S

    binchicken single

    Snakemake pipeline to discover samples for differential abundance binning for single-sample assembly based on co-occurrence of single-copy marker genes, excluding those genes present in reference genomes (e.g. previously recovered genomes).

    # Example: find relevant samples for differential coverage binning (no coassembly)
binchicken single --forward reads_1.1.fq ... --reverse reads_1.2.fq ...

# Example: run proposed assemblies through aviary with cluster submission
# Create snakemake profile at ~/.config/snakemake/qsub with cluster, cluster-status, cluster-cancel, etc.
# See https://snakemake.readthedocs.io/en/stable/executing/cli.html#profiles
binchicken single --forward reads_1.1.fq ... --reverse reads_1.2.fq ... --run-aviary \
  --snakemake-profile qsub --cluster-submission --local-cores 64 --cores 64

    Important options:

    • Maximum number of recovery samples for differential-abundance binning can be specified (--max-recovery-samples, default 20)
    • Genomes can be provided and matching marker genes will be excluded (--genomes)
    • Reads can be mapped to the matched bins with only unmapped reads being assembled (--assemble-unmapped).
    • Assembly and recovery running options:
      • Run directly through Aviary (--run-aviary)
      • Run Aviary commands manually (see coassemble/commands in output)
      • Run assemblies with differential-abundance-binning samples with the tool of your choice (see coassemble/target/elusive_clusters.tsv in output)
    • The taxa of the considered sequences can be filtered to target a specific taxon (e.g. --taxa-of-interest "p__Planctomycetota").

    Paired end reads of form reads_1.1.fq, reads_1_1.fq and reads_1_R1.fq, where reads_1 is the sample name are automatically detected and matched to their basename. Most intermediate files can be provided to skip intermediate steps (e.g. SingleM otu tables, read sizes or genome transcripts; see binchicken coassemble --full-help).

    Kmer preclustering

    Clustering groups of more than 1000 samples quickly leads to memory issues due to combinatorics. Kmer preclustering can be used (default if >1000 samples are provided, or use --kmer-precluster always) to reduce the number of combinations that are considered. This greatly reduces memory usage and allows scaling up to at least 250k samples. Kmer preclustering can be disabled with --kmer-precluster never.

    Cluster submission

    Snakemake profiles can be used to automatically submit jobs to HPC clusters (--snakemake-profile). Note that Aviary assemble commands are submitted to the cluster, while Aviary recover commands are run locally such that Aviary handles cluster submission. The --cluster-submission flag sets the local Aviary recover thread usage to 1, to enable multiple runs in parallel by setting --local-cores to greater than 1. This is required to prevent --local-cores from limiting the number of threads per submitted job.

    OPTIONS

    BASE INPUT ARGUMENTS

    --forward, --reads, --sequences FORWARD [FORWARD ...]

    input forward/unpaired nucleotide read sequence(s)

    --forward-list, --reads-list, --sequences-list FORWARD_LIST

    input forward/unpaired nucleotide read sequence(s) newline separated

    --reverse REVERSE [REVERSE ...]

    input reverse nucleotide read sequence(s)

    --reverse-list REVERSE_LIST

    input reverse nucleotide read sequence(s) newline separated

    --genomes GENOMES [GENOMES ...]

    Reference genomes for read mapping

    --genomes-list GENOMES_LIST

    Reference genomes for read mapping newline separated

    --coassembly-samples COASSEMBLY_SAMPLES [COASSEMBLY_SAMPLES ...]

    Restrict coassembly to these samples. Remaining samples will still be used for recovery [default: use all samples]

    --coassembly-samples-list COASSEMBLY_SAMPLES_LIST

    Restrict coassembly to these samples, newline separated. Remaining samples will still be used for recovery [default: use all samples]

    --singlem-metapackage SINGLEM_METAPACKAGE

    SingleM metapackage for sequence searching. [default: use path from SINGLEM_METAPACKAGE_PATH env variable]

    INTERMEDIATE RESULTS INPUT ARGUMENTS

    --sample-singlem SAMPLE_SINGLEM [SAMPLE_SINGLEM ...]

    SingleM otu tables for each sample, in the form "[sample name]_read.otu_table.tsv". If provided, SingleM pipe sample is skipped

    --sample-singlem-list SAMPLE_SINGLEM_LIST

    SingleM otu tables for each sample, in the form "[sample name]_read.otu_table.tsv" newline separated. If provided, SingleM pipe sample is skipped

    --sample-singlem-dir SAMPLE_SINGLEM_DIR

    Directory containing SingleM otu tables for each sample, in the form "[sample name]_read.otu_table.tsv". If provided, SingleM pipe sample is skipped

    --sample-query SAMPLE_QUERY [SAMPLE_QUERY ...]

    Queried SingleM otu tables for each sample against genome database, in the form "[sample name]_query.otu_table.tsv". If provided, SingleM pipe and appraise are skipped

    --sample-query-list SAMPLE_QUERY_LIST

    Queried SingleM otu tables for each sample against genome database, in the form "[sample name]_query.otu_table.tsv" newline separated. If provided, SingleM pipe and appraise are skipped

    --sample-query-dir SAMPLE_QUERY_DIR

    Directory containing Queried SingleM otu tables for each sample against genome database, in the form "[sample name]_query.otu_table.tsv". If provided, SingleM pipe and appraise are skipped

    --sample-read-size SAMPLE_READ_SIZE

    Comma separated list of sample name and size (bp). If provided, sample read counting is skipped

    --genome-transcripts GENOME_TRANSCRIPTS [GENOME_TRANSCRIPTS ...]

    Genome transcripts for reference database, in the form "[genome]_protein.fna"

    --genome-transcripts-list GENOME_TRANSCRIPTS_LIST

    Genome transcripts for reference database, in the form "[genome]_protein.fna" newline separated

    --genome-singlem GENOME_SINGLEM

    Combined SingleM otu tables for genome transcripts. If provided, genome SingleM is skipped

    CLUSTERING OPTIONS

    --taxa-of-interest TAXA_OF_INTEREST

    Only consider sequences from this GTDB taxa (e.g. p__Planctomycetota) [default: all]

    --appraise-sequence-identity APPRAISE_SEQUENCE_IDENTITY

    Minimum sequence identity for SingleM appraise against reference database [default: 86%, Genus-level]

    --min-sequence-coverage MIN_SEQUENCE_COVERAGE

    Minimum combined coverage for sequence inclusion [default: 10]

    --single-assembly

    Skip appraise to discover samples to differential abundance binning. Forces --num-coassembly-samples and --max-coassembly-samples to 1 and sets --max- coassembly-size to None

    --exclude-coassemblies EXCLUDE_COASSEMBLIES [EXCLUDE_COASSEMBLIES ...]

    List of coassemblies to exclude, space separated, in the form "sample_1,sample_2"

    --exclude-coassemblies-list EXCLUDE_COASSEMBLIES_LIST

    List of coassemblies to exclude, space separated, in the form "sample_1,sample_2", newline separated

    --num-coassembly-samples NUM_COASSEMBLY_SAMPLES

    Number of samples per coassembly cluster [default: 2]

    --max-coassembly-samples MAX_COASSEMBLY_SAMPLES

    Upper bound for number of samples per coassembly cluster [default: --num- coassembly-samples]

    --max-coassembly-size MAX_COASSEMBLY_SIZE

    Maximum size (Gbp) of coassembly cluster [default: 50Gbp]

    --max-recovery-samples MAX_RECOVERY_SAMPLES

    Upper bound for number of related samples to use for differential abundance binning [default: 20]

    --abundance-weighted

    Weight sequences by mean sample abundance when ranking clusters [default: False]

    --abundance-weighted-samples ABUNDANCE_WEIGHTED_SAMPLES [ABUNDANCE_WEIGHTED_SAMPLES ...]

    Restrict sequence weighting to these samples. Remaining samples will still be used for coassembly [default: use all samples]

    --abundance-weighted-samples-list ABUNDANCE_WEIGHTED_SAMPLES_LIST

    Restrict sequence weighting to these samples, newline separated. Remaining samples will still be used for coassembly [default: use all samples]

    --kmer-precluster {never,large,always}

    Run kmer preclustering using unbinned window sequences as kmers. [default: large; perform preclustering when given >1000 samples]

    --precluster-size PRECLUSTER_SIZE

    # of samples within each sample's precluster [default: 5 * max-recovery- samples]

    --prodigal-meta

    Use prodigal "-p meta" argument (for testing)

    COASSEMBLY OPTIONS

    --assemble-unmapped

    Only assemble reads that do not map to reference genomes

    --run-qc

    Run Fastp QC on reads

    --unmapping-min-appraised UNMAPPING_MIN_APPRAISED

    Minimum fraction of sequences binned to justify unmapping [default: 0.1]

    --unmapping-max-identity UNMAPPING_MAX_IDENTITY

    Maximum sequence identity of mapped sequences kept for coassembly [default: 99%]

    --unmapping-max-alignment UNMAPPING_MAX_ALIGNMENT

    Maximum percent alignment of mapped sequences kept for coassembly [default: 99%]

    --run-aviary

    Run Aviary commands for all identified coassemblies (unless specific coassemblies are chosen with --coassemblies) [default: do not]

    --cluster-submission

    Flag that cluster submission will occur through `--snakemake-profile`. This sets the local threads of Aviary recover to 1, allowing parallel job submission [default: do not]

    --aviary-speed {fast,comprehensive}

    Run Aviary recover in 'fast' or 'comprehensive' mode. Fast mode skips slow binners and refinement steps. [default: fast]

    --assembly-strategy {dynamic,metaspades,megahit}

    Assembly strategy to use with Aviary. [default: dynamic; attempts metaspades and if fails, switches to megahit]

    --aviary-gtdbtk-db AVIARY_GTDBTK_DB

    Path to GTDB-Tk database directory for Aviary. Only required if --aviary-speed is set to comprehensive [default: use path from GTDBTK_DATA_PATH env variable]

    --aviary-checkm2-db AVIARY_CHECKM2_DB

    Path to CheckM2 database directory for Aviary. [default: use path from CHECKM2DB env variable]

    --aviary-assemble-cores AVIARY_ASSEMBLE_CORES

    Maximum number of cores for Aviary assemble to use. [default: 64]

    --aviary-assemble-memory AVIARY_ASSEMBLE_MEMORY

    Maximum amount of memory for Aviary assemble to use (Gigabytes). [default: 500]

    --aviary-recover-cores AVIARY_RECOVER_CORES

    Maximum number of cores for Aviary recover to use. [default: 32]

    --aviary-recover-memory AVIARY_RECOVER_MEMORY

    Maximum amount of memory for Aviary recover to use (Gigabytes). [default: 250]

    GENERAL OPTIONS

    --output OUTPUT

    Output directory [default: .]

    --conda-prefix CONDA_PREFIX

    Path to conda environment install location. [default: Use path from CONDA_ENV_PATH env variable]

    --cores CORES

    Maximum number of cores to use [default: 1]

    --dryrun

    dry run workflow

    --snakemake-profile SNAKEMAKE_PROFILE

    Snakemake profile (see https://snakemake.readthedocs.io/en/v7.32.3/executing/cli.html#profiles). Can be used to submit rules as jobs to cluster engine (see https://snakemake.readthedocs.io/en/v7.32.3/executing/cluster.html).

    --local-cores LOCAL_CORES

    Maximum number of cores to use on localrules when running in cluster mode [default: 1]

    --cluster-retries CLUSTER_RETRIES

    Number of times to retry a failed job when using cluster submission (see `--snakemake-profile`) [default: 3].

    --snakemake-args SNAKEMAKE_ARGS

    Additional commands to be supplied to snakemake in the form of a space- prefixed single string e.g. " --quiet"

    --tmp-dir TMP_DIR

    Path to temporary directory. [default: no default]

    OTHER GENERAL OPTIONS

    --debug

    output debug information

    --version

    output version information and quit

    --quiet

    only output errors

    --full-help

    print longer help message

    --full-help-roff

    print longer help message in ROFF (manpage) format

    EXAMPLES

    find relevant samples for differential coverage binning (no coassembly)

    $ binchicken single --forward reads_1.1.fq ... --reverse reads_1.2.fq ...

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