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Updated: Apr 9, 2026
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Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
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FAVABEAN and FALAPhyl: Open-source pipelines for scalable 16s rRNA microbiome data processing and visualization.
Afnan Faridoun1, Ramon Carvalho2, Jacob Smith3
1Department of General Dental Practice, College of Dentistry, Kuwait University, Kuwait.
Plos One
|April 7, 2026
Summary
New containerized workflows, FAVABEAN and FALAPhyl, simplify 16S rRNA amplicon sequencing analysis for microbiome research. These tools automate complex bioinformatics tasks, enhancing reproducibility and accessibility for researchers studying microbial communities.
Area of Science:
- Microbiome Research
- Bioinformatics
- Computational Biology
Background:
- Analyzing 16S rRNA amplicon sequencing data is complex and requires significant bioinformatics expertise.
- Existing tools often lack compatibility and scalability, hindering reproducible microbiome research.
- There is a need for automated, user-friendly pipelines for 16S rRNA data analysis.
Purpose of the Study:
- To develop and validate containerized bioinformatics workflows for reproducible and scalable 16S rRNA amplicon data analysis.
- To address challenges in tool complexity, platform incompatibility, and bioinformatics expertise required for microbiome research.
- To provide automated preprocessing, variant annotation, phylogenetic analysis, and differential abundance testing.
Main Methods:
- Development of two Snakemake-based containerized pipelines: FAVABEAN (Fast Amplicon Variant Annotation, Binning, Error-correction And ANalysis) and FALAPhyl (Forays into Automating Laborious Analyses of Phylogeny).
- FAVABEAN automates ASV inference and taxonomic assignment using DADA2, FIGARO, and SMURF.
- FALAPhyl supports downstream analyses including diversity metrics, network analysis, differential abundance testing with DAtest, and provenance tracking.
- Validation using three oral microbiome datasets, including family member comparisons, dental aerosol analysis, and a mouthrinse trial.
Main Results:
- FAVABEAN and FALAPhyl provide reproducible and automated analysis of 16S rRNA amplicon data.
- Case studies demonstrated primer-dependent variability in oral microbiota and highlighted oral microbial stability.
- FALAPhyl's DAtest integration facilitated robust differential abundance inference.
- The pipelines are designed for flexible execution across local, cluster, and cloud environments.
Conclusions:
- FAVABEAN and FALAPhyl offer a significant advancement for microbiome research by simplifying complex 16S rRNA data analysis.
- The containerized and modular design enhances accessibility, reproducibility, and scientific rigor.
- These workflows empower researchers with limited bioinformatics expertise to conduct sophisticated microbiome analyses.
