Automated long-read metagenomics workflow, using either PacBio HiFi or Nanopore sequencing reads as input to generate characterized MAGs. The mmlong2 workflow is a continuation of mmlong.

Note: multiple large-scale databases are utilized by mmlong2 for genome bin analysis. If you are only interested in getting the MAGs, check out mmlong2-lite.

Overview of mmlong2 workflow in Nanopore-only mode:

Installation (Conda):
A local Conda environment containing all the required software dependencies can be created by using the code chunk posted below. To acquire microbial genome taxonomy and annotation results, databases will have to be setup.

conda create --prefix mmlong2 -c conda-forge -c bioconda snakemake=7.26.0 singularity=3.8.6 zenodo_get=1.3.4 pv=1.6.6 pigz=2.6 tar=1.34 -y
conda activate ./mmlong2 || source activate ./mmlong2 && zenodo_get -r 8027235 -o mmlong2/bin 
pv mmlong2/bin/sing-mmlong2-lite-*.tar.gz | pigz -dc - | tar xf - -C mmlong2/bin/.
pv mmlong2/bin/sing-mmlong2-proc-*.tar.gz | pigz -dc - | tar xf - -C mmlong2/bin/.
chmod +x mmlong2/bin/mmlong2

Quick-start (AAU bioserver users):

conda activate /projects/microflora_danica/mmlong2/conda/mmlong2-v0.9.2
mmlong2 -h

Usage example for Nanopore-only mode:

mmlong2 -np [Nanopore_reads.fastq] -p [Processes/Threads] -o [Output_dir]

Full usage:

MAIN INPUTS:
-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)
-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 binning taxonomy annotation variants)

ADDITIONAL INPUTS:
-tmp    --temporary_dir         Directory for temporary files (default: none)
-med1   --medaka_model_polish   Medaka polishing model (default: r1041_e82_400bps_sup_v4.2.0)
-med2	--medaka_model_variant	Medaka variant calling model (default: r1041_e82_400bps_sup_variant_v4.2.0)
-sem    --semibin_model         Binning model for SemiBin (default: global)
-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)
-mlc    --min_len_contig        Minimum assembly contig length (default: 3000)
-mlb    --min_len_bin           Minimum genomic bin size (default: 250000)
-slv    --silva                 Silva database to use (default: none)
-mds    --midas                 Midas database to use (default: none)
-gnc    --gunc                  Gunc database to use (default: none)
-bkt    --bakta                 Bakta database to use (default: none)
-kj     --kaiju                 Kaiju database to use (default: none)
-gdb    --gtdb                  GTDB-tk database to use (default: none)
-x1     --extra_inputs1         Extra inputs for the MAG production part of the Snakemake workflow (default: none)
-x2     --extra_inputs2         Extra inputs for the MAG processing part of the Snakemake workflow (default: none)

MISCELLANEOUS INPUTS:
-h      --help                  Print help information
-v      --version               Print workflow version number

Overview of result files:

• assembly.fasta - assembled and polished metagenome
• rRNA.fa - rRNA sequences, recovered from the polished metagenome
• rRNA_16S.fa - 16S rRNA sequences, recovered from the polished metagenome
• <name>_contigs.tsv - dataframe for metagenome contig metrics
• <name>_bins.tsv - dataframe for automated binning results
• <name>_general.tsv - workflow results, summarized into a single row
• dependencies.csv- list of dependencies used and their versions
• bins - directory for metagenome assembled genomes
• bakta - directory, containing bin annotation results from bakta

Additional documentation:

• Dataframe description
• Dependency list
• Database setup

Comments:

• The workflow assumes that the input reads have been quality-filtered and adapter/barcode sequences have been trimmed off.
• The workflow is long-read-based and requires either Nanopore or PacBio HiFi reads. It doesn't feature an Illumina-only mode.
• If the workflow crashes, it can be resumed by re-running the same command. Some of the intermediary files might have to be removed for compatibility.
• It is recommended to run the workflow from a screen session. This can be achieved with e.g. screen -R mmlong2 and then running the workflow.

Future improvements
Suggestions on improving the workflow or fixing bugs are always welcome.
Please use the GitHub Issues section or e-mail to mase@bio.aau.dk for providing feedback.