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.

Workflow description

Core features

• Snakemake workflow running dependencies from a Singularity container for enhanced reproducibility
• Bioinformatics tool and parameter optimizations for high complexity metagenomics samples
• 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

Schematic overview

Installation

Bioconda

The mmlong2-lite workflow is available through Bioconda:

mamba install -c bioconda mmlong2-lite

From source (Conda)

To create a local Conda environment for running mmlong2-lite workflow, just copy-paste the following:

mamba create --prefix mmlong2-lite -c conda-forge -c bioconda snakemake=8.2.3 singularity=3.8.6 zenodo_get=1.6.1 pv=1.6.6 pigz=2.6 tar=1.34 -y
mamba 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.

Running mmlong2-lite

Usage example

mmlong2-lite -np nanopore_reads.fastq.gz -o output_dir -p 100

Full usage

MAIN SETTINGS:
-np     --nanopore_reads        Path to Nanopore reads (default: none)
-pb     --pacbio_reads          Path to PacBio HiFi reads (default: none)
-o      --output_dir            Output directory name (default: mmlong2)
-p      --processes             Number of processes/multi-threading (default: 3)

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 polishing filtering singletons coverage)
-tmp    --temporary_dir         Directory for temporary files (default: none)
-dbg    --use_metamdbg          Use metaMDBG for assembly of PacBio reads (default: use metaFlye)
-med    --medaka_model          Medaka polishing model (default: r1041_e82_400bps_sup_v5.0.0)
-mo     --medaka_off            Do not run Medaka polishing with Nanopore assemblies (default: use Medaka)
-vmb    --use_vamb              Use VAMB for binning (default: use GraphMB)
-sem    --semibin_model         Binning model for SemiBin (default: global)
-mlc    --min_len_contig        Minimum assembly contig length (default: 3000)
-mlb    --min_len_bin           Minimum genomic bin size (default: 250000)
-h      --help                  Print help information
-v      --version               Print workflow version number

ADVANCED SETTINGS:
-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)
-env    --conda_envs_only       Use conda environments instead of container (default: use container)
-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

Using differential coverage binning

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.

Overview of result files

• <output_name>_assembly.fasta - assembled and polished metagenome
• <output_name>_bins.tsv - dataframe for automated binning results
• dependencies.csv- list of dependencies used and their versions
• bins - directory for metagenome assembled genomes

Additional documentation

• Dataframe description
• Dependency list

Citation

If you use mmlong2-lite in a publication, please cite:

Sereika M, Mussig AJ, Jiang C, Knudsen KS, Jensen TBN, Petriglieri F, et al. Recovery of highly contiguous genomes from complex terrestrial habitats reveals over 15,000 novel prokaryotic species and expands characterization of soil and sediment microbial communities. bioRxiv. 2024.12.19.629313. https://doi.org/10.1101/2024.12.19.629313