Provided by: vienna-rna_2.6.4+dfsg-1build2_amd64 bug

NAME

       RNAalifold - manual page for RNAalifold 2.6.4

SYNOPSIS

       RNAalifold [options] [<input0.aln>] [<input1.aln>]...

DESCRIPTION

       RNAalifold 2.6.4

       calculate secondary structures for a set of aligned RNAs

       Read  aligned  RNA  sequences  from  stdin  or  file.aln  and  calculate  their minimum free energy (mfe)
       structure, partition function (pf) and base pairing probability matrix. Currently, input alignments  have
       to  be  in CLUSTAL, Stockholm, FASTA, or MAF format. The input format must be set manually in interactive
       mode (default is Clustal), but will be determined automagically from the input file, if  not  expplicitly
       set.  It  returns the mfe structure in bracket notation, its energy, the free energy of the thermodynamic
       ensemble and the frequency of the mfe structure in the ensemble to stdout.  It also  produces  Postscript
       files  with  plots  of the resulting secondary structure graph ("alirna.ps") and a "dot plot" of the base
       pairing matrix ("alidot.ps").  The file "alifold.out" will contain a  list  of  likely  pairs  sorted  by
       credibility,  suitable  for  viewing   with  "AliDot.pl".  Be  warned that output file will overwrite any
       existing files of the same name.

       -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)

       -q, --quiet
              Be quiet.  (default=off)

              This option can be used to minimize the output of additional information and  non-severe  warnings
              which otherwise might spam stdout/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 particular 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').

       --mis  Output "most informative sequence" instead of simple consensus: For each column of  the  alignment
              output the set of nucleotides with frequency greater than average in IUPAC notation.

              (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 alignment at  a  time.
              Using  this  switch,  a user can instead start the computation for many alignments in the input in
              parallel. RNAalifold will create as many parallel computation slots as specified and assigns input
              alignments 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  RNAalifold 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, RNAalifold will not buffer individual results but print  them
              as soon as they have been computated.

       --noconv
              Do not automatically substitute nucleotide "T" with "U".

              (default=off)

       -n, --continuous-ids
              Use continuous alignment ID numbering when no alignment ID can be retrieved from input data.

              (default=off)

              Due  to  its  past,  RNAalifold  produces  a specific set of output file names for the first input
              alignment, "alirna.ps", "alidot.ps", etc. But for all further alignments in the input, it  usually
              adopts  a naming scheme based on IDs, which may be retrieved from the input alignment's meta-data,
              or generated by a prefix followed by an increasing counter. Setting this flag instructs RNAalifold
              to use the ID naming scheme also for the first alignment.

       --auto-id
              Automatically generate an ID for each alignment.

              (default=off)

              The default mode of RNAalifold 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  formatted  input. If this flag is active, RNAalifold 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), "prefix_xxxx_dp.ps" (dot-plot), "prefix_xxxx_aln.ps" (annotated alignment), 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 input alignment.

       --id-delim=CHAR
              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].

       --id-start=LONG
              Specify the first number in automatically generated alignment IDs.

              (default=`1')

              When  alignment  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
              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 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 '>'.

   Algorithms:
              Select additional algorithms which should be included in the calculations.

       -p, --partfunc[=INT]
              Calculate the partition function and base pairing  probability  matrix  in  addition  to  the  mfe
              structure. Default is calculation of mfe structure only.

              (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, as well as  the  centroid
              structure  derived  from  the pair probabilities together with its free energy and distance to the
              ensemble.  Finally it prints the frequency of the mfe structure.

              An  additionally  passed  value  to  this  option  changes  the  behavior  of  partition  function
              calculation:  -p0  deactivates  the  calculation  of  the  pair probabilities, saving about 50% in
              runtime. This prints the ensemble free energy 'dG=-kT ln(Z)'.

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

              The argument provided with this option is used to  scale  the  thermodynamic  temperature  in  the
              Boltzmann  factors independently from the temperature of the individual loop energy contributions.
              The Boltzmann factors then become 'exp(- dG/(kTn*betaScale))' where 'k' is the Boltzmann constant,
              'dG' the free energy contribution of the state, 'T' the absolute temperature and 'n' the number of
              sequences.

       -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.

       --MEA[=gamma]
              Compute MEA (maximum expected accuracy) structure.

              (default=`1.')

              The  expected  accuracy is computed from the pair probabilities: each base pair '(i,j)' receives a
              score '2*gamma*p_ij' and the score of an unpaired base is given by the probability of not  forming
              a  pair.  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.

       --sci  Compute the structure conservation index (SCI) for the MFE consensus structure of the alignment.

              (default=off)

       -c, --circ
              Assume a circular (instead of linear) RNA molecule.

              (default=off)

       --bppmThreshold=cutoff
              Set the threshold/cutoff for base pair probabilities included in the postscript output.

              (default=`1e-6')

              By  setting  the  threshold  the  base  pair  probabilities that are included in the output can be
              varied. By default only those exceeding '1e-6' 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)

       -s, --stochBT=INT
              Stochastic  backtrack.  Compute a certain number of random structures with a probability dependend
              on the partition function. See -p option in RNAsubopt.

       --stochBT_en=INT
              same as -s option but also print out the energies and probabilities of the backtraced structures.

       -N, --nonRedundant
              Enable non-redundant sampling strategy.

              (default=off)

   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.   The  constraining  structure  will  be  read  from
              'stdin', the alignment has to be given as a file name on the command line.

              (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 all alignment records.  (default=off)

              Usually,  constraints  provided  from  input file are only applied to a single sequence alignment.
              Therefore, RNAalifold will stop its computation and quit  after  the  first  input  alignment  was
              processed.  Using  this  switch,  RNAalifold  processes  all  sequence alignments in the input and
              applies the same provided constraints to each of them.

       --enforceConstraint
              Enforce base pairs given by round brackets '(' ')' in structure constraint.

              (default=off)

       --SS_cons
              Use consensus structures from Stockholm file ('#=GF SS_cons') as constraint.

              (default=off)

              Stockholm formatted alignment files have the possibility to store a secondary structure string  in
              one of if ('#=GC') column annotation meta tags. The corresponding tag name is usually 'SS_cons', a
              consensus  secondary  structure.   Activating this flag allows one to use this consensus secondary
              structure from the input file as structure constraint. Currently, only  the  following  characters
              are interpreted:

              '(' ')' [mathing parenthesis: column i pairs with column j]

              '<' '>' [matching angular brackets: column i pairs with column j]

              All other characters are not interpreted (yet).  Note: Activating this flag implies --constraint.

       --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 sequences 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 following parameters

       -d, --dangles=INT
              How to treat "dangling end" energies 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.

              The option -d0 ignores dangling ends altogether (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 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)

       --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')

       -E, --endgaps
              Score pairs with endgaps same as gap-gap pairs.

              (default=off)

       -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.

       --old  use old energy evaluation, treating gaps as characters.

              (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.

       -e, --energyModel=INT
              Set energy model.

              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.

       --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

       --color
              Produce a colored version of the consensus structure plot "alirna.ps" (default b&w only)

              (default=off)

       --aln  Produce  a  colored and structure annotated alignment in PostScript format in the file "aln.ps" in
              the current directory.

              (default=off)

       --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-stk[=prefix]
              Create a multi-Stockholm formatted output file.  (default=`RNAalifold_results')

              The default file name used for the output  is  "RNAalifold_results.stk".   Users  may  change  the
              filename to "prefix.stk" by specifying 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 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)

       --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.

       -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)

       Caveats:

       Sequences  are  not  weighted.  If  possible, do not mix very similar and dissimilar sequences. Duplicate
       sequences, for example, can distort the prediction.

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

       The  algorithm  is  a variant of the dynamic programming algorithms of M. Zuker and P. Stiegler (mfe) and
       J.S. McCaskill (pf) adapted for sets of aligned sequences with covariance information.

       Ivo L. Hofacker, Martin Fekete, and Peter F. Stadler (2002), "Secondary Structure Prediction for  Aligned
       RNA Sequences", J.Mol.Biol.: 319, pp 1059-1066.

       Stephan  H.  Bernhart,  Ivo  L. Hofacker, Sebastian Will, Andreas R. Gruber, and Peter F. Stadler (2008),
       "RNAalifold: Improved consensus structure prediction 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.  Susan,  M.  Zuker,  D.H.  Turner
       (2004),  "Incorporating  chemical  modification  constraints  into  a  dynamic  programming algorithm for
       prediction of RNA secondary 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", Nucleic Acids Research: 38, pp 280-282

EXAMPLES

       A simple call to compute consensus MFE structure, ensemble free energy, base pair probabilities, centroid
       structure, and MEA structure for a multiple sequence alignment (MSA) provided as Stockholm formatted file
       alignment.stk might look like:

         $ RNAalifold -p --MEA alignment.stk

       Consider the following MSA file for three sequences

         # STOCKHOLM 1.0

         #=GF AC   RF01293
         #=GF ID   ACA59
         #=GF DE   Small nucleolar RNA ACA59
         #=GF AU   Wilkinson A
         #=GF SE   Predicted; WAR; Wilkinson A
         #=GF SS   Predicted; WAR; Wilkinson A
         #=GF GA   43.00
         #=GF TC   44.90
         #=GF NC   40.30
         #=GF TP   Gene; snRNA; snoRNA; HACA-box;
         #=GF BM   cmbuild -F CM SEED
         #=GF CB   cmcalibrate --mpi CM
         #=GF SM   cmsearch --cpu 4 --verbose --nohmmonly -E 1000 -Z 549862.597050 CM SEQDB
         #=GF DR   snoRNABase; ACA59;
         #=GF DR   SO; 0001263; ncRNA_gene;
         #=GF DR   GO; 0006396; RNA processing;
         #=GF DR   GO; 0005730; nucleolus;
         #=GF RN   [1]
         #=GF RM   15199136
         #=GF RT   Human box H/ACA pseudouridylation guide RNA machinery.
         #=GF RA   Kiss AM, Jady BE, Bertrand E, Kiss T
         #=GF RL   Mol Cell Biol. 2004;24:5797-5807.
         #=GF WK   Small_nucleolar_RNA
         #=GF SQ   3

         AL031296.1/85969-86120     CUGCCUCACAACGUUUGUGCCUCAGUUACCCGUAGAUGUAGUGAGGGUAACAAUACUUACUCUCGUUGGUGAUAAGGAACAGCU
         AANU01225121.1/438-603     CUGCCUCACAACAUUUGUGCCUCAGUUACUCAUAGAUGUAGUGAGGGUGACAAUACUUACUCUCGUUGGUGAUAAGGAACAGCU
         AAWR02037329.1/29294-29150 ---CUCGACACCACU---GCCUCGGUUACCCAUCGGUGCAGUGCGGGUAGUAGUACCAAUGCUAAUUAGUUGUGAGGACCAACU
         #=GC SS_cons               -----((((,<<<<<<<<<___________>>>>>>>>>,,,,<<<<<<<______>>>>>>>,,,,,))))::::::::::::
         #=GC RF                    CUGCcccaCAaCacuuguGCCUCaGUUACcCauagguGuAGUGaGgGuggcAaUACccaCcCucgUUgGuggUaAGGAaCAgCU
         //

       Then, the above program call will produce this output:

         3 sequences; length of alignment 84.
         >ACA59
         CUGCCUCACAACAUUUGUGCCUCAGUUACCCAUAGAUGUAGUGAGGGUAACAAUACUUACUCUCGUUGGUGAUAAGGAACAGCU
         ...((((((.(((((((((...........))))))))).))))))..........(((((......)))))............ (-12.54 = -12.77 +   0.23)
         ...((((((.(((((((((...........))))))))).)))))){{,.......{{{{,......}))))............ [-14.38]
         ...((((((.(((((((((...........))))))))).))))))..........((((........))))............ {-12.44 = -12.33 +  -0.10 d=10.94}
         ...((((((.(((((((((...........))))))))).))))))..........((((........))))............ {-12.44 = -12.33 +  -0.10 MEA=66.65}
          frequency of mfe structure in ensemble 0.368739; ensemble diversity 17.77

       Here, the first line is written to stderr and simply states the number of sequences and the length of the
       alignment.  This  line  can  be  suppressed using the --quiet option.  The main output then consists of 7
       lines, where the first two resemble the FASTA header with the  ID  as  read  from  the  input  data  set,
       followed by the consensus sequence in the second line. The third line consists of the consensus secondary
       structure  in  dot-bracket  notation  followed  by  the averaged minimum free energy in parenthesis. This
       energy is composed of two major contributions, the actual free energies derived from the Nearest Neighbor
       model, and the covariance pseudo-energy term, which are both displayed after the equal sign.  The  fourth
       line  shows  the base pair propensity in pseudo dot-bracket notation followed by the ensemble free energy
       dG = -kT ln(Z) in square brackets.  Similarly, the next  two  lines  state  the  controid-  and  the  MEA
       structure  in  dot-bracket  notation, followed by their corresponding free energy contributions, the mean
       distance (d) to the ensemble as well as the maximum expected accuracy (MEA). Again, the free energies are
       split into Nearest Neighbor contribution and the covariance pseudo-energy term.

       Furthermore, RNAalifold will produce three output files: ACA59_ss.ps, ACA59_dp.ps, and ACA59_ali.out that
       contain the secondary structure drawing, the base pair probability dot-plot, and a detailed table of base
       pair probabilities, respectively.

THE ALIOUT FILE

       When computing base pair probabilities (--partfunc option), RNAalifold  will  produce  a  file  with  the
       suffix  `ali.out`.  This file contains the base pairing probabilities between different alignment columns
       together with some detailed statistics for the individual sequences within the alignment. The file  is  a
       simple  text file with a two line header that states the number of sequences and length of the alignment.
       The first couple of lines of this file may look like:

         3 sequence; length of alignment 84
         alifold output
            14    36  0  92.7%   0.212 CG:1    UA:2
            13    37  0  92.7%   0.218 GU:1    AU:2
            12    38  0  92.7%   0.231 CG:3
            15    35  0  91.9%   0.239 UG:3
            16    34  0  85.2%   0.434 UA:2    --:1
             8    42  0  80.7%   0.526 AU:3   +
             9    41  0  80.4%   0.542 CG:3   +
             7    43  1  80.1%   0.541 CG:2   +

       Starting with the third row, there are at least six and at  most  13  columns  separated  by  whitespaces
       stating:  (1)  the i-position and (2) the j-position of a potential base pair (i, j), followed by (3) the
       number of counter examples, i.e. the number of sequences in the alignment that  can't  form  a  canonical
       base  pair  with their respective sequence positions.  Next is (4) the base pair probabilitiy in percent,
       (5) a pseudo entropy measure S_ij = S_i + S_j - p_ij ln(p_ij), where  S_i  and  S_j  are  the  positional
       entropies for the two alignment columns i and j, and p_ij is the base pair probability. Finally, the last
       columns  (6-12)  state the number of particular base pairs for the individual sequences in the alignment.
       Here, we distinguish the base  pairs  "GC","CG","AU","UA","GU","UG",  and  the  special  case  "--"  that
       represents  gaps  at  both positions i and j.  Finally, base pairs that are not part of the MFE structure
       are marked by an additional "+" sign in the last column.

AUTHOR

       Ivo L Hofacker, Stephan Bernhart, 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

       The ALIDOT package http://www.tbi.univie.ac.at/RNA/Alidot/

RNAalifold 2.6.4                                  January 2025                                     RNAALIFOLD(1)