Provided by: vienna-rna_2.5.1+dfsg-1_amd64 bug

NAME

       RNAalifold - manual page for RNAalifold 2.5.1

SYNOPSIS

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

DESCRIPTION

       RNAalifold 2.5.1

       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

   General Options:
              Command line options which alter the general behavior of this program

       -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.

       -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)

       --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)

       -t, --layout-type=INT
              Choose the layout algorithm. Simple radial layout if 0, or naview if 1

              (default=`1')

       --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.

       -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).

       -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=prefix
              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=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].

       --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=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 '>'.

   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".

   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 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 -kT ln(Z).

       --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.

       --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)

       -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" but also print out the energies and probabilities  of  the  backtraced
              structures.

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

              (default=off)

       -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)

       --bppmThreshold=<value>
              Set the threshold 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-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)

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

              (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 -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.  The  matrix  is chosen according to the minimal and
              maximal pairwise identities of the sequences in the file.

              (default=off)

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

              (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/(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.

       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/