Journal: Microbiology Spectrum

Article Title: QIIME2 enhances multi-amplicon sequencing data analysis: a standardized and validated open-source pipeline for comprehensive 16S rRNA gene profiling

doi: 10.1128/spectrum.01673-25

Figure Lengend Snippet: Overall study design and bioinformatic analysis workflow. Overview of the experimental and analytical framework used in this study. Paired stool samples from children and their caregivers underwent DNA extraction, while the Zymo mock community, provided as ready-to-use DNA, served as a reference standard. All samples were prepared for sequencing using an Ion 16S metagenomics kit. Resulting FASTQ files were processed through a comparative pipeline assessment step, in which multiple V-region pipelines (V2–9) were evaluated to ensure robust performance. Following validation, the selected workflows were applied to the full data set, culminating in final analyses that encompassed microbial variant profiling, differential abundance testing, and ratio-based microbiome assessments.

Article Snippet: For this study, we generated independent 16S rRNA amplicon libraries from: (i) mock samples using the ZymoBIOMICS Microbial Community DNA Standard (Zymo Research); (ii) stool samples from a clinical study as validation.

Techniques: DNA Extraction, Sequencing, Biomarker Discovery, Variant Assay

Journal: Microbiology Spectrum

Article Title: QIIME2 enhances multi-amplicon sequencing data analysis: a standardized and validated open-source pipeline for comprehensive 16S rRNA gene profiling

doi: 10.1128/spectrum.01673-25

Figure Lengend Snippet: Comparative evaluation of microbial community profiling by Ion Reporter (IR) and QIIME 2 pipelines across multiple amplicon regions. Comparison of microbial profiles generated by IR and various QIIME 2 single- and multi-region pipelines. ( A ) Alpha diversity indices (Observed, Shannon, and inverse-Simpson) showing richness and evenness across the different pipelines, with statistical significance determined by pairwise Wilcoxon tests. ( B ) Bray-Curtis PCoA plot depicting the clustering of samples based on pipeline-derived microbial profiles. ( C ) Pearson correlation coefficients comparing IR vs QIIME 2 pipelines across samples. ( D ) Statistical significance (−log10 adjusted P values) of Pearson correlations, highlighting concordance differences between pipelines. ( C and D ) Paired samples are connected by gray lines.

Article Snippet: For this study, we generated independent 16S rRNA amplicon libraries from: (i) mock samples using the ZymoBIOMICS Microbial Community DNA Standard (Zymo Research); (ii) stool samples from a clinical study as validation.

Techniques: Amplification, Comparison, Generated, Derivative Assay

Journal: Microbiology Spectrum

Article Title: QIIME2 enhances multi-amplicon sequencing data analysis: a standardized and validated open-source pipeline for comprehensive 16S rRNA gene profiling

doi: 10.1128/spectrum.01673-25

Figure Lengend Snippet: Influence of caregiver microbiota on pediatric patient gut microbiome composition. ( A ) PCoA based on UniFrac distances, illustrating the gut microbial community structure of pediatric patients (triangles) and their caregivers (circles). Each child-caregiver pair is connected by a gray line. Pediatric samples were designated as high‐ (violet) or low‐similarity (green) depending on whether their correlation with the caregiver was significant ( P value ≤ 0.05) and their Euclidean distance in the PCoA space fell below 0.255. ( B ) Bubble plot showing the correlation strength (Pearson’s correlation) between child and caregiver microbiomes for each family. Bubble size indicates the –log( P value), reflecting statistical significance, while bubble color corresponds to whether each family falls into the high‐ (violet) or low‐similarity (green) group. ( C ) Box plots depicting two alpha diversity metrics (Faith’s phylogenetic diversity and observed features, i.e., ASV richness) for pediatric samples in the high‐ and low‐similarity groups. ( D ) Differential abundance analysis using DESeq2, comparing high‐ (violet) versus low‐similarity (green) groups. Significantly differentially abundant ASVs (|Log2FoldChange| > 1, FDR < 0.05) are listed by taxonomic affiliation alongside the number of ASVs per taxon. Blue‐highlighted species names indicate >99% sequence identity to entries in the NCBI 16S rRNA database, confirming species‐level identification. In the bubble plot, circle size represents –log10(FDR), and color indicates the similarity group in which the ASV is enriched.

Article Snippet: For this study, we generated independent 16S rRNA amplicon libraries from: (i) mock samples using the ZymoBIOMICS Microbial Community DNA Standard (Zymo Research); (ii) stool samples from a clinical study as validation.

Techniques: Sequencing