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DOI License: GPL v3 DOI

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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

mmlong2-lite-wf


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