--- /dev/null
+
+
+CLUSTAL-OMEGA is a general purpose multiple sequence alignment program
+for protein and DNA/RNA.
+
+
+
+INTRODUCTION
+
+Clustal-Omega is a general purpose multiple sequence alignment (MSA)
+program for protein and DNA/RNA. It produces high quality MSAs and is
+capable of handling data-sets of hundreds of thousands of sequences in
+reasonable time.
+
+In default mode, users give a file of sequences to be aligned and
+these are clustered to produce a guide tree and this is used to guide
+a "progressive alignment" of the sequences. There are also facilities
+for aligning existing alignments to each other, aligning a sequence to
+an alignment and for using a hidden Markov model (HMM) to help guide
+an alignment of new sequences that are homologous to the sequences
+used to make the HMM. This latter procedure is referred to as
+"external profile alignment" or EPA.
+
+Clustal-Omega uses HMMs for the alignment engine, based on the HHalign
+package from Johannes Soeding [1]. Guide trees are made using an
+enhanced version of mBed [2] which can cluster very large numbers of
+sequences in O(N*log(N)) time. Multiple alignment then proceeds by
+aligning larger and larger alignments using HHalign, following the
+clustering given by the guide tree.
+
+In its current form Clustal-Omega has been extensivly tested for
+protein sequences, DNA/RNA support has been added since version 1.1.0.
+
+
+
+SEQUENCE INPUT:
+
+-i, --in, --infile={<file>,-}
+ Multiple sequence input file (- for stdin)
+
+--hmm-in=<file>
+ HMM input files
+
+--dealign
+ Dealign input sequences
+
+--profile1, --p1=<file>
+ Pre-aligned multiple sequence file (aligned columns will be kept fixed)
+
+--profile2, --p2=<file>
+ Pre-aligned multiple sequence file (aligned columns will be kept fixed)
+
+--is-profile
+ disable check if profile, force profile (default no)
+
+-t, --seqtype={Protein, RNA, DNA}
+ Force a sequence type (default: auto)
+
+--infmt={a2m=fa[sta],clu[stal],msf,phy[lip],selex,st[ockholm],vie[nna]}
+ Forced sequence input file format (default: auto)
+
+
+For sequence and profile input Clustal-Omega uses the Squid library
+from Sean Eddy [3].
+
+
+Clustal-Omega accepts 3 types of sequence input: (i) a sequence file
+with un-aligned or aligned sequences, (ii) profiles (a multiple
+alignment in a file) of aligned sequences, (iii) a HMM. Valid
+combinations of the above are:
+
+(a) one file with un-aligned or aligned sequences (i); the sequences
+ will be aligned, and the alignment will be written out. For this
+ mode use the -i flag. If the sequences are aligned (all sequences
+ have the same length and at least one sequence has at least one
+ gap), then the alignment is turned into a HMM, the sequences are
+ de-aligned and the now un-aligned sequences are aligned using the
+ HMM as an External Profile for External Profile Alignment (EPA).
+ If no EPA is desired use the --dealign flag.
+
+ Use the above option to make a multiple alignment from a set of
+ sequences. A sequence file must contain more than one sequence (at
+ least two sequences).
+
+(b) two profiles (ii)+(ii); the columns in each profile will be kept
+ fixed and the alignment of the two profiles will be written
+ out. Use the --p1 and --p2 flags for this mode.
+
+ Use this option to align two alignments (profiles) together.
+
+
+(c) one file with un/aligned sequences (i) and one profile (ii); the
+ profile is converted into a HMM and the un-aligned sequences will
+ be multiply aligned (using the HMM background information) to form
+ a profile; this constructed profile is aligned with the input
+ profile; the columns in each profile (the original one and the one
+ created from the un-aligned sequences) will be kept fixed and the
+ alignment of the two profiles will be written out. Use the -i flag
+ in conjunction with the --p1 flag for this mode.
+ The un/aligned sequences file (i) must contain at least two
+ sequences. If a single sequence has to be aligned with a profile
+ the profile-profile option (b) has to be used.
+
+ Use the above option to add new sequences to an existing
+ alignment.
+
+(d) one file with un-aligned sequences (i) and one HMM (iii); the
+ un-aligned sequences will be aligned to form a profile, using the
+ HMM as an External Profile. So far only one HMM can be input and
+ only HMMer2 and HMMer3 formats are allowed. The alignment will be
+ written out; the HMM information is discarded. As, at the moment,
+ only one HMM can be used, no HMM is produced if the sequences are
+ already aligned. Use the -i flag in conjunction with the --hmm-in
+ flag for this mode. Multiple HMMs can be inputted, however, in the
+ current version all but the first HMM will be ignored.
+
+ Use this option to make a new multiple alignment of sequences from
+ the input file and use the HMM as a guide (EPA).
+
+Sequences that all have the same lengths but do not contain a single
+gap are by default not recognised as a profile. If these sequences are
+indeed a profile an not just a collection of unaligned sequences that
+happen to have the same length then specify the --is-profile flag.
+
+
+Invalid combinations of the above are:
+
+(v) an un/aligned sequence file containing just one sequence (i)
+
+(w) an un/aligned sequence file containing just one sequence and a profile
+ (i)+(ii)
+
+(x) an un/aligned sequence file containing just one sequence and a HMM
+ (i)+(iii)
+
+(y) two or more HMMs (iii)+(iii)+... cannot be aligned to one another.
+
+(z) one profile (ii) cannot be aligned with a HMM (iii)
+
+
+The following MSA file formats are allowed:
+
+ a2m=fasta, (vienna)
+ clustal,
+ msf,
+ phylip,
+ selex,
+ stockholm
+
+
+Prior to MSA, Clustal-Omega de-aligns all sequence input (i). However,
+alignment information is automatically converted into a HMM and used
+during MSA, unless the --dealign flag is specifically set. Profiles
+(ii) are not de-aligned.
+
+Since version 1.1.0 the Clustal-Omega alignment engine can process
+DNA/RNA. Clustal-Omega tries to guess the sequence type (protein,
+DNA/RNA), but this can be over-ruled with the --seqtype (-t) flag.
+
+
+CLUSTERING:
+
+ --distmat-in=<file>
+ Pairwise distance matrix input file (skips distance computation)
+
+ --distmat-out=<file>
+ Pairwise distance matrix output file
+
+ --guidetree-in=<file>
+ Guide tree input file
+ (skips distance computation and guide tree clustering step)
+
+ --guidetree-out=<file>
+ Guide tree output file
+
+ --full
+ Use full distance matrix for guide-tree calculation (slow; mBed is default)
+
+ --full-iter
+ Use full distance matrix for guide-tree calculation during iteration (mBed is default)
+
+ --cluster-size=<n>
+ soft maximum of sequences in sub-clusters
+
+ --clustering-out=<file>
+ Clustering output file
+
+ --use-kimura
+ use Kimura distance correction for aligned sequences (default no)
+
+ --percent-id
+ convert distances into percent identities (default no)
+
+In order to produce a multiple alignment Clustal-Omega requires a
+guide tree which defines the order in which sequences/profiles are
+aligned. A guide tree in turn is constructed, based on a distance
+matrix. Conventionally, this distance matrix is comprised of all the
+pair-wise distances of the sequences. The distance measure
+Clustal-Omega uses for pair-wise distances of un-aligned sequences is
+the k-tuple measure [4], which was also implemented in Clustal 1.83
+and ClustalW2 [5,6]. If the protein sequences inputted via -i are
+aligned, then Clustal-Omega uses pairwise aligned identities, these
+distances can be Kimura-corrected [7] by specifying --use-kimura. The
+distances between aligned DNA/RNA sequences are determined from the
+alignment, no Kimura correction can be used. The computational effort
+(time/memory) to calculate and store a full distance matrix grows
+quadratically with the number of sequences. Clustal-Omega can improve
+this scalability to N*log(N) by employing a fast clustering algorithm
+called mBed [2]; this option is automatically invoked (default). If a
+full distance matrix evaluation is desired, then the --full flag has
+to be set. The mBed mode calculates a reduced set of pair-wise
+distances. These distances are used in a k-means algorithm, that
+clusters at most 100 sequences. For each cluster a full distance
+matrix is calculated. No full distance matrix (of all input sequences)
+is calculated in mBed mode. If there are less than 100 sequences in
+the input, then in effect a full distance matrix is calculated in mBed
+mode, however, no distance matrix can be outputted (see below).
+
+
+Clustal-Omega uses Muscle's [8] fast UPGMA implementation to construct
+its guide trees from the distance matrix. By default, the distance
+matrix is used internally to construct the guide tree and is then
+discarded. By specifying --distmat-out the internal distance matrix
+can be written to file. This is only possible in --full or --full-iter
+mode. The guide trees by default are used internally to guide the
+multiple alignment and are then discarded. By specifying the
+--guidetree-out option these internal guide trees can be written out
+to file. Conversely, the distance calculation and/or guide tree
+building stage can be skipped, by reading in a pre-calculated distance
+matrix and/or pre-calculated guide tree. These options are invoked by
+specifying the --distmat-in and/or --guidetree-in flags,
+respectively. However, distance matrix reading is disabled in the
+current version. By default, distance matrix and guide tree files are
+not over-written, if a file with the specified name already exists. In
+this case Clustal-Omega aborts during the command-line processing
+stage. To force over-writing of already existing files use the --force
+flag (see MISCELLANEOUS). In mBed mode a full distance matrix cannot
+be outputted, distance matrix output is only possible in --full mode.
+mBed or --full distance mode do not affect the ability to write out
+guide-trees. It is possible to perform an initial mBed (not-full)
+distance calculation and a subsequent full distance calculation (see
+section ITERATION). In this case a distance matrix can be outputted.
+
+Guide trees can be iterated to refine the alignment (see section
+ITERATION). Clustal-Omega takes the alignment, that was produced
+initially and constructs a new distance matrix from this alignment.
+The distance measure used at this stage is a full alignment distance
+(as opposed the initial pairwise k-tuple distance); distances of
+protein sequences can be Kimura corrected [7], DNA/RNA distances are
+not. By default, Clustal-Omega constructs a reduced distance matrix at
+this stage using the mBed algorithm, which will then be used to create
+an improved (iterated) new guide tree. To turn off mBed-like
+clustering at this stage the --full-iter flag has to be set. While
+full alignment distances in general are much faster to calculate than
+k-tuple distances, time and memory requirements still scale
+quadratically with the number of sequences and --full-iter clustering
+should only be considered for smaller cases (<< 10,000 sequences) or
+if response time and resources are not an issue.
+
+The default cluster size in mBed mode is 100. This means that
+sequences are grouped into clusters with a soft maximum of 100
+sequences, full distance matrices are calculated for these clusters,
+guide-trees are calculated for the clusters and the clusters are then
+strung together with an over-arching guide-tree. It is possible to
+change the cluster-size with the --cluster-size flag. The clustering
+can be outputted to file. The output is comprised of the cluster
+index, a running index for the sequences within each cluster, the
+running index for the sequence within the input file, the name of the
+sequence and the bi-section sequence (see EXAMPLES).
+
+Clustal-Omega uses pair-distances. Between unaligned sequences these
+are so called k-tuple distance, between aligned sequences they are
+full alignment distances, as employed by Squid. These values range
+between 0.0 (identical) and 1.0 (completely different). The distances
+are used to construct the guide-tree and are by default outputted if
+--distmat-out is specified (and --full and/or --full-iter are
+set). For full alignment distances there is a so called Kimura
+correction [7] which more closely reflects evolutionary
+distance. Kimura-corrected distances range from 0.0 (identical) to
+theoretically infinity (completely different). In practice there
+appears to be a maximum value. In Clustal-Omega these Kimura-corrected
+distance can be outputted for protein if the --use-kimura flag is
+specified. Kimura correction is not available for DNA/RNA. Up to and
+including version 1.1.1 Kimura-corrected distances were outputted by
+default (where possible). Since version 1.2.0 the default is to output
+uncorrected distances.
+
+
+Pair-distances closely correspond to percentage pair-wise identities
+through i=100*(1-d), where i is the percentage pair-wise identity and
+d is the pair-wise distance. Percentage pair-wise identities can be
+outputted in Clustal-Omega instead of the distance matrix by
+specifying the --percent-id flag as well as --distmat-out, --full
+and/or --full-iter. Percentage pair-wise identities cannot be
+outputted if --use-kimura is specified.
+
+
+ALIGNMENT OUTPUT:
+
+ -o, --out, --outfile={file,-}
+ Multiple sequence alignment output file (default: stdout)
+
+ --outfmt={a2m=fa[sta],clu[stal],msf,phy[lip],selex,st[ockholm],vie[nna]}
+ MSA output file format (default: fasta)
+
+ --residuenumber, --resno
+ in Clustal format print residue numbers (default no)
+
+ --wrap=<n>
+ number of residues before line-wrap in output
+
+ --output-order={input-order,tree-order}
+ MSA output order like in input/guide-tree
+
+
+By default Clustal-Omega writes its results (alignments) to stdout. An
+output file can be specified with the -o flag. Output to stdout is not
+possible in verbose mode (-v, see MISCELLANEOUS) as verbose/debugging
+messages would interfere with the alignment output. By default,
+alignment files are not over-written, if a file with the specified
+name already exists. In this case Clustal-Omega aborts during the
+command-line processing stage. To force over-writing of already
+existing files use the --force flag (see MISCELLANEOUS).
+
+Clustal-Omega can output alignments in various formats by setting the
+--outfmt flag:
+
+ * for Fasta format set: --outfmt=a2m or --outfmt=fa or --outfmt=fasta
+
+ * for Clustal format set: --outfmt=clu or --outfmt=clustal
+
+ * for Msf format: set --outfmt= msf
+
+ * for Phylip format set: --outfmt=phy or --outfmt=phylip
+
+ * for Selex format set: --outfmt=selex
+
+ * for Stockholm format set: --outfmt=st or --outfmt=stockholm
+
+ * for Vienna format set: --outfmt=vie or --outfmt=vienna
+
+In ClustalW one could print the residue number of the last residue in
+each line in Clustal-Format. This feature can be turned on by setting
+the --resno or --residuenumber flag.
+
+The line lengths in Clustal Format is usually 60 residues, in Fasta
+format it is usually 60 or 80 residues. This value can be set using
+the --wrap flag.
+
+By default the order of sequences in the output is the same as in the
+input (--output-order=input-order). This can be changed to the order
+in which the sequences appear in the guide-tree by setting
+--output-order=tree-order.
+
+
+ITERATION:
+
+ --iterations, --iter=<n> Number of (combined guide tree/HMM) iterations
+
+ --max-guidetree-iterations=<n> Maximum guide tree iterations
+
+ --max-hmm-iterations=<n> Maximum number of HMM iterations
+
+
+By default, Clustal-Omega calculates (or reads in) a guide tree and
+performs a multiple alignment in the order specified by this guide
+tree. This alignment is then outputted. Clustal-Omega can 'iterate'
+its guide tree. The hope is that the full alignment distances, that
+can be derived from the initial alignment, will give rise to a better
+guide tree, and by extension, to a better alignment.
+
+A similar rationale applies to HMM-iteration. MSAs in general are very
+'vulnerable' at their early stages. Sequences that are aligned at an
+early stage remain fixed for the rest of the MSA. Another way of
+putting this is: 'once a gap, always a gap'. This behaviour can be
+mitigated by HMM iteration. An initial alignment is created and turned
+into a HMM. This HMM can help in a new round of MSA to 'anticipate'
+where residues should align. This is using the HMM as an External
+Profile and carrying out iterative EPA. In practice, individual
+sequences and profiles are aligned to the External HMM, derived after
+the initial alignment. Pseudo-count information is then transferred to
+the (internal) HMM, corresponding to the individual
+sequence/profile. The now somewhat 'softened' sequences/profiles are
+then in turn aligned in the order specified by the guide
+tree. Pseudo-count transfer is reduced with the size of the
+profile. Individual sequences attain the greatest pseudo-count
+transfer, larger profiles less so. Pseudo-count transfer to profiles
+larger than, say, 10 is negligible. The effect of HMM iteration is
+more pronounced in larger test sets (that is, with more sequences).
+
+Both, HMM- and guide tree-iteration come at a cost of increasing the
+run-time. One round of guide tree iteration adds on (roughly) the time
+it took to construct the initial alignment. If, for example, the
+initial alignment took 1min, then it will take (roughly) 2min to
+iterate the guide tree once, 3min to iterate the guide tree twice, and
+so on. HMM-iteration is more costly, as each round of iteration adds
+three times the time required for the alignment stage. For example, if
+the initial alignment took 1min, then each additional round of HMM
+iteration will add on 3min; so 4 iterations will take 13min
+(=1min+4*3min). The factor of 3 stems from the fact that at every
+stage both intermediate profiles have to be aligned with the
+background HMM, and finally the (softened) HMMs have to be aligned as
+well. All times are quoted for single processors.
+
+By default, guide tree iteration and HMM-iteration are coupled. This
+means, at each iteration step both, guide tree and HMM, are
+re-calculated. This is invoked by setting the --iter flag. For
+example, if --iter=1, then first an initial alignment is produced
+(without external HMM background information and using k-tuple
+distances to calculate the guide tree). This initial alignment is then
+used to re-calculate a new guide tree (using full alignment distances)
+and to create a HMM. The new guide tree and the HMM are then used to
+produce a new MSA.
+
+Iteration of guide tree and HMM can be de-coupled. This means that the
+number of guide tree iterations and HMM iterations can be
+different. This can be done by combining the --iter flag with the
+--max-guidetree-iterations and/or the --max-hmm-iterations flag. The
+number of guide tree iterations is the minimum of --iter and
+--max-guidetree-iterations, while the number of HMM iterations is the
+minimum of --iter and --max-hmm-iterations. If, for example, HMM
+iteration should be performed 5 times but guide tree iteration should
+be performed only 3 times, then one should set --iter=5 and
+--max-guidetree-iterations=3. All three flags can be specified at the
+same time (however, this makes no sense). It is not sufficient just to
+specify --max-guidetree-iterations and --max-hmm-iterations but not
+--iter. If any iteration is desired, then --iter has to be
+set. Conversely, if no alignment is desired but only distance
+calculation and tree construction, then --max-hmm-iterations=-1 will
+terminate the calculation before the alignment stage; --iter does not
+have to be specified in this case.
+
+
+LIMITS (will exit early, if exceeded):
+
+ --maxnumseq=<n> Maximum allowed number of sequences
+
+ --maxseqlen=<l> Maximum allowed sequence length
+
+Limits can be imposed on the number of sequences in the input file
+and/or the lengths of the sequences. This cap can be set with the
+--maxnumseq and --maxseqlen flags, respectively. Clustal-Omega will
+exit early, if these limits are exceeded.
+
+
+MISCELLANEOUS:
+
+ --auto Set options automatically (might overwrite some of your options)
+
+ --threads=<n> Number of processors to use
+
+ -l, --log=<file> Log all non-essential output to this file
+
+ -h, --help Print help and exit
+
+ -v, --verbose Verbose output (increases if given multiple times)
+
+ --version Print version information and exit
+
+ --long-version Print long version information and exit
+
+ --force Force file overwriting
+
+
+Users may feel unsure which options are appropriate in certain
+situations even though using ClustalO without any special options
+should give you the desired results. The --auto flag tries to
+alleviate this problem and selects accuracy/speed flags according to
+the number of sequences. For all cases will use mBed and thereby
+possibly overwrite the --full option. For more than 1,000 sequences
+the iteration is turned off as the effect of iteration is more
+noticeable for 'larger' problems. Otherwise iterations are set to 1 if
+not already set to a higher value by the user. Expert users may want
+to avoid this flag and exercise more fine tuned control by selecting
+the appropriate options manually.
+
+Certain parts of the MSA calculation have been parallelised. Most
+noticeably, the distance matrix calculation, and certain aspects of
+the HMM building stage. Clustal-Omega uses OpenMP. By default,
+Clustal-Omega will attempt to use as many threads as possible. For
+example, on a 4-core machine Clustal-Omega will attempt to use 4
+threads. The number of threads can be limited by setting the --threads
+flag. This may be desirable, for example, in the case of
+benchmarking/timing.
+
+Usually, non-essential (verbose) output is written to screen. This
+output can be written to file by specifying the --log flag.
+
+Help is available by specifying the -h flag.
+
+By default Clustal-Omega does not print any information to stdout
+(other than the final alignment, if no output file is
+specified). Information concerning the progress of the alignment can
+be obtained by specifying one verbosity flag (-v). This may be
+desirable, to verify what Clustal-Omega is actually doing at the
+moment. If two verbosity flags (-v -v) are specified, command-line
+flags (explicitly and implicitly set) are printed in addition to the
+progress report. Triple verbose level (-v -v -v) is the most verbose
+level. In addition to single- and double-verbose information much more
+information is displayed: input sequences and names, details of the
+tree construction and intermediate alignments. Tree construction
+information includes pairwise distances. The number of pairwise
+distances scales with the square of the number of sequences, and
+double verbose mode is probably only useful for a small number of
+sequences.
+
+The current version number of Clustal-Omega can be displayed by
+setting the --version flag.
+
+The current version number of Clustal-Omega as well as the code-name
+and the build date can be displayed by setting the --long-version
+flag.
+
+By default, Clustal-Omega does not over-write files. These can be (i)
+alignment output, (ii) distance matrix and (iii) guide
+tree. Overwriting can be forced by setting the --force flag.
+
+
+EXAMPLES:
+
+./clustalo -i globin.fa
+
+Clustal-Omega reads the sequence file globin.fa, aligns the sequences
+and prints the result to screen in fasta/a2m format.
+
+
+./clustalo -i globin.fa -o globin.sto --outfmt=st
+
+If the file globin.sto does not exist, then Clustal-Omega reads the
+sequence file globin.fa, aligns the sequences and prints the result to
+globin.sto in Stockholm format. If the file globin.sto does exist
+already, then Clustal-Omega terminates the alignment process before
+reading globin.fa.
+
+
+./clustalo -i globin.fa -o globin.aln --outfmt=clu --force
+
+Clustal-Omega reads the sequence file globin.fa, aligns the sequences
+and prints the result to globin.aln in Clustal format, overwriting the
+file globin.aln, if it already exists.
+
+
+./clustalo -i globin.fa --distmat-out=globin.mat --guidetree-out=globin.dnd --force
+
+Clustal-Omega reads the sequence file globin.fa, aligns the sequences,
+prints the result to screen in fasta/a2m format (default), the guide
+tree to globin.dnd and the distance matrix to globin.mat, overwriting
+those files if they already exist.
+
+
+./clustalo -i globin.fa --guidetree-in=globin.dnd
+
+Clustal-Omega reads the files globin.fa and globin.dnd, skipping
+distance calculation and guide tree creation, using instead the guide
+tree specified in globin.dnd.
+
+
+./clustalo -i globin.fa --hmm-in=PF00042.hmm
+
+Clustal-Omega reads the sequence file globin.fa and the HMM file
+PF00042.hmm (in HMMer2 or HMMer3 format). It then performs the
+alignment, transferring pseudo-count information contained in
+PF00042.hmm to the sequences/profiles during the MSA.
+
+
+./clustalo -i globin.sto
+
+Clustal-Omega reads the file globin.sto (of aligned sequences in
+Stockholm format). It converts the alignment into a HMM, de-aligns the
+sequences and re-aligns them, transferring pseudo-count information to
+the sequences/profiles during the MSA. The guide tree is constructed
+using a full distance matrix.
+
+
+./clustalo -i globin.sto --dealign
+
+Clustal-Omega reads the file globin.sto (of aligned sequences in
+Stockholm format). It de-aligns the sequences and then re-aligns
+them. No HMM is produced in the process, no pseudo-count information
+is transferred. Consequently, the output must be the same as for
+unaligned output (like in the first example ./clustalo -i globin.fa)
+
+
+./clustalo -i globin.fa --iter=2
+
+Clustal-Omega reads the file globin.fa, creates a UPGMA guide tree
+built from k-tuple distances, and performs an initial alignment. This
+initial alignment is converted into a HMM and a new guide tree is
+built from the (preliminary) full alignment distances of the initial
+alignment. The un-aligned sequences are then aligned (for the second
+time but this time) using pseudo-count information from the HMM
+created after the initial alignment (and using the new guide
+tree). This second alignment is then again converted into a HMM and a
+new guide tree is constructed. The un-aligned sequences are then
+aligned (for a third time), again using pseudo-count information of
+the HMM from the previous step and the most recent guide tree. The
+final alignment is written to screen.
+
+
+./clustalo -i globin.fa --iter=5 --max-guidetree-iterations=1
+
+Clustal-Omega reads the file globin.fa, creates a UPGMA guide tree
+built from k-tuple distances, and performs an initial alignment. This
+initial alignment is converted into a HMM and a new guide tree is
+built from the (preliminary) full alignment distances of the initial
+alignment. The un-aligned sequences are then aligned (for the second
+time but this time) using pseudo-count information from the HMM
+created after the initial alignment (and using the new guide
+tree). For the last 4 iterations the guide tree is left unchanged and
+only HMM iteration is performed. This means that intermediate
+alignments are converted to HMMs, and these intermediate HMMs are used
+to guide the MSA during subsequent iteration stages.
+
+
+./clustalo -i globin.fa -o globin.a2m -v
+
+In case the file globin.a2m does not exist, Clustal-Omega reads the
+file globin.fa, prints a progress report to screen and writes the
+alignment in (default) Fasta format to globin.a2m. The progress report
+consists of the number of threads used, the number of sequences read,
+the current progress in the k-tuple distance calculation, completion
+of the guide tree computation and current progress of the MSA stage.
+If the file globin.a2m already exists Clustal-Omega aborts before
+reading the file globin.fa. Note that in verbose mode an output file
+has to be specified, because progress/debugging information, which is
+printed to screen, would interfere with the alignment being printed to
+screen.
+
+
+./clustalo -i PF00042_full.fa --dealign --full --outfmt=vie -o PF00042_full.vie --force
+
+Clustal-Omega reads the file PF00042_full.fa. This file contains
+several thousand aligned sequences. --dealign tells Clustal-Omega to
+erase all alignment information and re-align the sequences from
+scratch. As there are several thousand sequences calculating a full
+distance matrix may be slow. Setting the --full flag specifically
+selects the full distance mode over the default mBed mode. The
+alignment is then written out in Vienna format (fasta format all on
+one line, no line breaks per sequence) to file PF00042_full.vie.
+
+
+./clustalo -i PF00042_full.fa --dealign --outfmt=vie -o PF00042_full.vie --force
+
+Clustal-Omega reads the file PF00042_full.fa. This file contains
+several thousand aligned sequences. --dealign tells Clustal-Omega to
+erase all alignment information and re-align the sequences from
+scratch. Calculating the distance matrix will be done by mBed
+(default). Clustal-Omega will calculate pairwise distances to a
+small number of reference sequences only. This will give a significant
+speed-up. The speed-up is greater for larger families (more
+sequences). The alignment is then written out in Vienna format (fasta
+format all on one line, no line breaks per sequence) to file
+PF00042_full.vie.
+
+
+./clustalo --p1=globin.sto --p2=PF00042_full.vie -o globin+pf00042.fa
+
+Clustal-Omega reads files globin.sto and PF00042_full.vie of aligned
+sequences (profiles). Both profiles are then aligned. The relative
+positions of residues in both profiles are not changed during this
+alignment, however, columns of gaps may be inserted into the profiles,
+respectively. The final alignment is written to file globin+pf00042.fa
+in fasta format.
+
+
+./clustalo -i globin.fa --p1=PF00042_full.vie -o pf00042+globin.fa
+
+Clustal-Omega reads file globin.fa of un-aligned sequences and the
+profile (of aligned sequences) in file PF00042_full.vie. A HMM is
+created from the profile. This HMM is used to guide the alignment of
+the un-aligned sequences in globin.fa. The profile that was generated
+during this alignment of un-aligned globin.fa sequences is then
+aligned to the input profile PF00042_full.vie. The relative positions
+of residues in profile PF00042_full.vie is not changed during this
+alignment, however, columns of gaps may be inserted into the
+profile. The final alignment is output to file pf00042+globin.fa in
+fasta format. The alignment in this example may be slightly different
+from the alignment in the previous example, because no HMM guidance
+was used generate the profile globin.sto. In this example HMM guidance
+was used to align the sequences in globin.fa; the hope being that this
+intermediate alignment will have profited from the bigger profile.
+
+
+./clustalo -i globin.fa --clustering-out=globin.aux --cluster-size=3
+
+globin.fa contains 7 sequences. Usually a full distance matrix is
+created for less than 100 sequences. This is over-written by
+specifying --cluster-size=3. Clustal-Omega attempts to create clusters
+of no more than 3 sequences. This clustering is recorded in the file
+globin.aux which looks like
+
+ Cluster 0: object 0 has index 0 (=seq P1|HBB_HUMAN ) 00
+ Cluster 0: object 1 has index 1 (=seq P1|HBB_HORSE ) 00
+ Cluster 1: object 0 has index 4 (=seq P1|MYG_PHYCA ) 1
+ Cluster 1: object 1 has index 5 (=seq P1|GLB5_PETMA ) 1
+ Cluster 1: object 2 has index 6 (=seq P1|LGB2_LUPLU ) 1
+ Cluster 2: object 0 has index 2 (=seq P1|HBA_HUMAN ) 01
+ Cluster 2: object 1 has index 3 (=seq P1|HBA_HORSE ) 01
+
+There are 3 clusters, named Cluster~0, Cluster~1 and
+Cluster~2. Cluster~0 has 2 sequences, which are sequence 0 and 1 from
+the input file, named P1|HBB_HUMAN and P1|HBB_HORSE. Cluster~1 has 3
+sequences, sequences 4,5,6 from the input file and Cluster~2 has 2
+sequences, sequences 2 and 3 from the input file. The binary string at
+the end of each line encode the bi-section that led to this
+clustering. The first digit indicated the initial split. The '0'
+indicates that in the first split sequences 0,1,2,3 were grouped
+together and the '1' that sequences 4,5,6 were grouped together. The
+size of Cluster~1 does not exceed --cluster-size, so it does need to
+be broken up. The Cluster (with the initial '0') containing sequences
+0,1,2,3 is comprised of 4 sequences; this number exceed
+--cluster-size, so that it will have to be broken up. This second
+split is indicated by the second digit of the binary string. The
+second '0' indicates that sequences 0,1 fall into one Cluster (which
+will ultimately be Cluster~0), and the second '1' indicates that
+sequences 2,3 fall into another cluster (ultimately Cluster~2).
+
+
+LITERATURE:
+
+[1] Johannes Soding (2005) Protein homology detection by HMM-HMM
+ comparison. Bioinformatics 21 (7): 951–960.
+
+[2] Blackshields G, Sievers F, Shi W, Wilm A, Higgins DG. Sequence
+ embedding for fast construction of guide trees for multiple
+ sequence alignment. Algorithms Mol Biol. 2010 May 14;5:21.
+
+[3] http://www.genetics.wustl.edu/eddy/software/#squid
+
+[4] Wilbur and Lipman, 1983; PMID 6572363
+
+[5] Thompson JD, Higgins DG, Gibson TJ. (1994). CLUSTAL W: improving
+ the sensitivity of progressive multiple sequence alignment through
+ sequence weighting, position-specific gap penalties and weight
+ matrix choice. Nucleic Acids Res., 22, 4673-4680.
+
+[6] Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA,
+ McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD,
+ Gibson TJ, Higgins DG. (2007). Clustal W and Clustal X version
+ 2.0. Bioinformatics, 23, 2947-2948.
+
+[7] Kimura M (1980). "A simple method for estimating evolutionary
+ rates of base substitutions through comparative studies of
+ nucleotide sequences". Journal of Molecular Evolution 16: 111–120.
+
+[8] Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high
+ accuracy and high throughput.Nucleic Acids Res. 32(5):1792-1797.
+
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