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RNAPLFOLD(1) User Commands RNAPLFOLD(1) NAME RNAplfold - manual page for RNAplfold 2.7.2 SYNOPSIS RNAplfold [OPTION]... DESCRIPTION RNAplfold 2.7.2 calculate locally stable secondary structure - pair probabilities Computes local pair probabilities for base pairs with a maximal span of L. The probabilities are averaged over all windows of size L that contain the base pair. For a sequence of length n and a window size of L the algorithm uses only O(n+L*L) memory and O(n*L*L) CPU time. Thus it is practical to "scan" very large genomes for short stable RNA structures. Output consists of a dot plot in postscript file, where the averaged pair probabilities can easily be parsed and visually inspected. The -u option makes i possible to compute the probability that a stretch of x consequtive nucleotides is unpaired, which is useful for predicting pos- sible binding sites. Again this probability is averaged over all windows containing the region. WARNING! Output format changed!! The output is a plain text matrix containing on each line a position i fol- lowed by the probability that i is unpaired, [i-1..i] is unpaired [i-2..i] is unpaired and so on to the probability that [i-x+1..i] is unpaired. -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 -c, --cutoff=FLOAT Report only base pairs with an average probability larger than 'cut- off' in the dot plot. (default=`0.01') -o, --print_onthefly Save memory by printing out everything during computation. (default=off) NOTE: activated per default for sequences over 1M bp. -O, --opening_energies Switch output from probabilities to their logarithms. (default=off) This is NOT exactly the mean energies needed to unfold the respec- tive stretch of bases! (implies --ulength option). --plex_output Create additional output files for RNAplex. (default=off) -b, --binaries Output accessibility profiles in binary format. (default=off) The binary files produced by RNAplfold do not need to be parsed by RNAplex, so that they are directly loaded into memory. This is useful when large sequences have to be searched for putative hybridization sites. Another advantage of the binary format is the 50% file size decrease. --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 RNAplfold 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, RNAplfold 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. Hence, the output files will obey the following naming scheme: "prefix_xxxx_dp.ps" (dot-plot), "prefix_xxxx_lunp" (unpaired probabilities), etc. Note: Setting this parameter 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_ss.ps" for secondary structure plots. With this flag set, no truncation of the output filenames is done, 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 --filename-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. (de- fault=`RNAplfold.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 and change parameters of (additional) algorithms which should be included in the calculations. -W, --winsize=size Average the pair probabilities over windows of given size. (default=`70') -L, --span=size Set the maximum allowed separation of a base pair to span. By setting the maximum base pair span no pairs (i,j) with j-i > span will be allowed. Defaults to winsize if parameter is omitted. -u, --ulength=length Compute the mean probability that regions of length 1 to a given length are unpaired. (default=`31') Output is saved in a '_lunp' file. --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. -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. Structure Constraints: Command line options to interact with the structure constraints fea- ture of this program --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) --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. 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 Specify "dangling end" model for bases adjacent to helices in free ends and multi-loops. (default=`2') With -d2 dangling energies will be added for the bases adjacent to a helix on both sides in any case while -d0 ignores dangling ends al- together (mostly for debugging). --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) --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. H. Bernhart, U. Mueckstein, and I.L. Hofacker (2011), "RNA Accessibility in cubic time", Algorithms Mol Biol. 6: 3. S. H. Bernhart, I.L. Hofacker, and P.F. Stadler (2006), "Local Base Pairing Probabilities in Large RNAs", Bioinformatics: 22, pp 614-615 A.F. Bompfuenewerer, R. Backofen, S.H. Bernhart, J. Hertel, I.L. Hofacker, P.F. Stadler, S. Will (2007), "Variations on RNA Folding and Alignment: Lessons from Benasque", J. Math. Biol. 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 Stephan H Bernhart, Ivo L Hofacker, Peter F Stadler, 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. SEE ALSO RNALfold(1) RNAplfold 2.7.2 December 2025 RNAPLFOLD(1)
NAME | SYNOPSIS | DESCRIPTION | REFERENCES | AUTHOR | REPORTING BUGS | SEE ALSO
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