Taxonomic Or FUnctional Metagenomic Assembly and PrOfiling = TOFU-MAaPO
Documentation about the pipeline can be found in the docs/ directory or under the links below:
Overview of TOFU-MAaPO 1.3.1
The pipeline analyses metagenomic short reads and can perform analysis for taxonomic profiling, abundance of microbial metabolic pathways and assembly of metagenomic genomes.
TOFU-MAaPO is a Nextflow pipeline for the processing and analysis of metagenomic short reads with a focus on gut metagenomes. The pipeline can run on any linux system and needs as dependencies only Nextflow and the container engine Singularity. An installation step is not needed as Nextflow can automatically download all needed files.
The pipeline can install needed databases on its own, see for this the usage documentation. The pipeline can therefor be downloaded and executed in one single command line call. It requires short single- or paired-end metagenomic shotgun sequencing FASTQ files as input, or a single csv file containing a list of samples and their associated FASTQ files or can download data from SRA by giving the project or sample id as an input.
TOFU-MAaPO processes the data for quality control and possible host decontamination (optional) and performs downstream analysis for taxonomic abundance profiles of each sample using Kraken2, Bracken, Salmon and/or Metaphlan4, metabolic pathway analysis using HUMAnN (v3.6) and assembly of metagenomic genomes (MAGs).
Genome assembly is done by generating contigs from the qc'ed reads with Megahit (single samples, grouped or all samples combined). The contigs are then catalogued and indexed using minimap2 and then binned with the option to use up to five binning tools (Metabat2, Concoct, Maxbin, Semibin2 and vamb). The resulting bins will then be refined and, where possible, combined with MAGScoT based on sets of single-copy microbial marker genes from the Genome Taxonomy Database. The profiles of present marker genes in each result from the different binning algorithms are compared, and new hybrid candidate bins are created if the bin sets share a user-adjustable proportion of marker genes. The results are also taxonomically annotated with GTDB-TK and quality checked with checkm. An estimated bin coverage per sample is generated as additional output.
Please install Conda. With it, you can easily install Singularity and Nextflow. After installation, make sure that the environment is activated and test that Singularity (now Apptainer) and Nextflow are working:
# Create a new conda environment for Singularity and Nextflow
conda create --name nf_env -c conda-forge -c bioconda singularity nextflow
# Activate environment
conda activate nf_env
# Check whether Singularity has been successfully installed
singularity --version
# Try a simple Nextflow demo
nextflow run hello
Please edit the configurations to your system needs as explained in the installation and configuration documentation.
The input to TOFU-MAaPO can be either locally stored fastq.gz files or SRA IDs (sample or project).
Running a workflow for gut metagenomes with QC (without host read removal) and MAG assembly with paired-end local files would be called like this:
nextflow run ikmb/TOFU-MAaPO \
-profile custom \
--reads '/path/to/fastqfiles/*_R{1,2}_001.fastq.gz' \
--assembly \
--updategtdbtk \
--gtdbtk_reference '/path/to/download/gtdbtk_db/to' \
--outdir results
For further usage options please see the usage documentation.
A workflow with QC (without host read removal) and MAG assembly for human gut metagenomes with SRA Run IDs as input would look like this:
nextflow run ikmb/TOFU-MAaPO \
-profile custom \
---sra 'SRX3105436' \
--assembly \
--updategtdbtk \
--apikey **YOUR_NCBI_API_KEY***
--gtdbtk_reference '/path/to/download/gtdbtk_db/to' \
--outdir results
You can get your NCBI API Key for your NCBI Account by going to NCBI -> Account -> Account Settings -> API Key Management.
The project was funded by the German Research Foundation (DFG) Research Unit 5042 - miTarget INF.