S

    galah process

    Runs both analyse and cluster in sequence: determines the MIMAG quality score for each genome, then dereplicates genomes into ANI-based clusters, using those quality scores to pick each cluster's representative. Supports bacteria, archaea, and eukaryotes in the same run.

    By default, galah runs isiteuk to classify each genome into its biological domain (Bacteria, Archaea, or Eukaryota), then selects the appropriate quality tool and marker gene search mode for that domain, exactly as analyse does:

    • Bacteria / Archaea: completeness and contamination estimated by CheckM2; rRNA genes found by Barrnap in bacterial/archaeal mode; tRNAs found by tRNAscan-SE in bacterial/archaeal mode.
    • Eukaryota: completeness and contamination estimated by EukCC; rRNA genes found by Barrnap in eukaryotic mode (finds 18S, 28S, 5.8S, 5S); tRNAs found by tRNAscan-SE in eukaryotic mode. If EukCC cannot estimate quality (e.g. no placement marker genes are found), completeness and contamination are recorded as 0.
    # Process a mixed set of genomes: isiteuk classifies domains automatically (default)
CHECKM2DB=CheckM2_database/uniref100.KO.1.dmnd \
ISITEUK_METAPACKAGE_PATH=/path/to/isiteuk.smpkg \
galah process \
    --genome-fasta-files genome1.fna genome2.fna genome3.fna \
    --output-cluster-definition clusters.tsv \
    --output-mimag-summary mimag_summary.tsv

# Process bacterial genomes only (skips isiteuk)
CHECKM2DB=CheckM2_database/uniref100.KO.1.dmnd \
galah process \
    --genome-fasta-files genome1.fna genome2.fna \
    --domain-choice bac \
    --output-cluster-definition clusters.tsv \
    --output-mimag-summary mimag_summary.tsv

# Process a mixed set using a pre-computed isiteuk classification and EukCC quality report
CHECKM2DB=CheckM2_database/uniref100.KO.1.dmnd \
galah process \
    --genome-fasta-files bacteria.fna archaea.fna euk.fna \
    --isiteuk-output isiteuk_results.tsv \
    --eukcc-quality-report eukcc_quality.tsv \
    --output-cluster-definition clusters.tsv \
    --output-mimag-summary mimag_summary.tsv

# Save the CheckM2 quality report produced during the run for later use
CHECKM2DB=CheckM2_database/uniref100.KO.1.dmnd \
galah process \
    --genome-fasta-files genome1.fna genome2.fna \
    --domain-choice bac \
    --output-quality-report quality_report.tsv \
    --output-cluster-definition clusters.tsv \
    --output-mimag-summary mimag_summary.tsv

# Save intermediate outputs to a directory; re-running reuses completed steps
CHECKM2DB=CheckM2_database/uniref100.KO.1.dmnd \
ISITEUK_METAPACKAGE_PATH=/path/to/isiteuk.smpkg \
galah process \
    --genome-fasta-list genomes.txt \
    --working-dir work/ \
    --output-cluster-definition clusters.tsv \
    --output-mimag-summary mimag_summary.tsv

    Domain choice

    The --domain-choice flag controls how galah determines the biological domain for each genome:

    ValueDescription
    isiteuk (default)Run isiteuk to classify each genome automatically
    bacTreat all genomes as Bacteria
    arcTreat all genomes as Archaea
    eukTreat all genomes as Eukaryota
    completenessRun CheckM2 and EukCC on every genome and keep whichever reports the higher completeness as a single row
    allRun CheckM2 and EukCC on every genome and report all three domains as separate rows

    --domain-choice all, which reports every domain as a separate row, is not supported by process: since it produces multiple rows per genome, there is no single quality value left to cluster on. Use galah analyse --domain-choice all instead if you need that view.

    MIMAG quality criteria

    Bacteria / Archaea:

    TierCriteria
    High qualityCompleteness ≥ 90%, contamination < 5%, ≥ 1 each of 5S/16S/23S rRNA, ≥ 18 tRNA types
    Medium qualityCompleteness ≥ 50%, contamination < 10% (and not High quality)
    Low qualityCompleteness < 50% or contamination ≥ 10%

    Eukaryota:

    TierCriteria
    High qualityCompleteness ≥ 90%, contamination < 5%, ≥ 1 eukaryotic rRNA (18S/28S/5.8S/5S), ≥ 18 tRNA types
    Medium qualityCompleteness ≥ 50%, contamination < 10% (and not High quality)
    Low qualityCompleteness < 50% or contamination ≥ 10%

    See galah analyse --full-help for a full description of the --output-mimag-summary output format, and galah cluster --full-help for clustering-specific parameters.

    GENOME INPUT

    -f, --genome-fasta-files PATH ..

    Path(s) to FASTA files of each genome e.g. pathA/genome1.fna pathB/genome2.fa.

    -d, --genome-fasta-directory PATH

    Directory containing FASTA files of each genome.

    -x, --genome-fasta-extension EXT

    File extension of genomes in the directory specified with -d/--genome-fasta-directory. [default: fna]

    --genome-fasta-list PATH

    File containing FASTA file paths, one per line.

    DOMAIN PARAMETERS

    --domain-choice CHOICE

    Method for determining genome domain. 'isiteuk' runs isiteuk to classify each genome (default). 'bac', 'arc', 'euk' fix all genomes to that domain. 'completeness' runs CheckM2 and EukCC (and matching rRNA/tRNA) for every genome and reports only the higher-completeness domain's result, one row per genome. 'all' runs CheckM2 and EukCC for every genome and reports all three domains as separate rows. [default: isiteuk]

    --isiteuk-output FILE

    Pre-computed isiteuk output TSV. If given, skips running isiteuk when --domain-choice isiteuk.

    --isiteuk-metapackage PATH

    Path to isiteuk metapackage. If not given, uses ISITEUK_METAPACKAGE_PATH environment variable.

    --isiteuk-bacteria-cutoff FLOAT

    Minimum isiteuk num_in_target_domain for Bacteria domain assignment. [default: 10]

    --isiteuk-archaea-cutoff FLOAT

    Minimum isiteuk num_in_target_domain for Archaea domain assignment. [default: 10]

    --isiteuk-eukaryota-cutoff FLOAT

    Minimum isiteuk num_in_target_domain for Eukaryota domain assignment. [default: 20]

    --eukcc-db-path PATH

    Path to EukCC database (required for eukaryotic quality assessment). If not given, uses EUKCC2_DB environment variable.

    --eukcc-quality-report FILE

    Pre-computed merged EukCC TSV with fasta/completeness/contamination columns. If given, skips running EukCC for eukaryotic genomes.

    QUALITY PARAMETERS

    --quality-method NAME

    method for finding genome quality. 'checkm2' for CheckM2. [default: checkm2]

    --checkm2-db-path PATH

    Path to CheckM2 database (required for CheckM2 quality method). If not given, will use CHECKM2DB environment variable if set.

    --checkm2-quality-report PATH

    Path to pre-generated CheckM2 quality_report.tsv file. If given, will use this file instead of running quality method.

    --checkm-tab-table PATH

    Path to pre-generated CheckM tab table file. If given, will use this file instead of running quality method.

    RNA PARAMETERS

    --rrna-method NAME

    method for finding rRNA genes. 'barrnap' for Barrnap. [default: barrnap]

    --trna-method NAME

    method for finding tRNA genes. 'trnascan' for tRNAscan-SE. [default: trnascan]

    --barrnap-gff-list PATH

    Path to two-column TSV file mapping genome paths (as given in input) to Barrnap GFF paths (no headers). If given, will use these files instead of running rRNA method.

    --trnascan-out-list PATH

    Path to two-column TSV file mapping genome paths (as given in input) to tRNAscan-SE output paths (no headers). If given, will use these files instead of running tRNA method.

    FILTERING PARAMETERS

    --checkm2-quality-report PATH

    CheckM version 2 quality_report.tsv (i.e. the quality_report.tsv in the output directory output of checkm2 predict ..) for defining genome quality, which is used both for filtering and to rank genomes during clustering.

    --checkm-tab-table PATH

    CheckM tab table (i.e. the output of checkm .. --tab_table -f PATH ..). The information contained is used like --checkm2-quality-report.

    --genome-info PATH

    dRep style genome info table for defining quality. The information contained is used like --checkm2-quality-report.

    --min-completeness FLOAT

    Ignore genomes with less completeness than this percentage. [default: not set]

    --max-contamination FLOAT

    Ignore genomes with more contamination than this percentage. [default: not set]

    --run-checkm2

    Run CheckM2 to generate quality scoring used for clustering. Requires --checkm2-db-path or CHECKM2DB env variable to be set.

    --checkm2-db-path DB_PATH

    Path to CheckM2 database (required for running CheckM2) [default: from CHECKM2DB environment variable]

    CLUSTERING PARAMETERS

    --ani FLOAT

    Overall ANI level to dereplicate at with the primary clusterer. [default: 95]

    --min-aligned-fraction FLOAT

    Min aligned fraction of two genomes for clustering. [default: 15]

    --small-genomes

    Use small-genomes settings in skani calculation. Recommended for sequences < 20kb.

    --fragment-length FLOAT

    Length of fragment used in FastANI calculation (i.e. --fragLen). [default: 3000]

    --quality-formula FORMULA

    Scoring function for genome quality [default: Parks2020_reduced]. One of:

    | formula | description | |:---|:---| | Parks2020_reduced | (default) A quality formula described in Parks et. al. 2020 https://doi.org/10.1038/s41587-020-0501-8 (Supplementary Table 19) but only including those scoring criteria that can be calculated from the sequence without homology searching: completeness-5*contamination-5*num_contigs/100-5*num_ambiguous_bases/100000 | | completeness-4contamination | completeness-4*contamination | | completeness-5contamination | completeness-5*contamination | | dRep | completeness-5*contamination+contamination*(strain_heterogeneity/100)+0.5*log10(N50) |

    --precluster-ani FLOAT

    Require at least this precluster-derived ANI for preclustering and to avoid primary clustering on distant lineages within preclusters. [default: 90]

    --precluster-method NAME

    method of calculating rough ANI for dereplication. 'finch' for finch MinHash, 'skani' for Skani. [default: skani]

    --cluster-method NAME

    method of calculating ANI. 'fastani' for FastANI, 'skani' for Skani. [default: skani]

    --cluster-contigs

    Cluster contigs within a fasta file instead of genomes. When used, either --small-contigs or --large-contigs must be specified.

    --small-contigs

    Use small-genomes settings in skani when clustering contigs. Recommended for contigs < 20kb. Mutually exclusive with --large-contigs.

    --large-contigs

    Do not use small-genomes settings in skani when clustering contigs. Recommended for contigs >= 20kb. Mutually exclusive with --small-contigs.

    --low-memory

    Reduce memory use by sketching to file and searching it instead.

    --skip-sanitise-headers

    Skip checking/rewriting FASTA headers that contain tab characters before running skani, passing genome paths straight through unchanged. Mainly useful for benchmarking against tools which do not perform this sanitizing step. If any input genome actually has a tab character in a header line, skani's TSV output will be silently corrupted, so only use this on genome sets already known not to have tabs in their headers.

    --reference-genomes PATH ...

    Reference genomes to cluster against. These should be pre-clustered at the chosen %ANI - reference-vs-reference comparisons are never made, so this is not checked. Input genomes (--genome-fasta-files etc.) are dereplicated amongst themselves first, as if no reference genomes were given at all, and only the resulting representative(s) are then compared against these reference genomes - so input genomes do not need to be pre-dereplicated beforehand. If quality is provided for representative selection, values for these genomes must also be provided. Genomes within the precluster ANI cutoff of each reference will be placed in the same precluster. Mutually exclusive with --reference-genomes-list.

    --reference-genomes-list PATH

    File containing paths to reference genomes (one per line). These should be pre-clustered at the chosen %ANI - reference-vs-reference comparisons are never made, so this is not checked. Input genomes (--genome-fasta-files etc.) are dereplicated amongst themselves first, as if no reference genomes were given at all, and only the resulting representative(s) are then compared against these reference genomes - so input genomes do not need to be pre-dereplicated beforehand. If quality is provided for representative selection, values for these genomes must also be provided. Genomes within the precluster ANI cutoff of each reference will be placed in the same precluster. Mutually exclusive with --reference-genomes.

    --skip-input-dereplication

    When used with --reference-genomes/--reference-genomes-list, skip dereplicating input genomes amongst themselves first: compare every input genome directly against the reference set instead, so near-duplicate input genomes are not merged before matching (restores the behaviour prior to this flag's introduction). Has no effect unless reference genomes are given.

    OUTPUT

    --output-mimag-summary PATH

    Output a tsv file summarising the MIMAG status for each genome.

    --output-quality-report PATH

    Output a CheckM2-format quality report TSV file.

    --output-cluster-definition PATH

    Output a file of representative<TAB>member lines.

    --output-representative-fasta-directory PATH

    Symlink representative genomes into this directory.

    --output-representative-fasta-directory-copy PATH

    Copy representative genomes into this directory.

    --output-representative-list PATH

    Print newline separated list of paths to representatives into this file.

    GENERAL PARAMETERS

    -t, --threads INT

    Number of threads. [default: 1]

    -v, --verbose

    Print extra debugging information

    -q, --quiet

    Unless there is an error, do not print log messages

    -h, --help

    Output a short usage message.

    --full-help

    Output a full help message and display in 'man'.

    --full-help-roff

    Output a full help message in raw ROFF format for conversion to other formats.

    EXIT STATUS

    0

    Successful program execution.

    1

    Unsuccessful program execution.

    101

    The program panicked.

    AUTHOR

    Ben J. Woodcroft, Centre for Microbiome Research, Queensland University of Technology <benjwoodcroft near gmail.com>

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