file names). (default=`_') This parameter can be used to change the default delimiter "_" be- tween the prefix string and the increasing number for automatically generated ID. --id-digits=INT Specify the number of digits of the counter in automatically gener- ated alignment IDs. (default=`4') When alignments IDs are automatically generated, they receive an in- creasing number, starting with 1. This number will always be left-padded by leading zeros, such that the number takes up a cer- tain width. Using this parameter, the width can be specified to the users need. We allow numbers in the range [1:18]. This option im- plies --auto-id. --id-start=LONG Specify the first number in automatically generated IDs. (default=`1') When sequence IDs are automatically generated, they receive an in- creasing number, usually starting with 1. Using this parameter, the first number can be specified to the users requirements. Note: nega- tive numbers are not allowed. Note: Setting this parameter implies to ignore any IDs retrieved from the input data, i.e. it activates the --auto-id flag. --filename-delim=CHAR Change the delimiting character used in sanitized filenames. (default=`ID-delimiter') This parameter can be used to change the delimiting character used while sanitizing filenames, i.e. replacing invalid characters. Note, that the default delimiter ALWAYS is the first character of the "ID delimiter" as supplied through the --id-delim option. If the delim- iter is a whitespace character or empty, invalid characters will be simply removed rather than substituted. Currently, we regard the following characters as illegal for use in filenames: backslash '\', slash '/', question mark '?', percent sign '%', asterisk '*', colon ':', pipe symbol '|', double quote '"', triangular brackets '<' and '>'. --filename-full Use full FASTA header to create filenames. (default=off) This parameter can be used to deactivate the default behavior of limiting output filenames to the first word of the sequence ID. Con- sider the following example: An input with FASTA header '>NM_0001 Homo Sapiens some gene' usually produces output files with the pre- fix "NM_0001" without the additional data available in the FASTA header, e.g. "NM_0001.sub". With this flag set, no truncation of the output filenames is performed, i.e. output filenames receive the full FASTA header data as prefixes. Note, however, that invalid characters (such as whitespace) will be substituted by a delimiting character or simply removed, (see also the parameter option --file- name-delim). --log-level=level Set log level threshold. (default=`2') By default, any log messages are filtered such that only warnings (level 2) or errors (level 3) are printed. This setting allows for specifying the log level threshold, where higher values result in fewer information. Log-level 5 turns off all messages, even errors and other critical information. --log-file[=filename] Print log messages to a file instead of stderr. (default=`RNAsub- opt.log') --log-time Include time stamp in log messages. (default=off) --log-call Include file and line of log calling function. (default=off) Algorithms: Select the algorithms which should be applied to the given RNA se- quence(s). -e, --deltaEnergy=range Compute suboptimal structures with energy in a certain range of the optimum (kcal/mol). Default is calculation of mfe structure only. --deltaEnergyPost=range Only print structures with energy within range of the mfe after post reevaluation of energies. Useful in conjunction with -logML, -d1 or -d3: while the -e option specifies the range before energies are re-evaluated, this option specifies the maximum energy after re-evaluation. -s, --sorted Sort the suboptimal structures by energy and lexicographical order. (default=off) Structures are first sorted by energy in ascending order. Within groups of the same energy, structures are then sorted in ascending in lexicographical order of their dot-bracket notation. See the --en-only flag to deactivate this second step. Note that sorting is done in memory, thus it can easily lead to exhaution of RAM! This is especially true if the number of structures produced becomes large or the RNA sequence is rather long. In such cases better use an ex- ternal sort method, such as UNIX "sort". --en-only Only sort structures by free energy. (default=off) In combination with --sorted, this flag deactivates the second sort- ing criteria and sorts structures solely by their free energy in- stead of additionally sorting by lexicographic order in each energy band. This might save some time during the sorting process in situa- tions where lexicographic order is not required. -p, --stochBT=number Randomly draw structures according to their probability in the Boltzmann ensemble. Instead of producing all suboptimals in an energy range, produce a random sample of suboptimal structures, drawn with probabilities equal to their Boltzmann weights via stochastic backtracking in the partition function. The -e and -p options are mutually exclusive. --stochBT_en=number Same as "--stochBT" but also print free energies and probabilities of the backtraced structures. --random-seed=INT Set the seed for the random number generator --betaScale=DOUBLE Set the scaling of the Boltzmann factors. (default=`1.') The argument provided with this option is used to scale the thermo- dynamic temperature in the Boltzmann factors independently from the temperature of the individual loop energy contributions. The Boltz- mann factors then become 'exp(- dG/(kT*betaScale))' where 'k' is the Boltzmann constant, 'dG' the free energy contribution of the state and 'T' the absolute temperature. -N, --nonRedundant Enable non-redundant sampling strategy. (default=off) -S, --pfScale=DOUBLE In the calculation of the pf use scale*mfe as an estimate for the ensemble free energy (used to avoid overflows). (default=`1.07') The default is 1.07, useful values are 1.0 to 1.2. Occasionally needed for long sequences. -c, --circ Assume a circular (instead of linear) RNA molecule. (default=off) -D, --dos Compute density of states instead of secondary structures. (default=off) This option enables the evaluation of the number of secondary struc- tures in certain energy bands around the MFE. -z, --zuker Compute Zuker suboptimals instead of all suboptimal structures within an energy band around the MFE. (default=off) -g, --gquad Incoorporate G-Quadruplex formation. (default=off) No support of G-quadruplex prediction for stochastic backtracking and Zuker-style suboptimals yet). Structure Constraints: Command line options to interact with the structure constraints fea- ture of this program --maxBPspan=INT Set the maximum base pair span. (default=`-1') -C, --constraint[=filename] Calculate structures subject to constraints. (default=`') The program reads first the sequence, then a string containing con- straints on the structure encoded with the symbols: '.' (no constraint for this base) '|' (the corresponding base has to be paired 'x' (the base is unpaired) '<' (base i is paired with a base j>i) '>' (base i is paired with a base j<i) and matching brackets '(' ')' (base i pairs base j) With the exception of '|', constraints will disallow all pairs con- flicting with the constraint. This is usually sufficient to enforce the constraint, but occasionally a base may stay unpaired in spite of constraints. PF folding ignores constraints of type '|'. --batch Use constraints for multiple sequences. (default=off) Usually, constraints provided from input file only apply to a single input sequence. Therefore, RNAsubopt will stop its computation and quit after the first input sequence was processed. Using this switch, RNAsubopt processes multiple input sequences and applies the same provided constraints to each of them. --canonicalBPonly Remove non-canonical base pairs from the structure constraint. (default=off) --enforceConstraint Enforce base pairs given by round brackets '(' ')' in structure con- straint. (default=off) --commands=filename Read additional commands from file Commands include hard and soft constraints, but also structure mo- tifs in hairpin and internal loops that need to be treeted differ- ently. Furthermore, commands can be set for unstructured and struc- tured domains. Experimental Structure Probing Data: The following arguments and siwtches control various implementations that allow for guiding the structure prediction with the help of ad- ditional (experimental) RNA structure probing data, such as SHAPE, DMS, etc. --sp-data=filename Read structure probing data from an input file and guide the predic- tions accordingly. Must precede the strategy, i.e. a data file must be specified before the corresponding --sp-strategy option! This option and its argument only specifies the file name with the (reactivity) data. What kind of data the file contains and how the data is actually used during the predictions can be specified by the --sp-strategy and --sp-preprocess options. --sp-strategy=strategy Select the strategy how the probing data is used to guide the struc- ture predictions. (default=`D') The following strategies (methods) are available to convert struc- ture probing data into pseudo energy contributions: 'D': Deigan et al 2009 strategy. Here, probing data is converted into pseudo energy terms using a linear model. The derived energies are then applied for every nucleotide involved in a stacked pair. The default slope and intercept vari- ables of the linear model are 'm=1.8' and 'b=-0.6', respectively. To change these parameters, e.g. to 'm=1.9' and 'b=-0.7', use a parame- ter string like this: --sp-strategy="Dm1.9b-0.7". Note the prece- dence of 'm' over 'b'. You may also provide only one of the two variables like: --sp-strategy="Dm1.9" or --sp-strategy="Db-0.7". 'E': Eddy 2014 strategy. This strategy distinguishes two structural states (classes) for each base, paired and unpaired. Based on a list of prior distributions for each of the classes, the posterior probability for observing a probing reactivity of a base is estimated and then converted into a respec- tive pseudo energy contribution. Thermodynamic temperature T is taken from the model settings as specified by --temp but can be changed to any other value, either in degrees Celcius ('c' or 't') or Kelvin ('k'). Use one of the following parameter strings to change the temperature to 25 degree Celcius: --sp-strategy="Ec=25" or --sp-strategy="Ek=298.15". By default, built-in prior distribu- tions are used with this strategy. This can be changed if the user passes additional probing data files via the --sp-data option and then selects the prior distribution specifier ('P') as strategy (see below). 'P': Use probing data as prior distribution for Eddy 2014 strategy. This indicates that the most recent probing data file actually con- tains a prior distribution of probing data for a particular structure class, i.e. unpaired or paired bases. The prior distribution then replaces the default priors in the Eddy 2014 ('E') strategy. Use the lower case characters 'u' and 'p' to indicate which class, unpaired or paired, the prior distribution represents, e.g. --sp-strategy="Pp" will be interpreted as prior distribution for paired nucleotides. Note, that this option must follow after the --sp-strategy="E" and before any further --sp-strategy options that do not specify other prior distributions! 'Z': Zarringhalam et al 2012 strategy. Here, the probing data is first converted into pairing probabilities and aberration from the observed pairing probabilities will be penalized during the folding recursion. The magnitude of the penalties can af- fected by adjusting the factor beta (e.g. --sp-strategy="Zb0.8"). 'W': Apply a given vector of perturbation energies to unpaired nu- cleotides according to Washietl et al 2012. Perturbation vectors can be calcu- lated by using RNApvmin. --sp-preprocess=method Select a method to pre-process the structure probing data before it undergoes conversion by a strategy. (default=`I') --shape=filename Use SHAPE reactivity data to guide structure predictions. --shapeMethod=method Select SHAPE reactivity data incorporation strategy. (default=`D') The following methods can be used to convert SHAPE reactivities into pseudo energy contributions. 'D': Convert by using the linear equation according to Deigan et al 2009. Derived pseudo energy terms will be applied for every nucleotide in- volved in a stacked pair. This method is recognized by a capital 'D' in the provided parameter, i.e.: --shapeMethod="D" is the default setting. The slope 'm' and the intercept 'b' can be set to a non-de- fault value if necessary, otherwise m=1.8 and b=-0.6. To alter these parameters, e.g. m=1.9 and b=-0.7, use a parameter string like this: --shapeMethod="Dm1.9b-0.7". You may also provide only one of the two parameters like: --shapeMethod="Dm1.9" or --shapeMethod="Db-0.7". 'Z': Convert SHAPE reactivities to pseudo energies according to Zarringhalam et al 2012. SHAPE reactivities will be converted to pairing proba- bilities by using linear mapping. Aberration from the observed pair- ing probabilities will be penalized during the folding recursion. The magnitude of the penalties can affected by adjusting the factor beta (e.g. --shapeMethod="Zb0.8"). 'W': Apply a given vector of perturbation energies to unpaired nu- cleotides according to Washietl et al 2012. Perturbation vectors can be calcu- lated by using RNApvmin. --shapeConversion=method Select method for SHAPE reactivity conversion. (default=`O') This parameter is useful when dealing with the SHAPE incorporation according to Zarringhalam et al. The following methods can be used to convert SHAPE reactivities into the probability for a certain nu- cleotide to be unpaired. 'M': Use linear mapping according to Zarringhalam et al. 'C': Use a cutoff-approach to divide into paired and unpaired nucleotides (e.g. "C0.25") 'S': Skip the normalizing step since the input data already represents probabilities for being unpaired rather than raw reactiv- ity values 'L': Use a linear model to convert the reactivity into a probability for being unpaired (e.g. "Ls0.68i0.2" to use a slope of 0.68 and an intercept of 0.2) 'O': Use a linear model to convert the log of the reactivity into a probability for being unpaired (e.g. "Os1.6i-2.29" to use a slope of 1.6 and an intercept of -2.29) Energy Parameters: Energy parameter sets can be adapted or loaded from user-provided input files -T, --temp=DOUBLE Rescale energy parameters to a temperature of temp C. Default is 37C. (default=`37.0') -P, --paramFile=paramfile Read energy parameters from paramfile, instead of using the default parameter set. Different sets of energy parameters for RNA and DNA should accompany your distribution. See the RNAlib documentation for details on the file format. The placeholder file name 'DNA' can be used to load DNA parameters without the need to actually specify any input file. -4, --noTetra Do not include special tabulated stabilizing energies for tri-, tetra- and hexaloop hairpins. (default=off) Mostly for testing. --salt=DOUBLE Set salt concentration in molar (M). Default is 1.021M. -m, --modifications[=STRING] Allow for modified bases within the RNA sequence string. (default=`7I6P9D') Treat modified bases within the RNA sequence differently, i.e. use corresponding energy corrections and/or pairing partner rules if available. For that, the modified bases in the input sequence must be marked by their corresponding one-letter code. If no additional arguments are supplied, all available corrections are performed. Otherwise, the user may limit the modifications to a particular sub- set of modifications, resp. one-letter codes, e.g. -mP6 will only correct for pseudouridine and m6A bases. Currently supported one-letter codes and energy corrections are: '7': 7-deaza-adenonsine (7DA) 'I': Inosine '6': N6-methyladenosine (m6A) 'P': Pseudouridine '9': Purine (a.k.a. nebularine) 'D': Dihydrouridine --mod-file=STRING Use additional modified base data from JSON file. Model Details: Tweak the energy model and pairing rules additionally using the fol- lowing parameters -d, --dangles=INT How to treat "dangling end" energies for bases adjacent to helices in free ends and multi-loops. (default=`2') With -d1 only unpaired bases can participate in at most one dangling end. With -d2 this check is ignored, dangling energies will be added for the bases adjacent to a helix on both sides in any case; this is the default for mfe and partition function folding (-p). The option -d0 ignores dangling ends altogether (mostly for debug- ging). With -d3 mfe folding will allow coaxial stacking of adjacent helices in multi-loops. At the moment the implementation will not allow coaxial stacking of the two enclosed pairs in a loop of degree 3 and works only for mfe folding. Note that with -d1 and -d3 only the MFE computations will be using this setting while partition function uses -d2 setting, i.e. dan- gling ends will be treated differently. --noLP Produce structures without lonely pairs (helices of length 1). (default=off) For partition function folding this only disallows pairs that can only occur isolated. Other pairs may still occasionally occur as he- lices of length 1. --noGU Do not allow GU pairs. (default=off) --noClosingGU Do not allow GU pairs at the end of helices. (default=off) --logML Recompute energies of structures using a logarithmic energy function for multi-loops before output. (default=off) This option does not effect structure generation, only the energies that are printed out. Since logML lowers energies somewhat, some structures may be missing. --nsp=STRING Allow other pairs in addition to the usual AU,GC,and GU pairs. Its argument is a comma separated list of additionally allowed pairs. If the first character is a "-" then AB will imply that AB and BA are allowed pairs, e.g. --nsp="-GA" will allow GA and AG pairs. Nonstandard pairs are given 0 stacking energy. --energyModel=INT Set energy model. Rarely used option to fold sequences from the artificial ABCD... al- phabet, where A pairs B, C-D etc. Use the energy parameters for GC (--energyModel 1) or AU (--energyModel 2) pairs. --helical-rise=FLOAT Set the helical rise of the helix in units of Angstrom. (default=`2.8') Use with caution! This value will be re-set automatically to 3.4 in case DNA parameters are loaded via -P DNA and no further value is provided. --backbone-length=FLOAT Set the average backbone length for looped regions in units of Angstrom. (default=`6.0') Use with caution! This value will be re-set automatically to 6.76 in case DNA parameters are loaded via -P DNA and no further value is provided. REFERENCES If you use this program in your work you might want to cite: R. Lorenz, S.H. Bernhart, C. Hoener zu Siederdissen, H. Tafer, C. Flamm, P.F. Stadler and I.L. Hofacker (2011), "ViennaRNA Package 2.0", Algorithms for Molecular Biology: 6:26 I.L. Hofacker, W. Fontana, P.F. Stadler, S. Bonhoeffer, M. Tacker, P. Schuster (1994), "Fast Folding and Comparison of RNA Secondary Structures", Monatshefte f. Chemie: 125, pp 167-188 R. Lorenz, I.L. Hofacker, P.F. Stadler (2016), "RNA folding with hard and soft constraints", Algorithms for Molecular Biology 11:1 pp 1-13 S. Wuchty, W. Fontana, I. L. Hofacker and P. Schuster (1999), "Complete Suboptimal Folding of RNA and the Stability of Secondary Structures", Biopolymers: 49, pp 145-165 M. Zuker (1989), "On Finding All Suboptimal Foldings of an RNA Molecule", Science 244.4900, pp 48-52 Y. Ding, and C.E. Lawrence (2003), "A statistical sampling algorithm for RNA secondary structure prediction", Nucleic Acids Research 31.24, pp 7280-7301 The energy parameters are taken from: D.H. Mathews, M.D. Disney, D. Matthew, J.L. Childs, S.J. Schroeder, J. Su- san, M. Zuker, D.H. Turner (2004), "Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA sec- ondary structure", Proc. Natl. Acad. Sci. USA: 101, pp 7287-7292 D.H Turner, D.H. Mathews (2009), "NNDB: The nearest neighbor parameter database for predicting stability of nucleic acid secondary structure", Nu- cleic Acids Research: 38, pp 280-282 AUTHOR Ivo L Hofacker, Stefan Wuchty, Walter Fontana, Ronny Lorenz REPORTING BUGS If in doubt our program is right, nature is at fault. Comments should be sent to rna@tbi.univie.ac.at. RNAsubopt 2.7.2 December 2025 RNASUBOPT(1)
NAME | SYNOPSIS | DESCRIPTION | REFERENCES | AUTHOR | REPORTING BUGS
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