Preprocessing steps for metagenomics reads

This is a Snakemake workflow to:

1. Check the quality of metagenomic reads using FastQC.
2. Trim low-quality bases and adapter sequences using Trimmomatic.
3. Assess the post-trimming quality of reads using FastQC.
4. Remove human DNA from the metagenomic dataset.

This workflow was used in:

1. Development of early life gut resistome and mobilome across gestational ages and microbiota-modifying treatments
   Authors: Ahmed Bargheet, Claus Klingenberg, Eirin Esaiassen, Erik Hjerde, Jorunn Pauline Cavanagh, Johan Bengtsson-Palme, Veronika Kuchařová Pettersen

2. Dynamics of the Gut Resistome and Mobilome in Early Life: A Meta-Analysis
   Authors: Ahmed Bargheet, Hanna Noordzij, Alise Ponsero, Ching Jian, Katri Korpela, Mireia Valles-Colomer, Justine Debelius, Alexander Kurilshikov, Veronika K. Pettersen

Installation and requirements

This pipeline requires the use of Snakemake, FastQC v0.11.9, Trimmomatic v=0.39, Bowtie 2 v2.4.5, SAMtools v1.17, and BEDTools v2.30.0.
If not previously installed run the following code:

# Clone the repository
git clone https://github.com/Ahmedbargheet/Snakemake_short_reads_preprocessing.git
cd Snakemake_short_reads_preprocessing

## Snakemake installation in a conda environment
conda env create --file envs/env_snakemake.yml

# Alternatively you can create the environment manually:
conda create -n snakemake_env -c bioconda snakemake fastqc=0.11.9 trimmomatic=0.39 bowtie2=2.4.5 samtools=1.17 bedtools=2.30.0
conda activate snakemake_env

Additionally, the human genome database should be downloaded from NCBI
Follow the following steps for downloading and indexing the human genome database

mkdir Human_database
cd Human_database
wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.40_GRCh38.p14/GCF_000001405.40_GRCh38.p14_genomic.fna.gz
gunzip GCF_000001405.40_GRCh38.p14_genomic.fna.gz
bowtie2-build GCF_000001405.40_GRCh38.p14_genomic.fna Human

Moreover, the Trimmomatic binary file should be downloaded
Follow the following steps

mkdir trimmomatic
cd trimmomatic
wget http://www.usadellab.org/cms/uploads/supplementary/Trimmomatic/Trimmomatic-0.39.zip
unzip Trimmomatic-0.39.zip

Overview of the pipeline

How to run the Snakemake pipeline

In the Snakefile, you will find samples variables. You can change ["sample_name"] to your actual sample name. The pipeline is designed to work with paired files {sample}_1.fastq.gz and {sample}_2.fastq.gz)

# run the pipeline
mkdir -p result/1.fastqc/
mkdir -p result/2.trimmomatic/
mkdir -p result/3.fastqc/
mkdir -p result/4.rm_dna/1.sort/
mkdir -p result/4.rm_dna/2.fq/
snakemake --cores 8

Note:

The main workflow is designed to remove adapters from the TruSeq kit. However, if the sequencing center used adapters from the Nextera kit, you can change this path:

/trimmomatic/Trimmomatic-0.39/adapters/TruSeq3-PE.fa

To

/trimmomatic/Trimmomatic-0.39/adapters/NexteraPE-PE.fa