nf-core pipeline ·
nf-core/multiplesequencealign
A Nextflow pipeline to run and systematically benchmark multiple sequence alignment methods and protein structures.
What nf-core/multiplesequencealign is
nf-core/multiplesequencealign is a bioinformatics analysis pipeline designed to execute and systematically benchmark multiple sequence alignment methods. The pipeline was originally written by Luisa Santus and Jose Espinosa-Carrasco from The Comparative Bioinformatics Group at The Centre for Genomic Regulation in Spain, with contributions from Leon Rauschning, Alessio Vignoli, Igor Trujnara, and Leila Mansouri.
Multiple sequence alignment is a foundational step in biological data analysis, but choosing the best tool and parameters can vary depending on sequence length, similarity, and structural features. This pipeline provides a unified framework to run tools such as FAMSA, FOLDMASON, T-Coffee, and REGRESSIVE across one or multiple sequence datasets, allowing researchers to directly compare alignment outputs under consistent computational conditions.
What the pipeline does
The workflow begins with optional input summarisation, computing statistics on the input data such as average sequence similarity, sequence length, and pLDDT scores when structural models are supplied. Users can optionally construct a guide tree using a specified tool and arguments to govern the sequence addition order during alignment. The core required step then performs sequence or structural alignment using the selected aligner.
Following alignment, an optional evaluation module calculates scores including Sum of Pairs (SoP), Total Column score (TC), intra-molecular root-mean-square deviation (iRMSD), and Total Consistency Score (TCS). Run metrics and outputs are subsequently compiled into a MultiQC summary table, an interactive Shiny application, and an optional Foldmason HTML alignment visualisation.
Key facts
- Pipeline framework
- Nextflow (nf-core)
- Latest release
- 1.1.1 (2025-05-27)
- Original authors
- Luisa Santus and Jose Espinosa-Carrasco
- Input formats
- FASTA, PDB directories, CSV samplesheets, CSV toolsheets
- Supported aligners
- FAMSA, FOLDMASON, TCOFFEE, REGRESSIVE
- Evaluation metrics
- Sum of Pairs (SoP), Total Column score (TC), iRMSD, Total Consistency Score (TCS)
- Output reports
- MultiQC summary table, Shiny app, Foldmason HTML visualisation
What you can use it for
- Single-tool sequence alignment
- Execute an individual aligner such as FAMSA on a FASTA file using simplified command-line arguments and preset deployment profiles.
- Protein structure alignment
- Align sets of three-dimensional protein structures contained in PDB directories using structure-aware tools like FOLDMASON.
- Multi-tool alignment benchmarking
- Benchmark several aligners and guide-tree combinations simultaneously across multiple sequence families using CSV samplesheets and toolsheets.
- Alignment accuracy evaluation
- Assess generated alignments against reference alignments or structural data using metrics such as SoP, TC, TCS, and iRMSD.
Run it here
The research agent runs nf-core/multiplesequencealign with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
Strengths and limits
Good at
- Supports both sequence-based FASTA inputs and structure-based PDB files in the same alignment and evaluation framework.
- Enables parallel benchmarking of different guide trees, aligners, and custom tool arguments using a structured toolsheet.
- Includes dedicated scoring metrics for both sequence consistency and structural deviation.
Watch out for
- Executing the alignment phase is mandatory; the pipeline cannot be run exclusively for guide-tree construction or input profiling.
- Benchmarking multiple datasets across varied tools requires defining two separate CSV configuration files (a samplesheet and a toolsheet).
Questions
Can I run nf-core/multiplesequencealign without my own cluster?
The research agent runs nf-core/multiplesequencealign with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
What licence is nf-core/multiplesequencealign under?
MIT, like every nf-core pipeline.
Can I supply both sequences and structures at the same time?
Yes. Providing both sequence files and PDB directories allows users to perform structural evaluations on sequence-based alignment tools.
What is the difference between a samplesheet and a toolsheet?
A samplesheet specifies the input datasets, reference alignments, and structural dependency files. A toolsheet defines which aligners and guide-tree tools will be deployed, along with any tool-specific command-line arguments.
Which evaluation metrics are supported?
The pipeline can calculate Sum of Pairs (SoP), Total Column score (TC), intra-molecular root-mean-square deviation (iRMSD), and Total Consistency Score (TCS).
How are alignment benchmarking outputs visualised?
Results are reported in a summary table within MultiQC, an interactive Shiny application, and an optional Foldmason HTML visual display.
How to cite it
10.5281/zenodo.13889386
Written from nf-core/multiplesequencealign's own README. Statements citing a figure that does not appear there are removed before publication. Licence and running details come from our registry.