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Tutorials - Explore MAGs with CoverM and Krona

Table of Contents

Description
Requirements
Data download
Bioconda: Installation of CoverM and Krona Plot
Mapping
Reformat the mapping output
Create Krona plot

Description

In this tutorial we will map a metagenomic library to the iMGMC mouse MAG collection to get species abundances and visualize the results with a Krona plot.

Requirements

Data-pre-processing

Data-Download

We select representative mMAG even with medium Quality (comp>50, con<10) to cover full diversity.

# download mMAG genomes and annotations
wget -O iMGMC-mMAGs-dereplicated_genomes.tar.gz "https://onedrive.live.com/download?cid=36ADEB4B3D109F6F&resid=36ADEB4B3D109F6F%2137126&authkey=ADFYgL1YRjtb-Vo"
wget -O "MAG-annotation_CheckM_dRep_GTDB-Tk.tar.gz" "https://onedrive.live.com/download?cid=36ADEB4B3D109F6F&resid=36ADEB4B3D109F6F%2137698&authkey=AL9RrHT7_3oj2vI"
tar -xzf iMGMC-mMAGs-dereplicated_genomes.tar.gz
tar -xzf MAG-annotation_CheckM_dRep_GTDB-Tk.tar.gz	

# download example data
wget -c ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR335/000/ERR3357550/ERR3357550_1.fastq.gz
wget -c ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR335/000/ERR3357550/ERR3357550_2.fastq.gz

# set sample name
SampleName=ERR3357550

Bioconda-Tool-Installation

We install CoverM and Krona Plot via Bioconda. Please be sure the you install and activate Bioconda before.

conda create -n coverm coverm
conda create -n krona krona

Mapping

For this tutorial we use CoverM for mapping the reads to MAG collection. Please see CoverM documentation for more details. Waring this process need around 32 GB of memory.

# bioconda activate enviroment
conda activate coverm
# run CoverM for Sample ($SampleName) with ReadR1 ($Fastq_R1) and ReadR2 ($Fastq_R2)
coverm genome --threads 24 \
--genome-fasta-directory dereplicated_genomes \
--genome-fasta-extension fa \
--coupled \
${SampleName}_1.fastq.gz \
${SampleName}_2.fastq.gz \
> abundances-${SampleName}.txt

coverm

Reformat-Mapping-Output

This will add taxonomy to the abundance profile and reformat output for Krona plotting with MAG-name

# add taxonomy and MAG-name
paste \
<( tail -n+3  abundances-${SampleName}.txt | sort ) \
<( cut -f1 abundances-${SampleName}.txt | sed "s/^/\^/" | grep -w -f - gtdbtk.bac120.summary.tsv | cut -f1,2 | sort ) | \
awk '{print $2"\t"$4"-"$1}' | tr -s ";" $"\t" > krona-input-MAGs-${SampleName}.txt

#add unmapped reads
grep unmapped abundances-${SampleName}.txt | cut -f2 >> krona-input-MAGs-${SampleName}.txt

reformat-krona.png

Create-Krona-Plot

# bioconda activate enviroment
conda activate krona
# run Krona
ktImportText -n MAG-mapping -o ${SampleName}.krona.html krona-input-MAGs-${SampleName}.txt

After running this steps you can open the resulting html file and explore the microbiota:

krona-plot