Multiple Sequence Alignments

This page is entirely dedicated to performing Multiple Sequence Alignments (MSA) with PanTools.


Performs multiple sequence alignment (MSA) with MAFFT on sets of sequences. These alignments can either be:

  • per homology group

  • multiple homology groups

  • regions

  • with all sequences containing a functional domain

Select a method
By default, this function will make a MSA for each homology group. (It can still be specified with --method=per-group.) Using another option from the list above requires use of the --method argument. For aligning multiple homology groups, please use --method=multiple-groups with the homology groups specified in a csv file on a single line (can be added with --homology-file=/path/to/hm.csv or --homology-groups=2,3,4). For aligning regions, please use --method=regions with a regions file that is added with --regions-file=/path/to/rf.txt. For aligning sequences based on a functional domain, please use --method=functions together with a functional domain that is added with --functions=<domain>.
Alignment behavior
Three flags exist to change the alignment behavior: --align-nucleotide, --align-protein, --align-variants. By default, only nucleotide sequences (CDS) are aligned for pangenomes and only protein sequences are aligned for panproteomes. If variants were added using add_variants, PanTools will align complete mRNA sequences (including all variants) instead of CDS sequences; see below for more information. PAV can be taken into account using the --pavs option.
By default, the MSA is performed in two rounds. In the first round, the sequences are aligned as is. Next, the sequences are trimmed based on the longest start and end gap of the initial alignment and aligned again. After the second round, the variable and parsimony informative sites are identified. When the --no-trimming argument is included, the variable and parsimony informative sites are identified from the initial alignment and no trimming is performed (thus, only one round of aligning).
For each round, a ML phylogeny is created with FastTree. The option --no-fasttree can be used to skip running FastTree, which is used for generating a tree from each alignment.
We know this behavior is not optimal and will be revised in future versions.
For phylogenetics, it is common to use a protein alignment for the trimming of nucleotide sequences for the nucleotide alignment. However, as this mixes two different types of data, it requires two separate runs using a pangenome. Please follow the following steps for this:
  1. Run the MSA with --align-protein.

  2. Run the MSA with --align-nucleotide and --trim-using-proteins.

NB: These two steps are also required for correctly running core_phylogeny and consensus_tree.

Identify phenotype shared or specific variation
Shared SNPs or amino acid substitutions can be found among the members of a phenotype when --phenotype is included. As homology groups can highly differ in size, the threshold for a phenotype shared or specific SNP/substitution is based on the number of sequences (from a certain phenotype) of an homology group instead of the number of genomes in the pangenome. For example, the pangenome holds 500 genomes but the homology group consists of only 100 sequences. The threshold can be lowered by including --phenotype-threshold=<phenotypeThreshold>, which lowers the original threshold by multiplying it to a given percentage.
Sequence identity and similarity
- The percentage identity of two sequences is calculated based on the number of exactly matching characters divided by the alignment length minus the positions were both sequences have a gap.
- The similarity (protein only) is calculated from the number of identical matches, increased by the number of similar amino acids (according to the BLOSUM 62 matrix), divided by the alignment length minus the shared gap positions. The calculated percentage of similarity is dependant on the BLOSUM matrix set by --blosum. Choose a larger BLOSUM number BLOSUM less divergent sequences.
More information on variants
With the --variants argument, gene variants as added to the pangenome with add_variants can be included in the alignment. For these variants, the consensus sequence for this variant is added to the input sequences for the alignment. Also, presence/absence variations as added to the pangenome with add_pavs will be used for determining whether or not to include a variant in the alignment. NB: This option uses full mRNA sequences for alignment, not CDS sequences.
Required software


Path to the database root directory.



Number of threads for MAFFT and IQ-tree, default is the number of cores or 8, whichever is lower.


Only include a selection of genomes.


Exclude a selection of genomes.


A text file with homology group node identifiers, separated by a comma. Default is all homology groups. (Mutually exclusive with --homology-groups.)


A comma separated list of homology group node identifiers. Default is all homology groups. (Mutually exclusive with --homology-file.)


A text file containing genome locations with on each line: a genome number, sequence number, begin and end position, separated by a space.


The kind of alignment to make. Can be either per-group, multiple-groups, regions or functions.


Align nucleotide sequences (default: depends on database type).


Align protein sequences (default: depends on database type).


For specifying one or multiple functional domains (Only used when --method=functions).


Identify phenotype shared/specific/exclusive positions in the alignment.


A BLOSUM matrix number to control MAFFT’s sensitivity and the similarity calculation. Allowed values are 45, 62 and 80 (default: 62).


Threshold for phenotype specific SNPs (default: 100%).


Align the sequences only once. Trimming is on by default.


Trim the (nucleotide) alignment using the protein alignment of a previous run. This option is only available when the database is a pangenome.


Run FastTree (default: true).


Use variants in the alignment. This option does not work with the --method=regions option. This option will include variant sequences in the alignment from pantools add_variants.


Use PAVs in the alignment. This option does not work with the --method=regions option and can only be used if --variants is set. This option will include PAVs as marked by pantools add_pavs.

Example regions file

Each line must have a genome number, sequence number, begin and end positions that are separated by a space. Place a minus symbol behind a region to extract the reverse complement sequence.

1 1 1 10000
195 1 477722 478426
71 10 17346 18056 -
138 47 159593 160300 -
Example commands
$ pantools msa tomato_DB
$ pantools msa --align-protein -H=hmgroups.txt tomato_DB
$ pantools msa --align-nucleotide --trim-using-proteins -H=hmgroups.txt tomato_DB
$ pantools msa --no-trimming --align-nucleotide -H=hmgroups.txt tomato_DB
$ pantools msa --phenotype --phenotype-threshold=99 -H=hmgroups.txt tomato_DB
$ pantools msa --method=multiple-groups tomato_DB
$ pantools msa --method=multiple-groups --align-protein -H=hmgroups.txt tomato_DB
$ pantools msa --method=multiple-groups --phenotype --phenotype-threshold=95 -H=hmgroups.txt tomato_DB
$ pantools msa --method=regions -R=regions.txt tomato_DB
$ pantools msa --method=functions --functions=PF10137 tomato_DB
$ pantools msa --variants tomato_DB
$ pantools msa --variants --pavs --threads=10 --method=functions --functions=PF10137 tomato_DB

Output files are stored in database_directory/alignments/msa_?/grouping_v?/ A separate directory is created for each alignment which holds the input and output files.

The ‘input’ directory contains the input files for the alignments.

  • prot/nuc/var(_trimmed).fasta, original and trimmed input sequences.

  • prot/nuc/, number of trimmed positions per sequence.

  •, relevant gene information of sequences in group: gene names, mRNA node id, address, strand orientation.

  •, information about the order of genomes in the alignment.

  • nuc/var.structure.tsv, nucleotide structure information of the alignment. Tab-separated file with the following columns: sequence_id, start, stop, strand, feature, mRNA.

  •, genomes that were skipped.

The alignments and output files are written to the ‘output’ directory.

  • prot/nuc/var(_trimmed).afa, the initial and second (trimmed) alignment in CLUSTAL format.

  • prot/nuc/var(_trimmed).fasta, the initial and second (trimmed) alignment in FASTA format.

  • prot/nuc/var(_trimmed).newick, FastTree ML tree inferred from the initial and second (trimmed) alignment.

  • prot/nuc/var(_trimmed), some statistics about the initial and second (trimmed) alignment: alignment length, number of conserved, variable and parsimony informative sites

Sequence identity and similarity output files.

  • prot/nuc/var(_trimmed)_identity.csv, table with the sequence identity scores.

  • prot(_trimmed)_similarity.csv, table with similarity of the protein sequences.

Variable and parsimony informative sites output files.

  • informative_prot/nuc/var(_trimmed)_distance.csv, table with distances between sequences based on parsimony informative sites in the alignment.

  • variable_prot/nuc/var(_trimmed)_distance.csv, table with distances between sequences based on variable sites in the alignment.

  • informative_prot/nuc/var(_trimmed)_sites.csv, table with the number shared parsimony informative sites between sequences.

  • variable_prot/nuc/var(_trimmed)_sites.csv, table with the number of shared variable sites between sequences.

When a --phenotype is included.

  • File named after phenotype property. A CSV table of all variable positions in the alignment, with counts of the letters per phenotype value and whether a letter is actually shared/specific/exclusive.

  • groups/functions/regions_with_phenotype_changes.txt, a summary of the alignments having at least one phenotype shared/specific/exclusive position. This file is generated once per analysis, not per alignment.