Lightweight workflow for microbial genome recovery using either Nanopore or PacBio HiFi reads.
mmlong2-lite is the microbial genome production part of the mmlong2 pipeline.
- Snakemake workflow running dependencies from a Singularity container for enhanced reproducibility
- Bioinformatics tool and parameter optimizations for high complexity metagenomics samples
- Automated metagenomic assembly curation for identification and removal of misassemblies
- Circular microbial genome extraction as separate genome bins
- Eukaryotic contig removal for reduced microbial genome contamination
- Differential coverage support for improved microbial genome recovery
- Iterative ensemble binning strategy for improved microbial genome recovery
The mmlong2-lite workflow is available through Bioconda:
conda create -c conda-forge -c bioconda mmlong2-lite
To create a local Conda environment for running mmlong2-lite workflow, just copy-paste the following:
conda create --prefix mmlong2-lite -c conda-forge -c bioconda snakemake=9.12.0 singularity=3.8.6 zenodo_get=1.6.1 pv=1.6.6 pigz=2.8 tar=1.35 rsync=3.4.1 -y
conda activate ./mmlong2-lite || source activate ./mmlong2-lite
git clone https://github.com/Serka-M/mmlong2-lite/ mmlong2-lite/repo
mv mmlong2-lite/repo/src/* mmlong2-lite/bin
chmod +x mmlong2-lite/bin/mmlong2-lite
mmlong2-lite -h
After setting up the virtual environment, the required software dependencies will be automatically installed when running the workflow for the first time.
mmlong2-lite -np nanopore_reads.fastq.gz -o output_dir -p 100
MAIN SETTINGS:
-np --nanopore_reads Path to Nanopore reads
-pb --pacbio_reads Path to PacBio HiFi reads
-o --output_dir Output directory name (default: mmlong2)
-p --processes Number of processes/multi-threading (default: 3)
-bin --binning_mode Run pipeline with a specified binning mode (e.g. fast default extended)
OPTIONAL SETTINGS:
-cov --coverage CSV dataframe for differential coverage binning (e.g. NP/PB/IL,/path/to/reads.fastq)
-run --run_until Run pipeline until a specified stage completes (e.g. assembly curation filtering singletons coverage binning)
-tmp --temporary_dir Directory for temporary files (default: $TMPDIR)
-fly --use_metaflye Use metaFlye for metagenomic assembly
-dbg --use_metamdbg Use metaMDBG for metagenomic assembly
-myl --use_myloasm Use myloasm for metagenomic assembly
-ca --custom_assembly Use a custom assembly
-cai --custom_assembly_info Optional assembly information file (metaFlye format) for custom assembly
-med --use_medaka Use Medaka for polishing Nanopore assemblies (default: skip Medaka)
-mm --medaka_model Medaka polishing model (default: r1041_e82_400bps_sup_v5.0.0)
-scr --skip_curation Skip assembly curation and removal of misassemblies (default: run curation)
-who --use_whokaryote Use Whokaryote for identifying eukaryotic contigs (default: use Tiara)
-sem --semibin_model Binning model for SemiBin (default: global)
-mcc --min_contig_cov Minimum assembly contig coverage for binning
-mlc --min_len_contig Minimum assembly contig length (default: 3000)
-mlb --min_len_bin Minimum genomic bin size (default: 250000)
-scl --skip_cleanup Skip cleanup of workflow intermediate files
-env --conda_envs_only Use conda environments instead of container (default: use container)
-h --help Print help information
-v --version Print workflow version number
ADVANCED SETTINGS:
-mmo --myloasm_min_ovlp Minimum overlap between reads used by myloasm assembler (default: 500)
-mmc --myloasm_min_cov Minimum contig coverage used by myloasm assembler (default: 1)
-xm --extra_myloasm Extra inputs for myloasm assembler (default: none)
-fmo --flye_min_ovlp Minimum overlap between reads used by Flye assembler (default: auto)
-fmc --flye_min_cov Minimum initial contig coverage used by Flye assembler (default: 3)
-n --dryrun Print summary of jobs for the Snakemake workflow
-t --touch Touch Snakemake output files
-r --rule Run specified Snakemake rule
-x --extra_inputs Extra inputs for Snakemake config file
To perform genome recovery with differential coverage, prepare a 2-column comma-separated dataframe, indicating the additional read datatype (NP for Nanopore, PB for PacBio, IL for short reads) and read file location.
Dataframe example:
PB,/path/to/your/reads/file1.fastq
NP,/path/to/your/reads/file2.fastq
IL,/path/to/your/reads/file3.fastq.gz
The prepared dataframe can be provided to the workflow through the -cov option.
<output_name>_assembly.fasta- metagenome assembly file<output_name>_bins.tsv- per-bin dataframe for the recovered genomes<output_name>_usage.tsv- dataframe for compute resource usagedependencies.csv- list of dependencies used and their versionsbins- directory for metagenome assembled genomes
If you use mmlong2-lite in a publication, please cite:
Sereika, M., Mussig, A.J., Jiang, C. et al. Genome-resolved long-read sequencing expands known microbial diversity across terrestrial habitats. Nat Microbiol (2025). https://doi.org/10.1038/s41564-025-02062-z