General Options:

Command line options which alter the general behavior of this program

-v, --verbose

Be verbose.

(default=off)

-j, --jobs[=number]

Split batch input into jobs and start processing in parallel using multiple threads. A value of 0 indicates to use as many parallel threads as computation cores are available.

(default=`0')

Default processing of input data is performed in a serial fashion, i.e. one sequence at a time. Using this switch, a user can instead start the computation for many sequences in the input in parallel. RNAfold will create as many parallel computation slots as specified and assigns input sequences of the input file(s) to the available slots. Note, that this increases memory consumption since input alignments have to be kept in memory until an empty compute slot is available and each running job requires its own dynamic programming matrices.

--unordered

Do not try to keep output in order with input while parallel processing is in place.

(default=off)

When parallel input processing (--jobs flag) is enabled, the order in which input is processed depends on the host machines job scheduler. Therefore, any output to stdout or files generated by this program will most likely not follow the order of the corresponding input data set. The default of RNAfold is to use a specialized data structure to still keep the results output in order with the input data. However, this comes with a trade-off in terms of memory consumption, since all output must be kept in memory for as long as no chunks of consecutive, ordered output are available. By setting this flag, RNAfold will not buffer individual results but print them as soon as they have been computated.

-i, --infile=<filename>

Read a file instead of reading from stdin

The default behavior of RNAfold is to read input from stdin or the file(s) that follow(s) the RNAfold command. Using this parameter the user can specify input file names where data is read from. Note, that any additional files supplied to RNAfold are still processed as well.

-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 "RNAfold_output.fold" if no FASTA header precedes the input sequences and the --auto-id feature is inactive. Otherwise, output files with the scheme "prefix.fold" are generated, where the "prefix" is taken from the sequence id, e.g. the FASTA header. The user may specify a single output file name for all data generated from the input by supplying a filename as argument following immediately after 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 directory path through this option (yet). (See also the "--filename-delim" parameter)

-t, --layout-type=INT

Choose the layout algorithm. (default=`1')

Select the layout algorithm that computes the nucleotide coordinates. Currently, the following algorithms are available:

0: simple radial layout

1: Naview layout (Bruccoleri et al. 1988)

2: circular layout

3: RNAturtle (Wiegreffe et al. 2018)

4: RNApuzzler (Wiegreffe et al. 2018)

--noPS

Do not produce postscript drawing of the mfe structure.

(default=off)

--noDP

Do not produce dot-plot postscript file containing base pair or stack probabilitities.

(default=off)

In combination with the -p option, this flag turns-off creation of individual dot-plot files. Consequently, computed base pair probability output is omitted but centroid and MEA structure prediction is still performed.

--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 RNAfold 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, RNAfold ignores any IDs retrieved 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=prefix

Prefix for automatically generated IDs (as used in output file names)

(default=`sequence')

If this parameter is set, each sequence will be prefixed with the provided string. Hence, the output files will obey the following naming scheme: "prefix_xxxx_ss.ps" (secondary structure plot), "prefix_xxxx_dp.ps" (dot-plot), "prefix_xxxx_dp2.ps" (stack probabilities), etc. where xxxx is the sequence number. Note: Setting this parameter implies --auto-id.

--id-delim=delimiter

Change the delimiter between prefix and increasing number for automatically generated IDs (as used in output file names)

(default=`_')

This parameter can be used to change the default delimiter "_" between

the prefix string and the increasing number for automatically generated ID.

--id-digits=INT

Specify the number of digits of the counter in automatically generated alignment IDs.

(default=`4')

When alignments IDs are automatically generated, they receive an increasing number, starting with 1. This number will always be left-padded by leading zeros, such that the number takes up a certain width. Using this parameter, the width can be specified to the users need. We allow numbers in the range [1:18]. This option implies --auto-id.

--id-start=LONG

Specify the first number in automatically generated alignment IDs.

(default=`1')

When sequence IDs are automatically generated, they receive an increasing number, usually starting with 1. Using this parameter, the first number can be specified to the users requirements. Note: negative 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=delimiter

Change the delimiting character that is used

for 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 delimiter 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. Consider the following example: An input with FASTA header ">NM_0001 Homo Sapiens some gene" usually produces output files with the prefix "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).

Structure Constraints:

Command line options to interact with the structure constraints feature 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 constraints 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 conflicting 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, RNAfold will stop its computation and quit after the first input sequence was processed. Using this switch, RNAfold 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 constraint

(default=off)

--shape=<filename>

Use SHAPE reactivity data to guide structure predictions

--shapeMethod=D|Z|W

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 a linear equation according to Deigan et al 2009.

Derived pseudo energy terms will be applied for every nucleotide involved 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-default 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 probabilities by using linear mapping. Aberration from the observed pairing 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 nucleotides

according to Washietl et al 2012. Perturbation vectors can be calculated by using RNApvmin.

--shapeConversion=M|C|S|L|O

Select method to convert SHAPE reactivities to

pairing probabilities.

(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 nucleotide 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 reactivity 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)

--motif=SEQUENCE,STRUCTURE,ENERGY

Specify stabilizing effect of ligand binding to

a particular sequence/structure motif.

Some ligands binding to RNAs require and/or induce particular sequence and structure motifs. For instance they bind to an interior loop, or small hairpin loop. If for such cases a binding free energy is known, the binding and therefore stabilizing effect of the ligand can be included in the folding recursions. Interior loop motifs are specified as concatenations of 5' and 3' motif, separated by an '&' character.

Energy contributions must be specified in kcal/mol.

See the manpage for an example usage of this option.

--commands=<filename>

Read additional commands from file

Commands include hard and soft constraints, but also structure motifs in hairpin and interior loops that need to be treeted differently. Furthermore, commands can be set for unstructured and structured domains.

Algorithms:

Select additional algorithms which should be included in the calculations. The Minimum free energy (MFE) and a structure representative are calculated in any case.

-p, --partfunc[=INT]

Calculate the partition function and base pairing probability matrix.

(default=`1')

In addition to the MFE structure we print a coarse representation of the pair probabilities in form of a pseudo bracket notation followed by the ensemble free energy. This notation makes use of the letters " . , | { } ( ) " denoting bases that are essentially unpaired, weakly paired, strongly paired without preference, weakly upstream (downstream) paired, or strongly up- (down-)stream paired bases, respectively. On the next line the centroid structure as derived from the pair probabilities together with its free energy and distance to the ensemble is shown. Finally it prints the frequency of the mfe structure, and the structural diversity (mean distance between the structures in the ensemble). See the description of pf_fold() and mean_bp_dist() and centroid() in the RNAlib documentation for details. Note that unless you also specify -d2 or -d0, the partition function and mfe calculations will use a slightly different energy model. See the discussion of dangling end options below.

An additionally passed value to this option changes the behavior of partition function calculation: -p0 Calculate the partition function but not the pair probabilities, saving about 50% in runtime. This prints the ensemble free energy -kT ln(Z). -p2 Compute stack probabilities, i.e. the probability that a pair (i,j) and the immediately interior pair (i+1,j-1) are formed simultaneously in addition to pair probabilities. A second postscript dot plot called "name_dp2.ps", or "dot2.ps" (if the sequence does not have a name), is produced that contains pair probabilities in the upper right half and stack probabilities in the lower left.

--MEA[=gamma]

Calculate an MEA (maximum expected accuracy) structure, where the expected accuracy is computed from the pair probabilities: each base pair (i,j) gets a score 2*gamma*p_ij and the score of an unpaired base is given by the probability of not forming a pair.

(default=`1.')

The parameter gamma tunes the importance of correctly predicted pairs versus unpaired bases. Thus, for small values of gamma the MEA structure will contain only pairs with very high probability. Using --MEA implies -p for computing the pair probabilities.

-S, --pfScale=scaling factor

In the calculation of the pf use scale*mfe as an estimate for the ensemble free energy (used to avoid overflows).

The default is 1.07, useful values are 1.0 to 1.2. Occasionally needed for long sequences. You can also recompile the program to use double precision (see the README file).

-c, --circ

Assume a circular (instead of linear) RNA molecule.

(default=off)

--ImFeelingLucky

Return exactly one stochastically backtracked structure

(default=off)

This function computes the partition function and returns exactly one secondary structure stochastically sampled from the Boltzmann equilibrium according to its probability in the ensemble

--bppmThreshold=<value>

Set the threshold for base pair probabilities included in the postscript output

(default=`1e-5')

By setting the threshold the base pair probabilities that are included in the output can be varied. By default only those exceeding 1e-5 in probability will be shown as squares in the dot plot. Changing the threshold to any other value allows for increase or decrease of data.

-g, --gquad

Incoorporate G-Quadruplex formation into the structure prediction algorithm.

(default=off)

Model Details:

-T, --temp=DOUBLE

Rescale energy parameters to a temperature of temp C. Default is 37C.

-4, --noTetra

Do not include special tabulated stabilizing energies for tri-, tetra- and hexaloop hairpins.

(default=off)

Mostly for testing.

-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 debugging). 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 interior 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. dangling 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 helices of length 1.

--noGU

Do not allow GU pairs

(default=off)

--noClosingGU

Do not allow GU pairs at the end of helices

(default=off)

-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. When passing the placeholder file name "DNA", DNA parameters are loaded without the need to actually specify any input 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. RNAfold -nsp -GA will allow GA and AG pairs. Nonstandard pairs are given 0 stacking energy.

-e, --energyModel=INT

Rarely used option to fold sequences from the artificial ABCD... alphabet, where A pairs B, C-D etc. Use the energy parameters for GC (-e 1) or AU (-e 2) pairs.

--betaScale=DOUBLE

Set the scaling of the Boltzmann factors (default=`1.')

The argument provided with this option enables to scale the thermodynamic temperature used in the Boltzmann factors independently from the temperature used to scale the individual energy contributions of the loop types. The Boltzmann 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.