metaBIOMx: Metagenomics pipeline for Microbial shot-gun sequencing data

main @ 240bd6c

Introduction metaBIOMx

The metagenomics microbiomics pipeline is a best-practice suite for the decontamination and annotation of sequencing data obtained via short-read shotgun sequencing. The pipeline contains NF-core modules and other local modules that are in the similar format. It can be runned via both docker and singularity containers.

Pipeline summary

The pipeline is able to perform different taxonomic annotation on either (single/paired) reads or contigs. The different subworkflows can be defined via --bypass flags, a full overview is shown by running --help. By default the pipeline will check if the right databases are present in the right formats, when the path is provided. If this is not the case, compatible databases will be automatically downloaded.

For both subworkflows the pipeline will perform read trimming via Trimmomatic and/or AdapterRemoval, followed by human removal via Kneaddata. Before and after each step the quality control will be assessed via fastqc and a multiqc report is created as output. Then taxonomy annotation is done as follows:

Read annotation

• paired reads are interleaved using BBTools.
• MetaPhlAn3 and HUMAnN3 are used for taxonomy and functional profiling.
• taxonomy profiles are merged into a single BIOM file using biom-format.

Contig annotation

• read assembly is performed via SPAdes.
• Quality assesment of contigs is done via Busco.
• taxonomy profiles are created using CAT.
• Read abundance estimation is performed on the contigs using Bowtie2 and BCFtools.
• Contigs are selected if a read can be aligned against a contig and a BIOM file is generated using biom-format.

Installation

[!NOTE] Make sure you have installed the latest nextflow version!

Clone the repository in a directory of your choice:

git clone https://github.com/CMG-GUTS/metabiomx.git

The pipeline is containerised, meaning it can be runned via docker or singularity images. No further actions need to be performed when using the docker profile, except a docker registery needs to be set on your local system, see docker. In case singularity is used, please specify the singularity.cacheDir in the nextflow.config so that singularity images are saved there and re-used again.

Usage

Since the latest version, metaBIOMx works with both a samplesheet (CSV) format or a path to the input files. Preferably, samplesheets should be provided.

nextflow run main.nf --input samplesheet.csv -work-dir work -profile singularity
nextflow run main.nf --input '*_{1,R1,2,R2}.{fq,fq.gz,fastq,fastq.gz}' -work-dir work -profile singularity

Automatic database setup

The pipeline requires a set of databases which are used by the different tools within this workflow. The user is required to specify the location in where the databases will be downloaded. It is also possible to download the databases manually. The configure subworkflow will evaluate the database format and presence of the compatible files automatically.

nextflow run main.nf \
    --bowtie_db path/to/db/bowtie2 \
    --metaphlan_db path/to/db/metaphlan \
    --humann_db path/to/db/humann \
    --cat_pack_db path/to/db/catpack \
    --busco_db path/to/db/busco_downloads \
    -work-dir  \
    -profile 

Manual database setup

HUMAnN3 and MetaPhlan3 DB

Make sure the path/to/db/humann should contain a chocophlan, uniref and utility_mapping directory. These can be obtained by the following command:

docker pull biobakery/humann:latest

docker run --rm -v $(pwd):/scripts biobakery/humann:latest \
    humann_databases --download chocophlan full ./path/to/db/humann \
    && humann_databases --download uniref uniref90_diamond ./path/to/db/humann \
    && humann_databases --download utility_mapping full ./path/to/db/humann

MetaPhlAn DB

wget http://cmprod1.cibio.unitn.it/biobakery4/metaphlan_databases/mpa_vJun23_CHOCOPhlAnSGB_202403.tar \
    && tar -xvf mpa_vJun23_CHOCOPhlAnSGB_202403.tar -C path/to/db/metaphlan \
    && rm mpa_vJun23_CHOCOPhlAnSGB_202403.tar

wget http://cmprod1.cibio.unitn.it/biobakery4/metaphlan_databases/bowtie2_indexes/mpa_vJun23_CHOCOPhlAnSGB_202403_bt2.tar \
    && tar -xvf mpa_vJun23_CHOCOPhlAnSGB_202403_bt2.tar -C path/to/db/metaphlan \
    && rm mpa_vJun23_CHOCOPhlAnSGB_202403_bt2.tar

echo 'mpa_vJun23_CHOCOPhlAnSGB_202403' > path/to/db/metaphlan/mpa_latest

Kneaddata DB

docker pull agusinac/kneaddata:latest

docker run --rm -v $(pwd):/scripts agusinac/kneaddata:latest \
    kneaddata_database \
        --download human_genome bowtie2 ./path/to/db/bowtie2

CAT_pack DB

A pre-constructed diamond database can be downloaded manually or by command:

docker pull agusinac/catpack:latest

docker run --rm -v $(pwd):/scripts agusinac/catpack:latest \
    CAT_pack download \
        --db nr \
        -o path/to/db/catpack

busco DB

BUSCO expects that the directory is called busco_downloads.

docker pull ezlabgva/busco:v5.8.2_cv1

docker run --rm -v $(pwd):/scripts ezlabgva/busco:v5.8.2_cv1 \
    busco \
        --download bacteria_odb12 \
        --download_path path/to/db/busco_downloads

Support

If you are having issues, please create an issue