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RNALALIFOLD(1) User Commands RNALALIFOLD(1) NAME RNALalifold - manual page for RNALalifold 2.7.2 SYNOPSIS RNALalifold [options] <file1.aln> DESCRIPTION RNALalifold 2.7.2 calculate locally stable secondary structures for a set of aligned RNAs reads aligned RNA sequences from stdin or file.aln and calculates locally stable RNA secondary structure with a maximal base pair span. For a se- quence of length n and a base pair span 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 RNA 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. -q, --quiet Be quiet. (default=off) This option can be used to minimize the output of additional infor- mation and non-severe warnings which otherwise might spam std- out/stderr. I/O Options: Command line options for input and output (pre-)processing -f, --input-format=C|S|F|M File format of the input multiple sequence alignment (MSA). If this parameter is set, the input is considered to be in a partic- ular file format. Otherwise, the program tries to determine the file format automatically, if an input file was provided in the set of parameters. In case the input MSA is provided in interactive mode, or from a terminal (TTY), the programs default is to assume CLUSTALW format. Currently, the following formats are available: ClustalW ('C'), Stockholm 1.0 ('S'), FASTA/Pearson ('F'), and MAF ('M'). --csv Create comma separated output (csv) (default=off) --aln[=prefix] Produce output alignments and secondary structure plots for each hit found. This option tells the program to produce, for each hit, a colored and structure annotated (sub)alignment and secondary structure plot in PostScript format. It also adds the subalignment hit into a multi-Stockholm formatted file "RNALalifold_results.stk". The post- script output file names are "aln_start_end.eps" and "ss_start_end.eps". All files will be created in the current direc- tory. The optional argument string can be used to set a specific prefix that is used to name the output files. The file names then become "prefix_aln_start_end.eps", "prefix_ss_start_end.eps", and "prefix.stk". Note: Any special characters in the prefix will be re- placed by the filename delimiter, hence there is no way to pass an entire directory path through this option yet. (See also the "--filename-delim" parameter) --aln-stk[=prefix] Add hits to a multi-Stockholm formatted output file. (default=`RNALalifold_results') The default file name used for the output is "RNALalifold_re- sults.stk". Users may change the filename to "prefix.stk" by speci- fying the prefix as optional argument. The file will be create in the current directory if it does not already exist. In case the file already exists, output will be appended to it. Note: Any special characters in the prefix will be replaced by the filename delimiter, hence there is no way to pass an entire directory path through this option yet. (See also the "--filename-delim" parameter) --mis Output "most informative sequence" instead of simple consensus: For each column of the alignment output the set of nucleotides with fre- quency greater than average in IUPAC notation. (default=off) --split-contributions Split the free energy contributions into separate parts (default=off) By default, only the total energy contribution for each hit is re- turned. Using this option, this contribution is split into individ- ual parts, i.e. the Nearest Neighbor model energy, the covariance pseudo energy, and if applicable, a remaining pseudo energy derived from special constraints, such as probing signals like SHAPE. --noconv Do not automatically substitute nucleotide "T" with "U". (default=off) --auto-id Automatically generate an ID for each alignment. (default=off) The default mode of RNALalifold is to automatically determine an ID from the input alignment if the input file format allows to do that. Alignment IDs are, for instance, usually given in Stockholm 1.0 for- matted input. If this flag is active, RNALalifold ignores any IDs retrieved from the input and automatically generates an ID for each alignment. --id-prefix=STRING Prefix for automatically generated IDs (as used in output file names). (default=`alignment') If this parameter is set, each alignment will be prefixed with the provided string. Hence, the output files will obey the following naming scheme: "prefix_xxxx_ss.ps" (secondary structure plot), "pre- fix_xxxx_dp.ps" (dot-plot), "prefix_xxxx_aln.ps" (annotated align- ment), etc. where xxxx is the alignment number beginning with the second alignment in the input. Use this setting in conjunction with the --continuous-ids flag to assign IDs beginning with the first in- put alignment. --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]. --id-start=LONG Specify the first number in automatically generated alignment IDs. (default=`1') When alignment 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 continuous alignment IDs, i.e. it activates the --continuous-ids 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 '>'. --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=`RNALali- fold.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 additional algorithms which should be included in the calcu- lations. The Minimum free energy (MFE) and a structure representa- tive are calculated in any case. -L, --maxBPspan=INT Set the maximum allowed separation of a base pair to span. I.e. no pairs (i,j) with j-i>span will be allowed. (default=`70') --threshold=DOUBLE Energy threshold in kcal/mol per nucleotide above which secondary structure hits are omitted in the output. (default=`-0.1') -g, --gquad Incoorporate G-Quadruplex formation into the structure prediction algorithm. (default=off) Structure Constraints: Command line options to interact with the structure constraints fea- ture of this program --shape=file1,file2 Use SHAPE reactivity data to guide structure predictions. Multiple shapefiles for the individual sequences in the alignment may be specified as a comma separated list. An optional association of particular shape files to a specific sequence in the alignment can be expressed by prepending the sequence number to the filename, e.g. "5=seq5.shape,3=seq3.shape" will assign the reactivity values from file seq5.shape to the fifth sequence in the alignment, and the values from file seq3.shape to sequence 3. If no assignment is specified, the reactivity values are assigned to corresponding se- quences in the order they are given. --shapeMethod=D[mX][bY] Specify the method how to convert SHAPE reactivity data to pseudo energy contributions. (default=`D') Currently, the only data conversion method available is that of to Deigan et al 2009. This method is the default and 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-default value if necessary. Otherwise m=1.8 and b=-0.6 as stated in the paper mentionen before. 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". 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. 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) --cfactor=DOUBLE Set the weight of the covariance term in the energy function (default=`1.0') --nfactor=DOUBLE Set the penalty for non-compatible sequences in the covariance term of the energy function (default=`1.0') -R, --ribosum_file=ribosumfile use specified Ribosum Matrix instead of normal energy model. Matrixes to use should be 6x6 matrices, the order of the terms is 'AU', 'CG', 'GC', 'GU', 'UA', 'UG'. -r, --ribosum_scoring use ribosum scoring matrix. (default=off) The matrix is chosen according to the minimal and maximal pairwise identities of the sequences in the file. --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. Plotting: Command line options for changing the default behavior of structure layout and pairing probability plots --aln-EPS[=prefix] Produce colored and structure annotated subalignment for each hit. The default file name used for the output is "aln_start_end.eps" where "start" and "end" denote the first and last column of the sub- alignment relative to the input (1-based). Users may change the filename to "prefix_aln_start_end.eps" by specifying the prefix as optional argument. Files will be create in the current directory. Note: Any special characters in the prefix 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) --aln-EPS-cols=INT Number of columns in colored EPS alignment output. (default=`60') A value less than 1 indicates that the output should not be wrapped at all. --aln-EPS-ss[=prefix] Produce colored consensus secondary structure plots in PostScript format. The default file name used for the output is "ss_start_end.eps" where "start" and "end" denote the first and last column of the sub- alignment relative to the input (1-based). Users may change the filename to "prefix_ss_start_end.eps" by specifying the prefix as optional argument. Files will be create in the current directory. Note: Any special characters in the prefix 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) --color-threshold=FLOAT Set the threshold of maximum counter examples for coloring consensus structure plot. (default=`2') Floating point numbers between 0 and 1 are treated as frequencies among all sequencesin the alignment. All other will be truncated to integer and used as absolute number of counter examples. --color-min-sat=FLOAT Set the minimum saturation for coloring consensus structure plot. (default=`0.2') Floating point number >= 0 and smaller than 1. 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 I.L. Hofacker, B. Priwitzer, and P.F. Stadler (2004), "Prediction of Lo- cally Stable RNA Secondary Structures for Genome-Wide Surveys", Bioinfor- matics: 20, pp 186-190 Stephan H. Bernhart, Ivo L. Hofacker, Sebastian Will, Andreas R. Gruber, and Peter F. Stadler (2008), "RNAalifold: Improved consensus structure pre- diction for RNA alignments", BMC Bioinformatics: 9, pp 474 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, 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. RNALalifold 2.7.2 December 2025 RNALALIFOLD(1)
NAME | SYNOPSIS | DESCRIPTION | REFERENCES | AUTHOR | REPORTING BUGS
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