MetaKraken2

MetaKraken2 is a Metagenomic pipeline for Kraken2 analysis and Krona report generation. It classifies each read with Kraken2 and then generates an interactive pie chart with Krona.

<p align="center"> <img src="Figures/MetaKraken2_pipeline.png" alt="drawing" width="300" title="MetaKraken2"> </p>

Getting started

Prerequisites

• Miniconda3. Tested with conda 4.9.2. which conda should return the path to the executable. If you don't have Miniconda3 installed, you could download and install it with:

wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh
chmod 755 Miniconda3-latest-Linux-x86_64.sh
./Miniconda3-latest-Linux-x86_64.sh

• A Kraken2-indexed database. You could download one precompiled database from here or generate one using Build_Kraken2_db.sh script.

• A fastq file containing sequences you want to analyse.

Installation

git clone https://github.com/MaestSi/MetaKraken2.git
cd MetaKraken2
chmod 755 *
./install.sh

A conda environment named MetaKraken2_env is created, where kraken2 and Krona are installed.

Usage

MetaKraken2.sh

Usage: ./MetaKraken2.sh -f <sample_name.fast*> -db <kraken2_db> -c <confidence_scoring> -t <threads>

Inputs:

• <sample_name.fast*>: fastq or fasta file containing sequences to be analysed; use double quotes to delimit the two files if working with paired-end reads
• <kraken2_db>: kraken2-indexed database
• <confidence_scoring>: confidence value in [0-1], defined as C/Q, where C is the number of k-mers mapped to LCA values in the clade rooted at the label, and Q is the number of k-mers in the sequence that lack an ambiguous nucleotide; higher values will result in more stringent classifications. Suggested values 0.05/0.1
• <threads>: maximum number of threads to use

Outputs:

• <sample_name>_confidence_<confidence_scoring>_kraken2_output.txt: Kraken2 output
• <sample_name>_confidence_<confidence_scoring>_kraken2_report.txt: Kraken2 report used by Krona for html report generation
• <sample_name>_confidence_<confidence_scoring>_kraken2_report_Krona_report.html: interatctive pie chart produced by Krona

Build_Kraken2_db.sh

Usage: ./Build_Kraken2_db.sh -db <kraken2_db> -t <threads>

Inputs:

• <kraken2_db>: kraken2 database you want to build. Possible choices are: standard, archaea, bacteria, plasmid, viral, human, fungi, plant, protozoa, nr, nt, UniVec, UniVec_Core. Except for standard database, you can include multiple libraries at once by delimiting them with double quotes (e.g. "archaea bacteria")
• <threads>: maximum number of threads to use

Output:

• <kraken2_db>_db: folder containing kraken2 database

Results visualization

After completing the analysis, you can open <sample_name>_kraken2_report_Krona_report.html with a browser.

<p align="center"> <img src="Figures/Krona_report_example.png" alt="drawing" width="30000" title="Krona report example"> </p>

Citations

Please refer to the following manuscripts for further information.

Kraken2: Wood DE, Lu J, Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019 Nov 28;20(1):257. doi: 10.1186/s13059-019-1891-0. PMID: 31779668; PMCID: PMC6883579.

Krona: Ondov BD, Bergman NH, Phillippy AM. Interactive metagenomic visualization in a Web browser. BMC Bioinformatics. 2011 Sep 30;12:385. doi: 10.1186/1471-2105-12-385. PMID: 21961884; PMCID: PMC3190407.