Pipeline consists of 3 chief scripts:

1. bbmap_index.nf - Makes indexes for your references and gives them appropriate identificator
2. main.nf - includes following submodules:
   • preprocessing
   • mapping
   • classify_reads
   • assembly
   • binning
3. additional.nf:
   • binrefinement
   • bins classification

All processes and their parameters can be found in subscripts/process.nf
All information about submodules - in according nf scripts. Each submodule can be launched on its own as separate script!

1. GTDB-Tk requires ~84G of external data that needs to be downloaded and unarchived:

wget https://data.gtdb.ecogenomic.org/releases/latest/auxillary_files/gtdbtk_data.tar.gz # mirror: https://data.ace.uq.edu.au/public/gtdb/data/releases/latest/auxillary_files/gtdbtk_data.tar.gz
tar xvzf gtdbtk_data.tar.gz

2. Link GTDB to the "gtdbtk_data" folder in pipeline:

ln -s /path/to/gtdbtk /path/to/pipeline/gtdbtk_data

1. CheckM DB requires ~1.4G of external data that needs to be downloaded and unarchived:

wget https://data.ace.uq.edu.au/public/CheckM_databases/checkm_data_2015_01_16.tar.gz
tar xvzf checkm_data_2015_01_16.tar.gz

2. Link CheckM database to the "checkm_data" folder in pipeline:

ln -s /path/to/checkm_data /path/to/pipeline/checkm_data

1. Kraken requires ~16Gb of external data that needs to be downloaded and unarchived:

wget https://genome-idx.s3.amazonaws.com/kraken/k2_standard_16gb_20231009.tar.gz
tar xvzf k2_standard_16gb_20231009.tar.gz

2. Link Kraken2 db to the "kraken2_data" folder in pipeline:

ln -s /path/to/kraken2_data /path/to/pipeline/kraken2_data

1. Install MetaPhlAn in the pipeline directory:

mamba create -p /path/to/pipeline/metaphlan -c bioconda metaphlan

2. It requires ~15Gb of external data that needs to be downloaded:

mamba run -p /path/to/pipeline/metaphlan metaphlan --install 

1. Install it in the pipeline directory:

mamba create -p /path/to/pipeline/mw-env -c ursky metawrap-mg=1.3.2

2. Make corrections to metaWRAP bin_refinement.sh script:

sed -i 's/(( $SIZE > 50000)) &&//g' /path/to/pipeline/mw-env/bin/metawrap-modules/bin_refinement.sh 

nextflow run bbmap_index.nf \
-with-report \
-with-conda \
--ref host.fna,human.fna

nextflow run main.nf \
-with-conda \
-with-report \
--ref host.fna,human.fna \
--ref_number 1,2 \
--reads 'pipeline/fq_check/*{1,2}.fq' \
--maxbin2 --concoct --metabat2 \ # specify at least one binner
--output pipeline_results

Optional parameters of main.nf with their default options:

1. fastp options
   • --compress_level 7
   • --poly_g_min_len 5
   • --poly_x_min_len 10
   • --cut_mean_quality 27
   • --n_base_limit 0
   • --average_qual 25
   • --length_required 80
   • --working_thread_n 12
2. kraken2, bracken options
   • --classification_lvl "S"
3. bbmap options
   • --host_dir "host"
   • --human_dir "human"
   • --bbmap_memory false
   • --bbmap_cpus 4
4. metawrap binning options
   • --concoct false
   • --metabat2 false
   • --maxbin2 false
5. metawrap bin refinement options
   • metawrap_completion 50
   • metawrap_contamination 30