# Quick start ## metaFun install and run ### 1. Install biocontainer If there is no conda or mamba in your system, follow the instructions and install conda or mamba. We reommend to install [miniconda](https://docs.anaconda.com/miniconda/miniconda-install/) or [mamba](https://mamba.readthedocs.io/en/latest/installation/mamba-installation.html). ```{code-block} bash :caption: Install miniconda # Suppose you are using Linux OS. wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh -O ~/miniconda3/miniconda.sh # you can indicate the installation path by replacing -p $PATH. $PATH is the base directory of your conda installation. bash miniconda.sh -b -u -p ~/miniconda3 rm miniconda.sh ``` ```{admonition} Install mamba We recommend to install mamba suitable for quick installation following instruction from [miniforge github](https://github.com/conda-forge/miniforge). ``` ### 2. Download metaFun from Bioconda ```{code-block} bash # we recommend create a new conda environment for metaFun conda create -n metafun bioconda::metafun # If you have mamba, you can install metaFun with mamba mamba create -n metafun bioconda::metafun mamba activate metafun ``` ### 3. Download databases to utilize metaFun ```{code-block} bash # download databases conda activate metafun (metafun) name $ metafun -module DOWNLOAD_DB ``` ````{admonition} module DOWNLOAD_DB execution :class: note Execution of `metafun -module DOWNLOAD_DB` is shown in the following figure. ```{figure} ../images/DownloadDB_execution_metafun_v011.png --- width: 100% figclass: margin-caption alt: metafun_pipeline name: download_db_metafun align: middle --- ``` ```` ````{admonition} module DOWNLOAD_DB execution :class: note Due to the huge size of databases (File size of raw tar gzipped files : ~683GB), it may take a while to download databasese depending on your network speed. Database information is available at [download repository](https://www.microbiome.re.kr/home_design/list_files.php?path=metafun%2Fv0.1%2FmetaFun_db_distribute). The integrity of downloaded databases is automatically checked by comparing sha256. If there are problems, please redonwload the datases. Typically you can find the database path by the following command. ```{code-block} bash ls -d $(find $(dirname $(which metafun)) -type d ! -name 'metafun')/../share/metafun/db ``` ```` ### 4. Run Modules of metaFun metaFun provides two main analysis workflows: #### Genome-based analysis path: RAWREAD_QC → ASSEMBLY_BINNING → BIN_ASSESSMENT → GENOME_SELECTOR → COMPARATIVE_ANNOTATION → INTERACTIVE_COMPARATIVE #### Read-based analysis path: For taxonomic composition RAWREAD_QC → WMS_TAXONOMY → INTERACTIVE_TAXONOMY For functional annotation RAWREAD_QC → WMS_FUNCTION For strain-level analysis RAWREAD_QC → WMS_TAXONOMY → WMS_STRAIN → INTERACTIVE_STRAIN For network analysis WMS_TAXONOMY → INTERACTIVE_NETWORK You can execute each module of metaFun using the following syntax: ```{code-block} bash metafun -module [options] ``` #### Available Modules ```{admonition} Important Workflow Information :class: warning - When specifying an input directory for RAWREAD_QC, subsequent modules will automatically use the output from previous steps as their input unless explicitly overridden. - Mandatory parameters are typically needed when specifying metadata files and their columns (sample ID or analysis columns). Each module that requires metadata will need these parameters explicitly defined. - For the most efficient usage, follow the color-coded workflow paths shown above. ``` ## RAWREAD_QC: Quality control of raw reads and host genome filtering **Required:** -i (input reads directory) ```{code-block} bash :caption: Example metafun -module RAWREAD_QC -i input_reads/ ``` ## ASSEMBLY_BINNING: Assembly and binning **Optional:** -i (filtered reads directory) (If you run RAWREAD_QC without output parameter, you can run it this module without `-i` parameter in this module.) ```{code-block} bash :caption: Example metafun -module ASSEMBLY_BINNING -i filtered_reads/ -p 40 ``` ## BIN_ASSESSMENT: Assess genome quality and taxonomy classification **Required:** -m -c ```{code-block} bash :caption: Example metafun -module BIN_ASSESSMENT -m metadata.txt -c 2 ``` ## GENOME_SELECTOR: Genome selection interface **Required:** -i ```{code-block} bash :caption: Example metafun -module GENOME_SELECTOR -i combined_metadata.csv ``` ## COMPARATIVE_ANNOTATION: Comparative genomic analysis **Required:** -i -m ```{code-block} bash :caption: Example (annotation only for INTERACTIVE_COMPARATIVE module) metafun -module COMPARATIVE_ANNOTATION --metadata metadata.csv --samplecol 1 ``` ```{code-block} bash :caption: Example (with static plots) This is deprecated. metafun -module COMPARATIVE_ANNOTATION -i genomes/ -m metadata.csv --samplecol 1 --metacol 2 ``` ## INTERACTIVE_COMPARATIVE: Interactive comparative analysis **Required:** -i -m ```{code-block} bash :caption: Example metafun -module INTERACTIVE_COMPARATIVE -i results/genomes -m metadata.csv ``` ## WMS_TAXONOMY: Taxonomic profiling of metagenomic reads **Required:** -m -s ```{admonition} Default selection is sylph. :class: note If you would like to utilize kraken2, please download kraken2 database and specify `--profiler kraken2`. `-s` option is accession column prefix of paired reads files of metagenomic data. `-i` option is input directory of metagenomic data. You need to specify this option if you did not run RAWREAD_QC module. ``` ```{code-block} bash :caption: Example metafun -module WMS_TAXONOMY -m meta.csv -s 1 ``` ## INTERACTIVE_TAXONOMY: Interactive taxonomy analysis **Required:** -i ```{code-block} bash :caption: Example metafun -module INTERACTIVE_TAXONOMY -i results/metagenome/WMS_TAXONOMY ``` ## WMS_FUNCTION: Functional analysis of metagenomic reads **Required:** -i -m -s -a ```{code-block} bash :caption: Example metafun -module WMS_FUNCTION -i filtered_reads/ -m metadata.csv -s 1 -a 2 ``` ## WMS_STRAIN: Strain-level microdiversity analysis **Required:** -i --phyloseq_object ```{admonition} Requires WMS_TAXONOMY output :class: note This module requires a phyloseq RDS object from WMS_TAXONOMY for selecting prevalent taxa to analyze at strain level. ``` ```{code-block} bash :caption: Example metafun -module WMS_STRAIN -i results/metagenome/RAWREAD_QC/read_filtered \ --phyloseq_object results/metagenome/WMS_TAXONOMY/phyloseq/phyloseq_object_sylph.RDS ``` ## INTERACTIVE_STRAIN: Interactive strain diversity analysis **Required:** -i ```{code-block} bash :caption: Example metafun -module INTERACTIVE_STRAIN -i results/metagenome/WMS_STRAIN ``` ## INTERACTIVE_NETWORK: Interactive microbial network analysis **Required:** -i ```{admonition} Requires WMS_TAXONOMY output :class: note This module requires a phyloseq RDS object from WMS_TAXONOMY for constructing co-occurrence networks. ``` ```{code-block} bash :caption: Example metafun -module INTERACTIVE_NETWORK -i results/metagenome/WMS_TAXONOMY/phyloseq/phyloseq_object_sylph.RDS ``` ## DOWNLOAD_DB: Download required databases ```{code-block} bash :caption: Example metafun -module DOWNLOAD_DB ``` ## PREPARE_CUSTOM_HOST: Prepare custom host genome index **Required:** -i -f ```{code-block} bash :caption: Example metafun -module PREPARE_CUSTOM_HOST -i genome.fasta -f mouse ``` ```{admonition} Any custom host genome can be used. :class: note If you would like to use custom host genome, please specify the path of fasta file and name of the host genome by `-f`. You need to specify `-f` option in RAWREAD_QC module to filter out your custom host genome. ``` ## Common Options - `-o, --output`: Output directory - `-p, --processors`: Number of processors to use - `-h, --help`: Show detailed help for a module For detailed usage and additional parameters for each module, use: ```{code-block} bash metafun -module -h ```