Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
October 15th, 2019
Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.
Profiling of microbiome is the first step toward defining the role of microbiome in health and disease.Here, we are describing a simple procedure for isolating high-quality bacterial DNA from fecal sample preparing library, performing 16SR RNA and a meta genomic sequencing and microbiome data analysis.This protocol will help researchers new to microbiome field, as well as researchers working in microbiome field in establishing the microbiome pipeline in their lab.This protocol can be extended for profiling of microbiome from other bio specimens such as nasal, oral, or skin samples of animal and human subjects.Bead picking is the most important steps as all downstream steps are depend on high quality DNA.Proper sealing of preserve plate is important as incomplete sealing might lead to the oppressing of amplified product resulting in low quality sequencing data.To begin, sterilize the divider boxes with 70%ethanol.Place the mice in sterilized divider boxes and allow them to defecate normally for up to one hour.Use sterile forceps to collect the fecal pellets in an empty pre-labeled 1.5 milliliter micro centrifuge tube.Enclose the tube securely.Sterilize the forceps with 70%ethanol, followed by a germicidal disposable wipe, after collecting fecal from each mouse.Weigh 200 milligrams of fecal pellets into a 1.5 milliliter micro centrifuge tube, with 0.1 milliliter glass beads, and place the bead tube into a bead beating homogenizer and homogenize the samples for 45 seconds at room temperature, at a speed of 4.5 meters per second.Extract DNA as per instructions of the kit manufacturer and elute DNA in a 1.5 milliliter micro centrifuge tube.After isolating DNA, quantify the isolated DNA by loading 1 microliter of the DNA on a fluorometer.Setup 16S ribosomal RNA gene amplification PCR1 in a 96-Well PCR plate, using a 25 microliter reaction volume.Using a multi-channel pipette, add 40 nano grams of DNA, 13.5 micro liters of 2X high-fidelity polymerase enzyme mix, containing buffer, plus DNTPs in forward and reverse primer.Seal the PCR plate properly from the top to the bottom in along all four edges.And centrifuge at 1000 times G at 20
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Overview
This article presents a detailed methodology for microbiome profiling through 16S rRNA metagenomic sequencing. Aimed at both novice and experienced researchers, the protocol offers insights into achieving accurate bacterial profiling using accessible tools.
Key Study Components
Research Area
- Microbiome analysis
- Metagenomic sequencing
- Bacterial profiling
Background
- The microbiome's role in health and disease
- Importance of high-quality DNA isolation
- Applications in various biological specimens
Methods Used
- 16S rRNA sequencing protocol
- Fecal sample preparation and DNA extraction
- Use of freely available bioinformatics tools for analysis
Main Results
- Successful isolation of high-quality bacterial DNA
- Robust library preparation for sequencing
- Detailed guidance for researchers to establish microbiome pipelines
Conclusions
- The study offers a comprehensive protocol for microbiome profiling.
- It enhances understanding of microbiome methodologies relevant to health research.
Frequently Asked Questions
Executive Industry Relevance
Microbiome profiling is a foundational step in target validation for inflammatory and metabolic diseases, enabling hypothesis generation about microbial contributions to pathogenesis. This protocol supports preclinical model development by providing high-resolution taxonomic data from complex biospecimens, facilitating mechanistic de-risking of microbiome-targeted interventions. The standardized workflow enhances translational continuity from discovery to lead identification in microbiome-modulating therapeutic programs.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by linking specific bacterial taxa to disease phenotypes in preclinical models.
- Operational Value: Provides reproducible microbial community profiling to support target prioritization and pathway clarification efforts.
Screening & Assay Development
- Scientific Value: Generates quantitative, high-resolution taxonomic outputs suitable for assay standardization and biomarker discovery initiatives.
- Operational Value: Establishes a scalable, reusable pipeline for screening microbiome alterations across diverse sample types and treatment conditions.
Translational & Preclinical Research
- Scientific Value: Supports disease-relevant system characterization by enabling longitudinal tracking of microbiome shifts in response to therapeutic candidates.
- Operational Value: Facilitates cross-functional collaboration through standardized data formats and analysis tools like DADA2 and Phyloseq.
Pipeline & Workflow Integration
The method integrates into early discovery workflows by delivering actionable microbiome data that informs target selection and mechanistic studies prior to lead optimization.
- Discovery Biology: Supports hypothesis testing and pathway clarification through precise taxonomic assignment at genus and species levels.
- Screening: Delivers assay-ready, reproducible microbial profiles that enable reliable comparison of experimental conditions.
- Analytics: Provides quantitative sequence variants and diversity metrics that support statistical comparison and biomarker correlation.
- Translational Research: Connects discovery findings to preclinical validation by enabling consistent microbiome monitoring across study phases.
- Enterprise Reuse: Establishes a standardized, cross-departmental capability for microbiome analysis applicable to multiple therapeutic areas.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity in host-microbe interactions.
- Operational Value: Ensures reproducibility and scalability through standardized two-step PCR and bioinformatics analysis.
- Strategic Value: Improves go/no-go decisions by enabling early detection of microbiome-mediated confounding factors in disease models.
- Portfolio Impact: Supports risk-adjusted advancement by providing data-driven insights into microbiome-targeted mechanism validity.
Implementation Considerations
- Requires expertise in molecular biology and bioinformatics for PCR setup and DADA2-based analysis.
- Depends on access to next-generation sequencing infrastructure and computational resources for metagenomic processing.
- Necessitates cross-team standardization of sample handling, DNA extraction, and sequencing library preparation.
- Involves adaptation considerations when extending the protocol to non-fecal biospecimens such as oral or skin samples.
- Includes practical limitations related to PCR bias and sequencing depth affecting low-abundance taxon detection.
Biopharma FAQs
Chapters in this video
0:04
Title
1:25
Extraction of DNA from Fecal Sample
2:29
16S rRNA Gene Amplification (PCR1) and DNA Library Construction (PCR2)
3:57
Clean-up of Indexed PCR (PCR2) and Quantification
6:03
Sequence Analysis
7:49
Results: Quality Profile and Statistical Analysis
9:29
Conclusion
