FAVABEAN and FALAPhyl: Open-Source Pipelines for Scalable 16s rRNA Microbiome Data Processing and Visualization

Reproducible and scalable analysis of 16S rRNA amplicon sequencing data remains a persistent challenge in microbiome research due to the complexity of available tools, incompatibilities between platforms, and the need for extensive bioinformatics expertise. We developed two containerized workflows—FAVABEAN (Fast Amplicon Variant Annotation, Binning, Error-correction And ANalysis) and FALAPhyl (Forays into Automating Laborious Analyses of Phylogeny)—to address these challenges. FAVABEAN and FALAPhyl are Snakemake-based pipelines designed for flexible execution across local, cluster, and cloud environments. FAVABEAN automates preprocessing, ASV inference, and taxonomic assignment using DADA2 and FIGARO, including primer averaging when samples are sequenced with multiple primers. FALAPhyl supports downstream analysis including alpha/beta diversity, network analysis, and differential abundance testing, with integrated provenance tracking. We validated both pipelines using three case studies involving oral microbiome datasets. In Case Study 1, we compared oral microbiota across family members and niches, showing primer-dependent variability in ASV-based similarity and minimal reseeding from familial sources after prophylaxis. Case Study 2 analyzed dental aerosol samples, revealing no significant microbial differences between pre-, intra-, and post-procedure air. Case Study 3, a randomized trial of a nitrate mouthrinse, demonstrated no significant microbiome shifts, highlighting oral microbial stability. FALAPhyl’s integration of DAtest enabled empirical evaluation of multiple statistical tests, aiding robust differential abundance inference. FAVABEAN and FALAPhyl offer a reproducible, automated solution for 16S rRNA amplicon data analysis. Their modular design, containerization, and provenance tracking enhance accessibility and scientific rigor in microbiome research.