Align multiple protein or nucleotide sequences with high-accuracy PPP refinement. Learn more
Run
5 credits
Output
Configure inputs to begin
Set options on the left, then click “Align Sequences”.
Align multiple protein or nucleotide sequences with high-accuracy PPP refinement. Learn more
Configure inputs to begin
Set options on the left, then click “Align Sequences”.
Align multiple protein or nucleotide sequences with high-accuracy PPP refinement. Learn more
Configure inputs to begin
Set options on the left, then click “Align Sequences”.
MUSCLE5 is a multiple sequence alignment method for proteins and nucleic acids. Version 5 introduced improved guide-tree construction, the high-accuracy PPP algorithm, a scalable Super5 mode, and alignment ensembles for measuring uncertainty.
ProteinIQ runs the verified MUSCLE 5.3 Linux release. MUSCLE detects the sequence alphabet itself, matching the source program's default behavior.
| Mode | Best for | Behavior |
|---|---|---|
| PPP | Routine and accuracy-focused alignment | Native align mode and the default in MUSCLE 5.3. |
| Super5 | Large datasets, especially around 1,000 sequences or more | Faster, source-provided approximation. Super5 returns one alignment and does not support PPP ensembles. |
MUSCLE does not automatically switch from PPP to Super5. Select Super5 explicitly when runtime or dataset size makes PPP impractical.
The default result is one alignment. PPP mode also supports two native ensemble strategies:
Ensemble members have approximately comparable expected accuracy; their order is not a quality ranking. Use agreement across members to identify robust columns and disagreement to identify alignment uncertainty.
Ensembles consume compute in proportion to the number of alignments generated, so the credit estimate scales by the requested ensemble size.
Submit one FASTA record or a multi-record FASTA file. Each record begins with > and a sequence name, followed by one or more sequence lines.
>human_insulin
MALWMRLLPLLAVTFLAGCGAKSQVQLVESGGGLVQPGGSLRLSCAASGFTFSGYY
>mouse_insulin
MALWMRLLPLLAVTFLAGCGAKSSVQLLESGGGLVQPGGSLRLSCAASGFTFSGYY
>zebrafish_insulin
MQLWMRLPPLAVTFLVLCGAKSSVQLVESGGGLVQPGGSLRLSCAASGFTFSGYYMUSCLE 5.3 accepts a single sequence and returns it unchanged as a valid one-record alignment. Protein, DNA, and RNA inputs are supported. Do not mix different alphabets in one job.
| Setting | Default | Notes |
|---|---|---|
| Alignment mode | PPP | Select Super5 for a faster large-dataset alignment. |
| Alignment result | Single alignment | Stratified and diversified ensembles are available in PPP mode. |
| Replicates per permutation | 4 | Stratified mode creates four times this number of alignments; ProteinIQ allows 2-10 because MUSCLE 5.3 treats 1 as a single-alignment request. |
| Ensemble size | 100 | Diversified mode; ProteinIQ allows 2-100. |
| Guide-tree permutation | Native default | Optional abc, acb, or bca permutation for a single alignment. |
| Perturbation seed | 0 | A positive integer applies native guide-tree perturbation. |
| Preserve input order | Off | Keeps submitted sequence order instead of MUSCLE tree order. |
A single-alignment job returns:
.afa) file used by the MSA viewer and available to downstream workflows;An ensemble job additionally returns:
.efa) archive;The first ensemble member opens in the MSA viewer for convenience. It is not a best-scoring or recommended member.
Jobs run with 8 CPU cores and 8 GB of memory. The native process can run for up to 115 minutes within a 120-minute job limit. Diversified ensembles and large PPP alignments can take substantially longer than one alignment; use Super5 or reduce the dataset if a job approaches the limit.
Accepted file extensions are .fasta, .fa, .fas, and .txt, with a 50 MB upload limit. Account-level text and job limits also apply.
To keep ensemble result bundles reliable, the estimated and generated combined EFA archive is limited to 50 MB, and the EFA plus split AFA files and diagnostics are limited to 100 MB in total. Reduce the input size or ensemble count if a request would exceed either output budget.
For a different speed-accuracy tradeoff or additional output formats, use MAFFT or Clustal Omega. For database-scale sequence search, use MMseqs2.
Edgar RC. MUSCLE v5 enables improved estimates of phylogenetic tree confidence by ensemble bootstrapping. Nature Communications 13, 6968 (2022). https://doi.org/10.1038/s41467-022-34630-w
Perform multiple sequence alignment using MAFFT (Multiple Alignment using Fast Fourier Transform). Supports multiple algorithms from fast progressive to highly accurate iterative methods.
Calculate batched Levenshtein distances and Needleman–Wunsch or Smith–Waterman sequence scores with StringZilla v5 CPU acceleration.
Ultra-fast sequence search and clustering. 10,000x faster than BLAST for database searches, with powerful sequence clustering capabilities for proteins and nucleotides.
USAlign (Universal Structure Alignment) aligns protein, RNA, and DNA structures to compute TM-scores and generate superposed structures. Compare 3D structures to assess structural similarity.
Perform multiple sequence alignment on protein or nucleotide sequences using the Clustal Omega algorithm.
Infer approximately-maximum-likelihood phylogenetic trees from alignments of nucleotide or protein sequences.
Build phylogenetic trees using maximum likelihood with automatic model selection (ModelFinder) and ultrafast bootstrap support.
Rapidly align and compare DNA sequences using MUMmer4 nucmer. Perform pairwise genome comparisons to identify SNPs, indels, and structural variants between reference and query genomes.
Analyze immunoglobulin (antibody) and T cell receptor variable domain sequences. Identifies V/D/J gene segments, delineates CDR regions, and analyzes rearrangement junctions.
Fast protein structure search, comparison, and clustering. Search your structure against 200M+ AlphaFold predictions, compare 2 structures, or cluster up to 2500.
MUSCLE5 is a multiple sequence alignment method for proteins and nucleic acids. Version 5 introduced improved guide-tree construction, the high-accuracy PPP algorithm, a scalable Super5 mode, and alignment ensembles for measuring uncertainty.
ProteinIQ runs the verified MUSCLE 5.3 Linux release. MUSCLE detects the sequence alphabet itself, matching the source program's default behavior.
| Mode | Best for | Behavior |
|---|---|---|
| PPP | Routine and accuracy-focused alignment | Native align mode and the default in MUSCLE 5.3. |
| Super5 | Large datasets, especially around 1,000 sequences or more | Faster, source-provided approximation. Super5 returns one alignment and does not support PPP ensembles. |
MUSCLE does not automatically switch from PPP to Super5. Select Super5 explicitly when runtime or dataset size makes PPP impractical.
The default result is one alignment. PPP mode also supports two native ensemble strategies:
Ensemble members have approximately comparable expected accuracy; their order is not a quality ranking. Use agreement across members to identify robust columns and disagreement to identify alignment uncertainty.
Ensembles consume compute in proportion to the number of alignments generated, so the credit estimate scales by the requested ensemble size.
Submit one FASTA record or a multi-record FASTA file. Each record begins with > and a sequence name, followed by one or more sequence lines.
>human_insulin
MALWMRLLPLLAVTFLAGCGAKSQVQLVESGGGLVQPGGSLRLSCAASGFTFSGYY
>mouse_insulin
MALWMRLLPLLAVTFLAGCGAKSSVQLLESGGGLVQPGGSLRLSCAASGFTFSGYY
>zebrafish_insulin
MQLWMRLPPLAVTFLVLCGAKSSVQLVESGGGLVQPGGSLRLSCAASGFTFSGYYMUSCLE 5.3 accepts a single sequence and returns it unchanged as a valid one-record alignment. Protein, DNA, and RNA inputs are supported. Do not mix different alphabets in one job.
| Setting | Default | Notes |
|---|---|---|
| Alignment mode | PPP | Select Super5 for a faster large-dataset alignment. |
| Alignment result | Single alignment | Stratified and diversified ensembles are available in PPP mode. |
| Replicates per permutation | 4 | Stratified mode creates four times this number of alignments; ProteinIQ allows 2-10 because MUSCLE 5.3 treats 1 as a single-alignment request. |
| Ensemble size | 100 | Diversified mode; ProteinIQ allows 2-100. |
| Guide-tree permutation | Native default | Optional abc, acb, or bca permutation for a single alignment. |
| Perturbation seed | 0 | A positive integer applies native guide-tree perturbation. |
| Preserve input order | Off | Keeps submitted sequence order instead of MUSCLE tree order. |
A single-alignment job returns:
.afa) file used by the MSA viewer and available to downstream workflows;An ensemble job additionally returns:
.efa) archive;The first ensemble member opens in the MSA viewer for convenience. It is not a best-scoring or recommended member.
Jobs run with 8 CPU cores and 8 GB of memory. The native process can run for up to 115 minutes within a 120-minute job limit. Diversified ensembles and large PPP alignments can take substantially longer than one alignment; use Super5 or reduce the dataset if a job approaches the limit.
Accepted file extensions are .fasta, .fa, .fas, and .txt, with a 50 MB upload limit. Account-level text and job limits also apply.
To keep ensemble result bundles reliable, the estimated and generated combined EFA archive is limited to 50 MB, and the EFA plus split AFA files and diagnostics are limited to 100 MB in total. Reduce the input size or ensemble count if a request would exceed either output budget.
For a different speed-accuracy tradeoff or additional output formats, use MAFFT or Clustal Omega. For database-scale sequence search, use MMseqs2.
Edgar RC. MUSCLE v5 enables improved estimates of phylogenetic tree confidence by ensemble bootstrapping. Nature Communications 13, 6968 (2022). https://doi.org/10.1038/s41467-022-34630-w
Perform multiple sequence alignment using MAFFT (Multiple Alignment using Fast Fourier Transform). Supports multiple algorithms from fast progressive to highly accurate iterative methods.
Calculate batched Levenshtein distances and Needleman–Wunsch or Smith–Waterman sequence scores with StringZilla v5 CPU acceleration.
Ultra-fast sequence search and clustering. 10,000x faster than BLAST for database searches, with powerful sequence clustering capabilities for proteins and nucleotides.
USAlign (Universal Structure Alignment) aligns protein, RNA, and DNA structures to compute TM-scores and generate superposed structures. Compare 3D structures to assess structural similarity.
Perform multiple sequence alignment on protein or nucleotide sequences using the Clustal Omega algorithm.
Infer approximately-maximum-likelihood phylogenetic trees from alignments of nucleotide or protein sequences.
Build phylogenetic trees using maximum likelihood with automatic model selection (ModelFinder) and ultrafast bootstrap support.
Rapidly align and compare DNA sequences using MUMmer4 nucmer. Perform pairwise genome comparisons to identify SNPs, indels, and structural variants between reference and query genomes.
Analyze immunoglobulin (antibody) and T cell receptor variable domain sequences. Identifies V/D/J gene segments, delineates CDR regions, and analyzes rearrangement junctions.
Fast protein structure search, comparison, and clustering. Search your structure against 200M+ AlphaFold predictions, compare 2 structures, or cluster up to 2500.