From 9f81a03cf11728726a45917773e3c42195b471c2 Mon Sep 17 00:00:00 2001 From: pvtroshin Date: Fri, 12 Aug 2011 13:34:38 +0000 Subject: [PATCH] adding clustalO documentation git-svn-id: link to svn.lifesci.dundee.ac.uk/svn/barton/ptroshin/JABA2@4507 e3abac25-378b-4346-85de-24260fe3988d --- website/prog_docs/clustalo.txt | 619 ++++++++++++++++++++++++++++++++++++++++ 1 file changed, 619 insertions(+) create mode 100644 website/prog_docs/clustalo.txt diff --git a/website/prog_docs/clustalo.txt b/website/prog_docs/clustalo.txt new file mode 100644 index 0000000..109f65e --- /dev/null +++ b/website/prog_docs/clustalo.txt @@ -0,0 +1,619 @@ + + +CLUSTAL-OMEGA is a general purpose multiple sequence alignment program +for proteins. + + + +INTRODUCTION + +Clustal-Omega is a general purpose multiple sequence alignment (MSA) +program for proteins. 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 can only align protein sequences but +not DNA/RNA sequences. It is envisioned that DNA/RNA will become +available in a future version. + + + +SEQUENCE INPUT: + +-i, --in, --infile={,-} + Multiple sequence input file (- for stdin) + +--hmm-in= + HMM input files + +--dealign + Dealign input sequences + +--profile1, --p1= + Pre-aligned multiple sequence file (aligned columns will be kept fixed) + +--profile2, --p2= + Pre-aligned multiple sequence file (aligned columns will be kept fixed) + + +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). + + +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. + +The Clustal-Omega alignment engine can at the moment not process +DNA/RNA. If a sequence input file (i) or a profile (ii) is interpreted +as DNA/RNA the program will terminate during the file input stage. + + + +CLUSTERING: + + --distmat-in= + Pairwise distance matrix input file (skips distance computation) + + --distmat-out= + Pairwise distance matrix output file + + --guidetree-in= + Guide tree input file + (skips distance computation and guide tree clustering step) + + --guidetree-out= + 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) + + +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 sequences inputted via -i are aligned +Clustal-Omega uses the Kimura-corrected pairwise aligned identities +[7]. 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 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. + +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 the Kimura distance [7]. 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 Kimura +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). + + + +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) + + +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 + + +ITERATION: + + --iterations, --iter= Number of (combined guide tree/HMM) iterations + + --max-guidetree-iterations= Maximum guide tree iterations + + --max-hmm-iterations= 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 (Kimura) 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 Kimura 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 --iter has to be set. + + +LIMITS (will exit early, if exceeded): + + --maxnumseq= Maximum allowed number of sequences + + --maxseqlen= 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= Number of processors to use + + -l, --log= 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 of Kimura distances. + + +./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 Kimura 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 Kimura 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. + + + +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. + -- 1.7.10.2