Related Experiment Video
Updated: Aug 5, 2026
Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging
Published on: July 9, 2021
Optimized protocol for profiling mucosa-associated microbiota from formalin-fixed paraffin-embedded gut tissues from
Noora Al-Ali1, Haifa Al-Awadhi2, Maya Hassane1
1Department of Medical Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.
Background:
Formalin-fixed, paraffin-embedded (FFPE) tissues are yet underutilized resources for microbiome studies. Data on the mucosa-associated microbiota (MAM) of patients with Crohn's disease (CD) are scarce, due to several methodological limitations. In this study, we aimed to develop and validate a new optimized amplicon-based workflow to profile MAM from FFPE gut biopsies of pediatric CD patients.
Methods:
We examined 68 FFPE samples, including 34 biopsies from treatment-naïve patients with CD and 34 from healthy controls (HC). V3-V7 regions of the 16S rRNA gene were amplified and sequenced on the Oxford Nanopore platform. Two protocols were tested: Protocol 1 (P1), consisting of a single PCR amplification and purification step, and Protocol 2 (P2), including two sequential PCR amplifications with purification after each round. The second amplification and purification steps were introduced to increase sequencing yield and improve microbiota detection.
Results:
P2 consistently outperformed P1, yielding significantly higher DNA concentration and purity, reducing human DNA contamination and sustaining pore performance. P2 also generated more microbial reads and recovered a richer, more taxonomically diverse community, including increased detection of species with low abundance. More taxa were enriched in P2 across all levels, enhancing species-level resolution. P2 enabled comprehensive detection of pathogenic genera, such as Escherichia, Mycobacterium, and Klebsiella, which were significantly enriched in the P2 samples compared to the P1 samples. Alpha diversity analysis showed increased richness and reduced evenness in P2 compared to P1, with a significant difference in beta diversity, while maintaining community structure in both CD and HC.
Conclusions:
The optimized workflow with a two-step strategy improved sequencing performance and enhanced microbiota detection in FFPE tissues. This approach enabled successful profiling of MAM, providing a novel method for retrospective characterization of the microbiome from archival tissues and providing a scalable platform for clinical biomarker discovery.
Insights
A new two-step PCR method enhances microbiome analysis in FFPE tissues, improving detection of mucosa-associated microbiota in pediatric Crohn's disease patients.
Area of Science:
- Microbiome research
- Molecular biology
- Genomics
Background:
- Formalin-fixed, paraffin-embedded (FFPE) tissues are valuable but underutilized for microbiome studies.
- Data on mucosa-associated microbiota (MAM) in Crohn's disease (CD) are limited due to methodological challenges.
Purpose of the Study:
- To develop and validate an optimized amplicon-based workflow for profiling MAM from pediatric CD patient FFPE gut biopsies.
- To improve the detection and characterization of the gut microbiome in archival tissue samples.
Main Methods:
- Compared two protocols (single vs. two-step PCR) for 16S rRNA gene amplification and sequencing on the Oxford Nanopore platform.
- Analyzed 68 FFPE gut biopsy samples from pediatric CD patients and healthy controls.
- Optimized protocol involved two sequential PCR amplifications with purification steps to enhance yield and purity.
Main Results:
- The two-step protocol (P2) significantly outperformed the single-step protocol (P1).
- P2 yielded higher DNA concentration, reduced human DNA contamination, and improved pore performance.
- P2 detected a richer, more diverse microbial community, including low-abundance species and pathogenic genera like *Escherichia* and *Klebsiella*.
Conclusions:
- The optimized two-step workflow enhances sequencing performance and microbiota detection in FFPE tissues.
- This method enables retrospective microbiome characterization from archival samples.
- The approach offers a scalable platform for clinical biomarker discovery in diseases like CD.
