Multiple Sequence Alignments
This page is entirely dedicated to performing Multiple Sequence Alignments (MSA) with PanTools.
MSA
Performs multiple sequence alignments 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
The alignment consists of two rounds: After the initial alignment, protein sequences are trimmed based on the longest start and end gap of the alignment. The number of trimmed amino acids is multiplied by three to trim the correct number of nucleotides. If only nucleotide sequences are aligned, the nucleotide sequence alignment is used for trimming. The trimmed sequences are aligned a second time to identify variable and parsimony informative sites. For each round, a ML phylogeny will be created with FastTree.
--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=2,3,4,4).
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>.--align-nucleotide and --align-protein. 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). The option --no-fasttree can be used
to skip running FastTree, which is used for generating a tree from the
alignment.--phenotype=<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.--blosum.
Choose a larger BLOSUM number BLOSUM less divergent sequences.--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.Required software
Parameters
<databaseDirectory> |
Path to the database root directory. |
Options
|
Number of threads for MAFFT and IQ-tree, default is the number of cores or 8, whichever is lower. |
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Only include a selection of genomes. |
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Exclude a selection of genomes. |
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A text file with homology group node identifiers, separated by a comma.
Default is all homology groups. (Mutually exclusive with
|
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A comma separated list of homology group node identifiers.
Default is all homology groups. (Mutually exclusive with
|
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A text file containing genome locations with on each line: a genome number, sequence number, begin and end position, separated by a space. |
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The kind of alignment to make. Can be either |
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Align nucleotide sequences (default: depends on database type). |
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Align protein sequences (default: depends on database type). |
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For specifying one or multiple functional domains (Only used when
|
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Identify phenotype shared/specific/exclusive positions in the alignment. |
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A BLOSUM matrix number to control MAFFT’s sensitivity and the similarity calculation. Allowed values are 45, 62 and 80 (default: 62). |
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Threshold for phenotype specific SNPs (default: 100%). |
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Align the sequences only once. Trimming is on by default. |
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Run FastTree (default: true). |
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Use variants in the alignment. This option does not work with the
|
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Use PAVs in the alignment. This option does not work with the
|
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 --no-trimming --align-nucleotide -H=hmgroups.txt tomato_DB
$ pantools msa --phenotype=resistance --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=resistance --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
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.
trimmed.info, number of trimmed positions per sequence.
sequences.info, relevant gene information of sequences in group: gene names, mRNA node id, address, strand orientation.
genome_order.info, 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.skipped_genomes.info, 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)_alignment.info, 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.