Clustal Omega

What is Clustal Omega?

Clustal Omega is a multiple sequence alignment (MSA) program that aligns protein or nucleotide sequences to reveal conserved regions, evolutionary relationships, and functional motifs. MSA is a foundational step in many bioinformatics workflows—from phylogenetic analysis to structure prediction to primer design.

Clustal Omega can handle datasets ranging from a handful of sequences to tens of thousands, making it suitable for both focused studies and large-scale comparative genomics. Once you have an alignment, you can use FastTree to build a phylogenetic tree from it.

Inputs and modes

ProteinIQ runs Clustal Omega 1.2.4. Choose Align sequences, Align two profiles, or Add sequences to a profile. A profile is an existing alignment whose columns are retained during profile alignment. The sequence mode requires at least two sequences; requested distance-matrix or guide-tree files require at least three because the native two-sequence shortcut does not emit them.

Sequence and profile inputs accept FASTA/A2M, Clustal, Phylip, MSF, Stockholm, SELEX, and Vienna-style single-line FASTA. Gzip FASTA uploads are expanded automatically. Each input file has a 50 MiB limit, including expanded gzip content. Native format detection is the default. The advanced input-format control passes your explicit choice to Clustal Omega; files still need to follow that reader's grammar.

Optional inputs provide an existing distance matrix, a Newick guide tree, HMMER-format HMMs, a per-sequence HMM mapping, or native pseudocount parameters. HMM mappings use a sequence identifier followed by uploaded HMM filenames, separated by spaces. Every referenced file must be included in HMM files, and filenames must be distinct. Clustal Omega's global HMM input uses its native behavior; adding multiple files does not mean all models contribute to every sequence.

The native pseudocount file alternates a parameter name and its numeric value on separate lines, for example gapb followed by 0.5, then pca followed by 0.5. Names and values on one line do not follow the 1.2.4 parser's grammar.

Ordinary FASTA descriptions and repeated identifiers are retained. The sequence input also accepts >name SEQUENCE shorthand entries and # comment lines in FASTA. Profile files pass through unchanged. For reproducible interchange, use ordinary multiline FASTA with unique identifiers.

Credits

The existing residue-based price uses a five-credit minimum. The exact quote appears before submission. Sequence and profile residues contribute to the quote; distance matrices, guide trees, HMMs, mapping files, and pseudocount files do not. Selecting another output format or advanced native controls does not change the current tariff.

How does Clustal Omega work?

Clustal Omega uses a progressive alignment strategy: it first estimates how similar sequences are to each other, builds a guide tree from those similarities, then aligns sequences following the tree order. The key innovations are the mBed algorithm for scalability and HMM-based profile alignment for accuracy.

The mBed algorithm

Traditional pairwise distance calculation scales as $O(N^2)$, which becomes prohibitive for large datasets. The mBed algorithm reduces this to $O(N \log N)$ by "embedding" each sequence into a low-dimensional space.

Instead of comparing every sequence to every other sequence, mBed selects a small set of reference sequences and represents each sequence as a vector of distances to these references. These vectors can be clustered rapidly using k-means, with a default cluster size of 100 sequences. The advanced cluster-size control changes that value. Full distance matrices are only computed within clusters, not across the entire dataset.

HMM-based profile alignment

When combining two groups of aligned sequences (profiles), Clustal Omega uses hidden Markov model alignment via the HHalign package. Each profile is converted to an HMM with match, insert, and delete states. Aligning two HMMs rather than simple position-specific scoring matrices improves sensitivity for distantly related sequences.

Guide tree and progressive alignment

The distance matrix (partial or full) is used to construct a guide tree via UPGMA. This tree determines the order of pairwise alignments: closely related sequences are aligned first, then progressively merged with more distant groups until all sequences are incorporated.

Alignment settings

Sequence type

Clustal Omega auto-detects whether your sequences are protein or nucleotide by examining character composition. Manual selection (Protein, DNA, or RNA) is useful when auto-detection might be ambiguous—for example, with very short sequences or sequences containing unusual characters.

Output format

• FASTA: Aligned sequences with gaps represented as -. Most compatible with downstream tools including FastTree.
• Clustal: Traditional format showing alignment blocks with conservation symbols. Good for visual inspection.
• Phylip: Fixed-width format used by phylogenetic programs.
• MSF: GCG format, useful for legacy software.
• Stockholm: Annotated format used by Pfam and Rfam databases.
• SELEX: Named alignment rows, with optional annotations.
• Vienna: FASTA records with each sequence on one line.

Refinement iterations

After the initial alignment, Clustal Omega can refine it by rebuilding the guide tree from the alignment itself (rather than pairwise distances) and realigning. Each iteration uses the preceding alignment to construct the next guide tree.

ProteinIQ supports integer values from 0 to 5. Use 1-2 iterations when you want to evaluate whether refinement helps your dataset. For exploratory work or very large datasets, skip refinement (0 iterations, the native default) to save time.

Full distance matrix

By default, mBed approximates the information needed for scalable guide-tree construction. Enabling the full distance matrix computes every pairwise distance at the cost of $O(N^2)$ time and memory.

The full matrix is useful when you specifically need all pairwise distances or want to compare guide-tree construction methods. It does not guarantee a better biological alignment. For large datasets, mBed is usually the practical choice.

Output order

Controls whether sequences in the output follow your original input order or are reordered by the guide tree. Tree order groups similar sequences together, which can make visual inspection easier and is the natural order for phylogenetic workflows. Input order (the default) preserves whatever order you provided.

Remove existing alignment

Clustal Omega can use an existing input alignment to build an HMM before it realigns the underlying sequences. Enable this option when you want to discard that existing alignment information and realign the ungapped sequences from scratch.

Output distance matrix

Produces an additional output file containing Clustal Omega's native pair distances. By default, values range from 0 for identical aligned residues to 1 when no aligned residues are identical. Advanced controls can request Kimura correction for aligned protein sequences or percent identities instead. These two controls cannot be combined. The result records which matrix convention was requested.

Output guide tree

Produces an additional Newick-format tree file showing the guide tree used to order the progressive alignment. This is the UPGMA tree built from pairwise distances (or mBed-approximated distances). It is an operational alignment tree, not a phylogenetic inference.

Advanced native controls

Control	Default	Behavior
Native input format	Auto-detect	Select the native sequence/profile reader explicitly.
Treat sequences as a profile	Off	Treat aligned input as a profile, including alignments without gaps.
Full matrices during refinement	Off	Use full pairwise matrices during guide-tree refinement.
mBed cluster size	100	Set the native positive integer cluster-size parameter.
Pileup alignment	Off	Select the native pileup path.
Transitivity	0	Pass the native transitivity integer.
Maximum guide-tree iterations	2147483647	Independently cap guide-tree iterations. The default is the native unlimited sentinel.
Maximum HMM iterations	2147483647	Independently cap HMM iterations. A negative value skips writing the alignment in sequence modes; tree, matrix, diagnostic, and log files may still be returned. Profile-profile mode follows its native behavior and writes an alignment.
Kimura-corrected distances	Off	Request native correction for aligned protein sequences.
Percent identity matrix	Off	Return percent identities instead of distances.
Native automatic parameters	Off	Let Clustal Omega choose parameters, including overrides of matrix and iteration settings. Requested values remain in the log; the summary leaves automatically selected iteration/matrix values unspecified unless an emitted matrix establishes full-matrix computation.
Output line width	60	Set the native output wrapping width.
Show residue numbers	Off	Add numbering where the selected native output writer supports it.
Output clustering diagnostic	Off	Download the clustering diagnostic if the native path emits it.
Output posterior probabilities	Off	Download the native posterior output if available.

The Files tab contains the alignment, every emitted auxiliary file, and a run log with requested settings plus native stdout/stderr. Some native modes do not emit clustering or posterior diagnostics; those fields are marked omitted. Requested distance-matrix and guide-tree files must be produced for the run to complete successfully.

Native reader/writer quirks are preserved. For example, some MSF files emitted by this version contain weights its reader rejects. Use FASTA or Stockholm for straightforward reuse. The native explicit Vienna reader can reject files that automatic detection accepts. See the Clustal Omega documentation for file grammars and expert parameters.

Understanding the results

The output is a multiple sequence alignment where:

• Columns represent homologous positions across sequences
• Gap characters (-) indicate insertions or deletions relative to other sequences
• Conserved columns (same residue across all sequences) suggest functional or structural importance

The alignment length reported is the number of columns, which will be longer than any individual sequence due to gaps. High-quality alignments have fewer scattered gaps and more continuous aligned blocks.

Clustal Omega examples

Vertebrate beta-globins in a refined protein alignment

This example aligns beta-globin sequences from human, mouse, and chicken to show how Clustal Omega presents conserved positions and lineage-specific substitutions across homologous proteins.

• Inputs: UniProt P68871 (human HBB), P02088 (mouse HBB-B1), and P02112 (chicken HBB)
• Non-default settings: Sequence type = Protein, Output format = Clustal, and Refinement iterations = 1

The MSA viewer reports three sequences across 147 columns. Long conserved blocks are visible across the globin sequences, with substitutions distributed through the alignment. These shared columns are useful candidates for further functional or structural investigation, but the alignment alone does not establish residue function or a phylogenetic history.

E. coli lac operators with exact distances and a guide tree

This short-DNA example compares the submitted 5′→3′ lacO1, lacO2, and lacO3 operator strings. Manual DNA selection avoids ambiguity for 21-nucleotide inputs, while the additional outputs demonstrate the distance and guide-tree workflow.

• Inputs: E. coli lacO1 primary operator and lacO2/lacO3 auxiliary operators
• Non-default settings: Sequence type = DNA, Use full distance matrix = enabled, Output order = Tree order, Output distance matrix = enabled, and Output guide tree = enabled

The 21-column alignment groups the three operators in tree order and makes their conserved core pattern and substitutions directly inspectable. The output order reflects Clustal Omega's operational guide tree; it is not evidence of regulatory strength or evolutionary ancestry.

The Files view confirms that the run returned the aligned FASTA, the native uncorrected pair-distance matrix, and the Newick guide tree. The matrix values measure sequence difference in this alignment, not binding affinity, and the UPGMA tree is an alignment aid rather than a phylogenetic inference.

Human let-7 microRNAs in Stockholm format

This example aligns five mature human let-7 5p sequences to demonstrate explicit RNA handling, short-sequence conservation, and Stockholm output for downstream RNA-family workflows.

• Inputs: miRBase MIMAT0000062, MIMAT0000063, MIMAT0000064, MIMAT0000065, and MIMAT0000066
• Non-default settings: Sequence type = RNA, Output format = Stockholm, and Refinement iterations = 2

All five mature miRNAs fit within a 22-column alignment, with a strongly conserved 5′ region and family-specific substitutions visible in the viewer. This pattern supports positional comparison within the submitted family, but it does not by itself prove shared targets, expression, or biological function.

Common workflows

Clustal Omega is typically the first step in a multi-tool pipeline:

1. Phylogenetic analysis: Align sequences with Clustal Omega → Build tree with FastTree
2. Conservation analysis: Align sequences → Identify conserved regions for mutagenesis targets
3. Homology modeling: Align target to templates → Use alignment for structure prediction
4. Primer design: Align variants → Design primers in conserved regions

Limitations

Clustal Omega assumes sequences are homologous and alignable. It will produce an alignment even for unrelated sequences, but the result will be meaningless. Always verify that your sequences share evolutionary or functional relationships before aligning.

Very divergent sequences (below ~20% identity for proteins) may not align reliably with any progressive alignment method. Consider structure-based alignment for such cases.