Taxonomic profiling of metagenomes from diverse environments with mOTUs4

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Project description

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mOTUs profiler

The mOTUs profiler is a computational tool that estimates taxonomic abundance of microbial community members from known and currently unknown (uncultured) species using metagenomic shotgun sequencing data.

The current version of the mOTUs profiler is built on top of the mOTUs database (motus-db) which is constructed from 919K isolate and single cell-amplified (SAGs) genomes and 2.98M metagenome-assembled genomes (MAGs) generated from ~120k metagenomic samples spanning diverse microbiomes, which include (in addition to the human and ocean microbiome) soil, freshwater and gastrointestinal tract microbiomes of ruminants and other animals, environments we found to be greatly underrepresented by reference genomes.

In the current version, 124,295 species-level taxonomic units (mOTUs) were constructed using sequences of 10 single-copy marker genes recovered from these genomes. 30,256 mOTUs are represented by an isolate genome, whereas 94,039 mOTUs are represented by MAGs only.

Please cite the paper(s) corresponding to the version(s) you use:

Version	Journal	Year	DOI
mOTUs v1	Nature Methods	2013	10.1038/nmeth.2693
mOTUs v2	Nature Communications	2019	10.1038/s41467-019-08844-4
mOTUs v3 (profiler)	Microbiome	2022	10.1186/s40168-022-01410-z
mOTUs v3 (protocol)	Current Protocols	2022	10.1002/cpz1.218
mOTUs database (v4)	Nucleic Acids Research	2025	10.1093/nar/gkae1004

📦 Installation

The mOTUs profiler, written in Python 3 (>=3.12), can be executed on a 64-bit Linux or macOS system. It requires external dependencies (bwa>=0.7.19, vsearch>=2.30.4) which must be available on PATH.

Package managers automate this: they resolve dependency graphs, manage binary versions, and provide isolated environments so mOTUs and its dependencies don't conflict with system packages. Below are the recommended approaches.

Installation with Pixi (Recommended)

Pixi is a fast, dependency-free package manager. Install mOTUs globally:

pixi global install motus
motus profile -h

Or add to an existing workspace:

pixi workspace channel add conda-forge
pixi workspace channel add bioconda
pixi add motus
pixi run motus profile -h

Installation with Conda

mOTUs is available in bioconda:

# First time: add channels
conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge

# Create and activate environment
conda create -n mOTUs4 motus
conda activate mOTUs4

# Verify installation
motus profile -h

🚀 Usage

📖 Full documentation

After installation, you can test whether the tool was installed correctly by executing:

motus --help

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Usage:
        motus <command> [options]


    Commands:

        -- Taxonomic profiling

            profile       Perform taxonomic profiling (map_tax + calc_mgc + calc_motu) in a single step

            map_tax       Map reads to the marker gene database
            calc_mgc      Calculate marker gene cluster (MGC) abundance
            calc_motu     Summarize MGC abundances into a mOTU profile


        -- Tool utilities

            downloadMGDB  Download the mOTUs marker gene database
            merge         Merge multiple taxonomic profiling results into one table
            classify      Classify user genomes into mOTUs
            prep_long     Prepare long reads to be profiled by mOTUs


        -- Genome accession

            genomes       Search the mOTUs-db by keyword (taxonomic, functional)
            download      Download sequence files from mOTUs-db


    Type motus <command> to print the help menu for a specific command

Commands

The profile function in mOTUs is the main function that executes map_tax, calc_mgc, and calc_motu in sequence. It takes short read metagenomic sequencing data as input and generates a taxonomic profile.

Additionally, the tool includes six helper functions:

merge: Combines multiple taxonomic profiles into a single file.
classify: Assigns user-submitted genomes to existing mOTUs.
genomes: Finds genomes by functional or taxonomic annotation.
prep_long: Finds genomes by functional or taxonomic annotation.
download: Provides programmatic access to the ~4 million genomes in the motus-db.
downloadMGDB: Downloads the mOTUs marker gene database.

Profile

📖 Full documentation

Produces a taxonomic profile from short read metagenomic sequencing data by executing map_tax, calc_mgc, and calc_motu in succession.

motus profile

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The profile command in mOTUs is the main function that executes map_tax, calc_mgc,
        and calc_motu in sequence. It takes short read metagenomic sequencing data as input
        and generates a taxonomic profile.


    Usage:
       motus profile -f FILE [FILE ...] -r FILE [FILE ...] -s FILE [FILE ...] -o FILE [options]
       motus profile -f FILE [FILE ...] -r FILE [FILE ...] -o FILE [options]
       motus profile -s FILE [FILE ...] -o FILE [options]


    Input options:
        -f, --forward  FILE [FILE ...]
            Input file(s) for reads in forward orientation, fastQ/A(.gz)-formatted

        -r, --reverse  FILE [FILE ...]
            Input file(s) for reads in reverse orientation, fastQ/A(.gz)-formatted

        -s, --single  FILE [FILE ...]
            Input file(s) for unpaired reads, fastQ/A(.gz)-formatted

        -n, --sample-name  STR
            Sample name (default: 'unnamed sample')

        -db  PATH
            Alternative path for the mOTUs marker gene database

    Output options:
        -o, --output-file  FILE
            Output file name [required]

    Algorithm options:
        -g, --marker-genes  INT
            Required number of marker genes for a mOTU to be called present:
            1=higher recall, 6=higher precision, 10=maximum (default: 3)

        -l, --alignment-length  INT
            Minimum length of the alignment (bp) (default: 75)

        -t, --threads  INT
            Number of threads (default: 1)

        -y, --counting-mode  STR
            Which scale the abundances are reported in (default: INSERT_SCALED)
            Choices: [INSERT_RAW, INSERT_NORM, INSERT_SCALED, BASE_RAW, BASE_NORM]

        --skip-pair-check
            Skip validation that forward and reverse read headers match.
            Use when reads are unsorted or contain singletons.

Map Tax

📖 Full documentation

Maps short reads against the mOTUs marker gene database.

motus map_tax

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The map_tax command takes short read metagenomic sequencing data as input and
        maps reads to the mOTUs marker gene database.


    Usage:
        motus map_tax -f FILE [FILE ...] -r FILE [FILE ...] -s FILE [FILE ...] -o FILE [options]
        motus map_tax -f FILE [FILE ...] -r FILE [FILE ...] -o FILE [options]
        motus map_tax -s FILE [FILE ...] -o FILE [options]


    Input options:
        -f, --forward  FILE [FILE ...]
            Input file(s) for reads in forward orientation, fastQ/A(.gz)-formatted

        -r, --reverse  FILE [FILE ...]
            Input file(s) for reads in reverse orientation, fastQ/A(.gz)-formatted

        -s, --single  FILE [FILE ...]
            Input file(s) for unpaired reads, fastQ/A(.gz)-formatted

        -db  PATH
            Alternative path for the mOTUs marker gene database

    Output options:
        -o, --output-file  FILE
            Output file name [required]

    Algorithm options:
        -l, --alignment-length  INT
            Minimum length of the alignment (bp) (default: 75)

        -t, --threads  INT
            Number of threads (default: 1)

        --skip-pair-check
            Skip validation that forward and reverse read headers match.
            Use when reads are unsorted or contain singletons.

Calc MGC

📖 Full documentation

Calculates the number of inserts mapping to each marker gene cluster within the mOTUs marker gene database.

motus calc_mgc

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The calc_mgc command takes a file storing the alignments of sequencing reads
        to the mOTUs marker gene database and calculates marker gene cluster abundances.


    Usage:
        motus calc_mgc -i FILE -o FILE [options]


    Input options:
        -i, --input-file  FILE
            Path to BAM file generated after running the motus map_tax command [required]

        -db  PATH
            Alternative path for the mOTUs marker gene database

    Output options:
        -o, --output-file  FILE
            Output file name [required]

    Algorithm options:
        -l, --alignment-length  INT
            Minimum length of the alignment (bp) (default: 75)

Calc mOTU

📖 Full documentation

Calculates the taxonomic profile based on the number of inserts mapped to the corresponding marker gene clusters.

motus calc_motu

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The calc_motu command takes a file containing marker gene cluster
        abundances and generates a taxonomic profile.


    Usage:
        motus calc_motu -i FILE -o FILE [options]


    Input options:
        -i, --input-file  FILE
            MGC abundance table generated by the calc_mgc command [required]

        -n, --sample-name  STR
            Sample name (default: 'unnamed sample')

        -db  PATH
            Alternative path for the mOTUs marker gene database

    Output options:
        -o, --output-file  FILE
            Output file name [required]

    Algorithm options:
        -g, --marker-genes  INT
            Required number of marker genes for a mOTU to be called present:
            1=higher recall, 6=higher precision, 10=maximum (default: 3)

        -y, --counting-mode  STR
            Which scale the abundances are reported in (default: INSERT_SCALED)
            Choices: [INSERT_RAW, INSERT_NORM, INSERT_SCALED, BASE_RAW, BASE_NORM]

merge

📖 Full documentation

Merges taxonomic profiles from multiple samples into one (tab-separated) table.

motus merge

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The merge command takes multiple profiles produced after running the
        profile command and combines them into a single table.


    Usage:
        motus merge -i FILE [FILE ...] -o FILE


    Input options:
        -i, --input-files  FILE [FILE ...]
            A list of mOTUs profile files or a text file containing the list of profile
            files to be merged, with one line per file [required]

        -db  PATH
            Alternative path for the mOTUs marker gene database

    Output options:
        -o, --output-file  FILE
            Output file name [required]

downloadMGDB

📖 Full documentation

Downloads the marker gene reference database required for profiling.

motus downloadMGDB

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The downloadMGDB command downloads the marker gene reference database used
        by the profile and map_tax commands.


    Usage:
        motus downloadMGDB [options]


    Options:
        -f, --force
            Force download even when database is already present

        --toy
            Download the lightweight toy database (v4.1-toy) instead of the full database.
            Useful for testing and development. Note: the genomes and download commands
            are not available with the toy database.

        -db  PATH
            Alternative path for the mOTUs marker gene database

classify

📖 Full documentation

Assigns provided genomes to a mOTU if the corresponding taxon is present within the database.

motus classify

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The classify command takes a list of genome sequence files as input and
        assigns these genomes to existing mOTUs in the database.
        Requires vsearch to be installed and on PATH.


    Usage:
        motus classify -i FILE -o FILE [options]


    Input options:
        -i, --input-file  FILE
            Text file listing genome sequence files in fastA(.gz) format to classify.
            One line per genome file [required]

        -db  PATH
            Alternative path for the mOTUs marker gene database

    Output options:
        -o, --output-file  FILE
            Output file name [required]

    Algorithm options:
        -t, --threads  INT
            Number of threads (default: 1)

prep_long

📖 Full documentation

Prepares long reads for profiling by splitting them into ~300 bp fragments.

motus prep_long

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The prep_long command takes long-read sequencing data and converts it
        into the appropriate input format to be used by the profile and map_tax commands.


    Usage:
        motus prep_long -i FILE -o FILE [options]


    Input options:
        -i, --input-file  FILE
            Long-read sequencing file to convert, can be in fastQ/A(.gz) format [required]

    Output options:
        -o, --output-file  FILE
            Output file name. This converted file is ready to be used by motus profile [required]

    Algorithm options:
        -sl, --splitting-length  INT
            Target fragment length (in bp) for splitting long reads (default: 300)

        -ml, --minimum-length  INT
            Minimum read length after splitting. Shorter reads are discarded (default: 50)

download

📖 Full documentation

Downloads sequences for indicated genomes from the mOTUs genome database.

motus download

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The download command downloads listed genome files from mOTUs-db.


    Usage:
        motus download -i FILE -o PATH [options]
        motus download -i STR [STR ...] -o PATH [options]


    Input options:
        -i, --input-genomes  FILE/STR
            Can be either a list of genome identifiers separated by spaces or a text file
            listing the identifiers of genomes for download. One line per genome. The output of
            the motus genomes command can be used as input for this command [required]

        -db  PATH
            Alternative path for the mOTUs marker gene database

    Output options:
        -o, --output-folder  PATH
            Path to output folder where the downloaded sequences will be saved [required]

        -r, --representatives
            Download only sequences from representative genomes.

        -t, --file-type  STR
            File type to download (default: genome)
            Choices: [genome, gene_fna, gene_faa, gene_gff, antismash, pfam, eggnog, kegg, trna, rrna]

genomes

📖 Full documentation

Queries the mOTUs genome database to find genomes matching indicated mOTU identifiers, taxonomic clades, or functional annotations.

motus genomes

Show options

Program: motus - a tool for marker gene-based OTU (mOTU) profiling
    Version: 4.1.0


    References:
        Profiler: Ruscheweyh, Milanese et al. Cultivation-independent genomes greatly expand
        taxonomic-profiling capabilities of mOTUs across various environments. Microbiome (2022).
        doi: https://doi.org/10.1186/s40168-022-01410-z

        Database: Dmitrijeva, Ruscheweyh et al. The mOTUs online database provides web-accessible
        genomic context to taxonomic profiling of microbial communities. Nucleic Acids Research (2025).
        doi: https://doi.org/10.1093/nar/gkae1004


    Summary:
        The genomes command queries the mOTUs-db based on identifiers, functional,
        or taxonomic annotations and returns a list of genomes matching indicated query.


    Usage:
        motus genomes -i FILE -o FILE [options]
        motus genomes -i STR [STR ...] -o FILE [options]
        motus genomes -l GENOME|TAXONOMY|PFAM|KEGG|EGGNOG -o FILE [options]


    Input options:
        -i, --input-queries  FILE/STR
            Can be either a list of search queries or a text file listing search queries
            with one line per query. Queries can be genome or mOTUs identifiers, PFAM, KEGG, EGGNOG,
            or GTDB taxonomy names. If the query does not exactly match any database entry,
            alternative queries will be suggested [required unless -l is used]

        -l, --list  STR
            List all searchable entries for a given category and write them to -o.
            Choose from [GENOME, TAXONOMY, PFAM, KEGG, EGGNOG]. When used, -i is not required.

        -db  PATH
            Alternative path for the mOTUs database

    Output options:
        -o, --output-file  FILE
            Output file containing a list of genome identifiers matching search queries and their
            annotations as indicated by the -d parameter. This output file can be used as input
            for the motus download command [required]

        -d, --details  STR [STR ...]
            List of annotations to report. Choose any combination of [KEGG, PFAM, EGGNOG, TAXONOMY],
            for example, -d KEGG PFAM.

❓ Need Help?

Report an issue: GitHub Issues — for bugs, feature requests, or technical questions
Join the community: Discord — chat with other mOTUs users and developers

📋 Changelog

v4.1.0

Database

Default marker gene database updated to v4.1. Version v4.0 still supported. Clustering didn't change but ~150k MAGs from underexplored environments were added and associated with existing mOTUs.
Annotation database (used by genomes) is now version-matched to the installed marker gene DB; v4.0 and v4.1 annotation DBs are downloaded and stored separately
GTDB taxonomy bumped to R226 from R220
Toy database added (downloadMGDB --toy): lightweight database for testing; disables genomes and download commands

classify

Output columns changed to GENOME, CLOSEST_MOTU, SIMILARITY, ASSIGNED_TO_MOTU, TAXONOMY, #MGs
Reports one best mOTU per genome with GTDB taxonomy; ASSIGNED_TO_MOTU is True if similarity ≥96.5%
Unclassified genomes reported as no_mOTU (hits found but all below threshold) or no_mOTU_<6MGs (fewer than 6 marker genes extracted)

CLI

-db PATH flag added to all commands to specify a custom database parent folder
--skip-pair-check added to profile and map_tax for unsorted inputs or inputs containing singletons. Use as last resort!
motus genomes -l GENOME|TAXONOMY|PFAM|KEGG|EGGNOG lists all searchable entries of a given type without requiring -i
Tool checks at startup whether bwa is on PATH (hard error if missing) and whether vsearch is on PATH (warning if missing)

Bug fixes

Database download: incomplete downloads (content-length mismatch) no longer leave a broken DB with a valid completion marker
merge: blank lines in a profile list file no longer cause a cryptic FileNotFoundError
Multimapper resolution: replaced non-deterministic random.choice with sorted()[0] for reproducible MG selection within an MGC
Edge correction: fixed inaccurate weight distribution in inverse padding

Algorithm

INSERT_NORM and INSERT_SCALED calculation corrected. INSERT_NORM now reports length normalised insert counts without any scaling factor. Full scaling was moved to INSERT_SCALED.

v4.0.4

Initial public release of mOTUs4 with database v4.0 (124,295 mOTUs)
Three-stage pipeline: map_tax → calc_mgc → calc_motu; profile runs all three in sequence
classify command: genome-to-mOTU assignment via fetchMGs + vsearch
genomes and download commands for programmatic access to mOTUs-db genome sequences
merge command for combining multiple single-sample profiles into one table
prep_long command: splits long reads into ~300 bp fragments for profiling
Read name normalisation: /1//2 suffixes stripped and re-appended as BAM qname tags to track orientation through the pipeline

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Jul 17, 2026

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Dec 11, 2025

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motus-tool 4.1.0

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mOTUs profiler

📦 Installation

Installation with Pixi (Recommended)

Installation with Conda

🚀 Usage

Commands

Profile

Map Tax

Calc MGC

Calc mOTU

merge

downloadMGDB

classify

prep_long

download

genomes

❓ Need Help?

📋 Changelog

v4.1.0

v4.0.4

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