OmicsBox Metagenomics Module

The OmicsBox Metagenomics Module, developed by BioBam, is a comprehensive bioinformatics solution for end-to-end microbiome data analysis. It enables the flexible and intuitive combination of all necessary steps — from raw read quality control through to taxonomic classification, metagenomic assembly, gene prediction, and functional annotation — within a single, user-friendly platform. The module is designed for researchers working with both 16S/ITS amplicon and whole-genome shotgun (WGS) sequencing data, and runs on any standard PC or laptop running Windows, Linux, or macOS.

All computationally intensive tasks are executed transparently in the BioBam Bioinformatics Cloud, meaning an ordinary computer is sufficient to carry out even large-scale analyses. Custom pipelines can be designed to suit individual analysis strategies, making the module equally accessible to bioinformatics novices and experienced researchers.

Quality Control and Assessment

• FastQC is used to generate quality assessment reports for sequencing samples.
• Trimmomatic is applied to filter reads and remove low-quality bases.
• Contaminant removal is performed using Bowtie2 against existing or user-provided target genomes.
• rRNA removal with SortMeRNA is available to prepare metatranscriptomic NGS data for downstream analysis.

Taxonomic Classification

• Kraken 2 is used to identify Bacteria, Archaea, Fungi, Protozoa, and Viruses down to strain level.
• The Kraken 2 database includes WGS RefSeq genomes of Archaea, Bacteria, Protozoa, Fungi, and Viruses, as well as common host organisms including Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, Sus scrofa, Drosophila melanogaster, and Arabidopsis thaliana.
• New Kraken databases are available, including UNITE Fungi and MiDAS 5.
• New taxonomic classifications are supported via Silva, Greengenes, and GTDB.
• The main result table displays all identified OTUs for each provided sample.
• Results can be sorted, filtered, and reads belonging to selected taxonomic units can be extracted.
• A PDF report provides a clean overview of the most abundant OTUs at different taxonomic levels for each sample.

Rich Visualizations for Taxonomic Results

• Stacked bar charts enable comparison of samples at specific taxonomic levels.
• Radial cladograms in the form of Krona charts allow exploration of OTU abundances within each sample.
• Rarefaction curves assess sequencing depth at different taxonomic levels.
• Chao1 species richness curves evaluate species diversity across different levels with sample selection options.
• Principal Coordinates Analysis (PCoA) plots reveal how samples and groups separate and help identify outliers, with configurable PCoA options.
• Interactive bar charts support inter-sample comparison at the genus or species level, providing a quick overview of taxonomic abundance compositions.
• Colorful and interactive charts allow intuitive exploration of relative species abundances and confidence scores within complex metagenomic classification hierarchies.

Differential Abundance Testing of Taxa

• OTU differential abundance testing is performed using edgeR to identify over- and underrepresented OTUs between samples and conditions.
• Results are accompanied by a PDF report, heatmaps, and additional visualizations to support interpretation.

Metagenomic Assembly

• MetaSPAdes and MEGAHIT are both available for assembling large metagenomic datasets quickly and efficiently in the cloud.

Gene Prediction

• FragGeneScan is used for gene prediction directly from plain reads.
• Prodigal is applied to assembled metagenomic data to identify and extract possible genes and proteins.

Functional Analysis

• High-throughput functional annotation is achieved using EggNOG-Mapper and PfamScan.
• Metagenomic GO-Slim analysis is supported.
• Comparative analysis of functions is visualised with bar charts and GO graphs.
• Differential abundance testing of functions is available for Pfam Domains and Families, KEGG Maps, and EggNOG COGs.
• Results are presented as spreadsheets and can be filtered and visualised with hierarchical bar and graph charts, as well as bubble charts.

Example Workflows

• Taxonomic Classification Workflow: Reads are preprocessed with Trimmomatic, quality reports are generated with FastQC, and Kraken 2 is used to identify and count all OTUs. Results include a filterable spreadsheet, a PDF report of the most abundant OTUs, and intra- and inter-sample comparison charts. The entire workflow can be launched within a few clicks.
• Functional Characterization Workflow: Combines MEGAHIT assembly with Prodigal gene prediction, followed by high-throughput functional annotation using EggNOG-Mapper and PfamScan. This streamlined workflow is designed to handle resource-demanding assembly and annotation steps for large metagenomic datasets.

OmicsBox Metagenomics Module is suitable for both targeted sequencing and WGS metagenomics projects. A benchmark analysis of cyanobacterial bloom in lakes demonstrated processing of 6.7 million input reads, prediction of 250,000 genes, and 90% high-quality annotation within 6 hours. The module is available for a free trial or via a custom demo, and operates out of the box on Windows, Linux, and Mac systems.