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Updated: Sep 13, 2025
11:22
Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
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QIIME2 enhances multi-amplicon sequencing data analysis: a standardized and validated open-source pipeline for
Armando G Licata1, Marica Zoppi1, Chiara Dossena1
1Integrated Biology of Rare Tumors, Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
Microbiology Spectrum
|July 25, 2025
Summary
This study developed a validated, open-source QIIME2 and R pipeline for multi-amplicon 16S rRNA gene sequencing. The pipeline offers standardized and reproducible microbial profiling, crucial for clinical and research applications.
Area of Science:
- Microbiology and Bioinformatics
- Genomic Sequencing and Analysis
Background:
- Multi-amplicon 16S rRNA gene sequencing provides comprehensive microbial diversity insights but faces challenges due to limited documentation and validation of open-source pipelines.
- Lack of standardization in bioinformatics workflows hinders reproducibility in clinical and experimental microbiome research.
Purpose of the Study:
- To develop and benchmark a standardized, open-source QIIME2 and R pipeline for 16S rRNA gene profiling using semiconductor sequencing.
- To compare the open-source pipeline against a commercial, closed-source software for accuracy and resolution.
- To validate the pipeline's effectiveness in a pediatric cancer cohort, investigating parental influence on child microbiomes.
Main Methods:
- Development of a QIIME2 and R-based bioinformatics pipeline for multi-amplicon 16S rRNA gene sequencing.
- Benchmarking using 5 mock microbial communities and 12 child-caregiver fecal sample pairs.
- Comparison of multi-region (V2-9) sequencing approach with single-region analyses and commercial software.
Main Results:
- The multi-region open-source pipeline produced microbial profiles nearly identical to proprietary software outputs, with enhanced sequencing depth and taxonomic resolution.
- Both the open-source and commercial approaches showed similar microbial richness, accurate mock community reconstruction, and high reproducibility (R = 0.99, P < 0.0001).
- Application to pediatric samples identified distinct microbial variants, with closer resemblance between children and caregivers, suggesting parental influence.
Conclusions:
- A validated, open-source QIIME2 and R pipeline for multi-amplicon 16S rRNA sequencing has been established, offering a standardized and reproducible framework.
- The pipeline effectively leverages multi-amplicon data for detailed microbial variant analysis, surpassing traditional genus-level limitations.
- Adoption of open-source solutions is essential for ensuring scientific reproducibility and adaptability in microbiome research.
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