RNALALIFOLD(1)

📦
version 2.7.0 · 5 versions
, in ubuntu resolute, ubuntu stonking

🦋 RNALalifold(1) 🦋

User Commands 💅

✨ NAME ✨

RNALalifold - manual page for RNALalifold 2.7.0 🌸

✨ SYNOPSIS ✨

RNALalifold [options] <file1.aln>

✨ DESCRIPTION ✨

RNALalifold 2.7.0 💖 is all about one thing: calculating locally stable secondary structures for a set of aligned RNAs 🧚

Okay so it reads aligned RNA sequences from stdin or from a file.aln, then calculates locally stable RNA secondary structure with a maximal base pair span. For a sequence of length n and a base pair span of L, the algorithm only uses O(n+L*L) memory and O(n*L*L) CPU time. That's literally why it's 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) 📢
    • It lowers the log level so even INFO messages get passed through.
  • -q, --quiet — Be quiet (default=off) 🤫
    • This one keeps the extra info and non-severe warnings from spamming stdout/stderr.

✨ I/O Options ✨

Command line options for input and output (pre-)processing 🎀

  • -f, --input-format=C|S|F|M — Tell it the file format of the input multiple sequence alignment (MSA) 📄
    • If you set this, the input is treated as that particular format.
    • If you don't, it tries to figure out the format automatically, as long as an input file was given.
    • When the input MSA comes in interactive mode or from a terminal (TTY), the program defaults to assuming CLUSTALW format.
    • Available formats: 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 🎨
    • For every hit, you get a colored, structure-annotated (sub)alignment and a secondary structure plot, both in PostScript format.
    • The subalignment hit also gets added into a multi-Stockholm file called RNALalifold_results.stk.
    • The PostScript files are named aln_start_end.eps and ss_start_end.eps, and they all land in the current directory.
    • The optional prefix changes the names to prefix_aln_start_end.eps, prefix_ss_start_end.eps, and prefix.stk.
    • > Heads up: any special characters in the prefix get replaced by the filename delimiter, so there's no way to pass a whole directory path through this option yet. (See also --filename-delim.)
  • --aln-stk[=prefix] — Add hits to a multi-Stockholm formatted output file (default=RNALalifold_results) 🗂️
    • The default file name is RNALalifold_results.stk. Pass a prefix and it becomes prefix.stk instead.
    • The file gets created in the current directory if it doesn't already exist. If it does exist, the output gets appended to it.
    • > Same warning as above: special characters in the prefix get replaced by the filename delimiter, so no full directory paths yet. (See also --filename-delim.)
  • --mis — Output the "most informative sequence" instead of the simple consensus (default=off) 🧠
    • For each column of the alignment, you get the set of nucleotides whose frequency is greater than average, written in IUPAC notation.
  • --split-contributions — Split the free energy contributions into separate parts (default=off) 🍰
    • By default you only get the total energy contribution for each hit.
    • With this option, that total gets split into the Nearest Neighbor model energy, the covariance pseudo energy, and, if it applies, a remaining pseudo energy from special constraints like probing signals such as SHAPE.
  • --noconv — Don't automatically swap T for U (default=off) 🙅‍♀️
  • --auto-id — Automatically generate an ID for each alignment (default=off) 🪪
    • The default vibe is that RNALalifold figures out an ID from the input alignment, as long as the input format allows it. Stockholm 1.0 formatted input usually comes with IDs like that.
    • If you turn this flag on, RNALalifold ignores any IDs from the input and generates one for each alignment on its own.
  • --id-prefix=STRING — Prefix for automatically generated IDs (as used in output file names) (default=alignment) 🏷️
    • Each alignment gets prefixed with the string you provide, so output files follow this naming scheme: prefix_xxxx_ss.ps (secondary structure plot), prefix_xxxx_dp.ps (dot-plot), prefix_xxxx_aln.ps (annotated alignment), and so on.
    • In those names, xxxx is the alignment number, starting with the second alignment in the input.
    • Pair this with --continuous-ids if you want IDs to start from the first input alignment.
  • --id-delim=CHAR — Change the delimiter between the prefix and the increasing number for automatically generated IDs (as used in output file names) (default=_) ✂️
    • The default _ is what sits between the prefix string and the counter. Swap it out here if you like.
  • --id-digits=INT — Set how many digits the counter has in automatically generated alignment IDs (default=4) 🔢
    • Numbers start at 1 and always get left-padded with zeros so they fill a set width.
    • Pick the width you want, within the range [1:18].
  • --id-start=LONG — Set the first number in automatically generated alignment IDs (default=1) 🚀
    • Alignment IDs usually start at 1, but you can choose your own starting number here.
    • > Negative numbers are not allowed.
    • Setting this implies continuous alignment IDs, which means it turns on the --continuous-ids flag.
  • --filename-delim=CHAR — Change the delimiting character used in sanitized filenames (default=ID-delimiter) 🧼
    • This is the character that replaces invalid characters while filenames get sanitized.
    • The default delimiter is ALWAYS the first character of the "ID delimiter" you pass through --id-delim.
    • If the delimiter is a whitespace character or empty, invalid characters are just removed instead of being substituted.
    • These characters count as illegal in filenames: backslash \, slash /, question mark ?, percent sign %, asterisk *, colon :, pipe symbol |, double quote ", and triangular brackets < and >.
  • --log-level=level — Set the log level threshold (default=2) 📉
    • By default only warnings (level 2) or errors (level 3) get printed. Higher values mean fewer messages.
    • > 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=RNALalifold.log) 📝
  • --log-time — Include a time stamp in log messages (default=off) ⏰
  • --log-call — Include the file and line of the log-calling function (default=off) 🔍

✨ 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 🧮

  • -L, --maxBPspan=INT — Set the maximum allowed separation of a base pair to span (default=70) 📏
    • In other words, no pairs (i,j) with j-i>span will be allowed.
  • --threshold=DOUBLE — Set the energy threshold in kcal/mol per nucleotide (default=-0.1) 🌡️
    • Secondary structure hits above this threshold get left out of the output.
  • -g, --gquad — Incorporate G-Quadruplex formation into the structure prediction algorithm (default=off) 💎

✨ Structure Constraints ✨

Command line options to interact with the structure constraints feature of this program 🦋

  • --shape=file1,file2 — Use SHAPE reactivity data to guide structure predictions 🧪
    • You can specify multiple shapefiles for the individual sequences in the alignment as a comma separated list.
    • You can also link a particular shape file to a specific sequence by putting the sequence number in front of the filename. For example, 5=seq5.shape,3=seq3.shape assigns the reactivity values from seq5.shape to the fifth sequence in the alignment, and the values from seq3.shape to sequence 3.
    • If you don't specify any assignment, the reactivity values get assigned to the corresponding sequences in the order they're given.
  • --shapeMethod=D[mX][bY] — Specify the method for converting SHAPE reactivity data into pseudo energy contributions (default=D) ⚡

Okay so the shape method bit is giving main character energy 💅 Right now there's only ONE data conversion method on the menu, and it's the one from Deigan et al 2009 🦋 It's the default, and you can spot it by the capital D in the parameter, so --shapeMethod="D" is what you get if you don't touch anything 💖 You can also tweak the slope m and intercept b if the defaults aren't vibing with you. Otherwise they're m=1.8 and b=-0.6, same as in the paper I mentioned earlier 📄 To change them, like m=1.9 and b=-0.7, pass a string like --shapeMethod="Dm1.9b-0.7" ✨ You can also set just one of them, like --shapeMethod="Dm1.9" or --shapeMethod="Db-0.7" 🌸

🎀 Energy Parameters 🎀

Energy parameter sets can be tweaked or loaded from your own input files, so you're in control 💅

  • -T, --temp=DOUBLE — Rescale the energy parameters to a temperature of temp C. The 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 come with your distribution. Check the RNAlib documentation for the file format details. The placeholder file name DNA lets you load DNA parameters without actually specifying any input file.
  • -4, --noTetra — Don't include the special tabulated stabilizing energies for tri-, tetra- and hexaloop hairpins 🙈 (default=off) Mostly for testing.
  • --salt=DOUBLE — Set the salt concentration in molar (M). The default is 1.021M.

🌈 Model Details 🌈

Tweak the energy model and pairing rules even more with these 💜

  • -d, --dangles=INT — This decides how to treat "dangling end" energies for bases sitting next 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, that check gets skipped, and dangling energies are added for bases adjacent to a helix on both sides no matter what. This is the default for mfe and partition function folding (-p). -d0 ignores dangling ends altogether (mostly for debugging 🛠️). With -d3, mfe folding allows coaxial stacking of adjacent helices in multi-loops. Right now the implementation won't allow coaxial stacking of the two enclosed pairs in a loop of degree 3, and it only works for mfe folding.
Heads up: with -d1 and -d3, only the MFE computations use this setting. The partition function uses the -d2 setting, so dangling ends get treated differently between the two.
  • --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 show up as helices of length 1.
  • --noGU — Don't allow GU pairs 🙅‍♀️ (default=off)
  • --noClosingGU — Don't 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 the specified Ribosum Matrix instead of the normal energy model 🧬 Matrices should be 6x6 matrices, and the order of the terms is 'AU', 'CG', 'GC', 'GU', 'UA', 'UG'.
  • -r, --ribosum_scoring — Use the ribosum scoring matrix 🔥 (default=off) The matrix is picked based on the minimal and maximal pairwise identities of the sequences in the file.
  • --nsp=STRING — Allow other pairs on top of the usual AU, GC, and GU pairs 🌟 The argument is a comma separated list of additionally allowed pairs. If the first character is a -, then AB implies that AB and BA are both allowed pairs, so --nsp="-GA" allows GA and AG pairs. Nonstandard pairs get 0 stacking energy.
  • --energyModel=INT — Set the energy model. This is a rarely used option for folding sequences from the artificial ABCD... alphabet, where A pairs with B, C with D, and so on. 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 gets re-set automatically to 3.4 if 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 gets re-set automatically to 6.76 if DNA parameters are loaded via -P DNA and no further value is provided.

💖 Plotting 💖

Command line options for changing the default behaviour of structure layout and pairing probability plots 📊

  • --aln-EPS[=prefix] — Produce colored, structure annotated subalignments for each hit 🎨 The default file name is aln_start_end.eps, where start and end are the first and last column of the subalignment relative to the input (1-based). You can change the filename to prefix_aln_start_end.eps by giving a prefix as the optional argument. Files get created in the current directory. Any special characters in the prefix get replaced by the filename delimiter, so there's no way to pass a whole directory path through this option yet 🙃 (See also the --filename-delim parameter.)
  • --aln-EPS-cols=INT — Set the number of columns in colored EPS alignment output. (default=`60') A value less than 1 means the output won't be wrapped at all.
  • --aln-EPS-ss[=prefix] — Produce colored consensus secondary structure plots in PostScript format ✨ The default file name is ss_start_end.eps, where start and end are the first and last column of the subalignment relative to the input (1-based). You can change it to prefix_ss_start_end.eps by giving a prefix as the optional argument. Files get created in the current directory. Any special characters in the prefix get replaced by the filename delimiter, so no full directory paths through this option yet 🙃 (See also the --filename-delim parameter.)
  • --color-threshold=FLOAT — Set the threshold of maximum counter examples for coloring the consensus structure plot. (default=`2') Floating point numbers between 0 and 1 are treated as frequencies among all sequences in the alignment. Everything else gets truncated to an integer and used as an absolute number of counter examples.
  • --color-min-sat=FLOAT — Set the minimum saturation for coloring the consensus structure plot. (default=`0.2') This is a floating point number that's >= 0 and smaller than 1.

📚 REFERENCES 📚

If you use this program in your work, you might want to cite these bestie 🫶

  • 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 Locally Stable RNA Secondary Structures for Genome-Wide Surveys", Bioinformatics: 20, pp 186-190
  • 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

👑 AUTHOR 👑

Ivo L Hofacker, Ronny Lorenz 💜

🐛 REPORTING BUGS 🐛

If in doubt, our program is right and nature is at fault 🙈 Comments should be sent to rna@tbi.univie.ac.at.

That's the whole page, bestie 🫶 This was the RNALalifold 2.7.0 edition from February 2026, so you're now officially up to date ✨ Go be iconic 💅🌈

📦 where this page comes from