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Updated: Jun 24, 2026
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Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
High-throughput quantitative analysis of the human intestinal microbiota with a phylogenetic microarray
Oleg Paliy1, Harshavardhan Kenche, Frank Abernathy
1Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435, USA. oleg.paliy@wright.edu
Applied and Environmental Microbiology
|April 14, 2009
Summary
Researchers developed a custom microarray to identify hundreds of intestinal bacteria species. This tool revealed differences in gut microbiota composition between healthy children and adults, with adults showing more Clostridia and fewer Bacteroidetes.
Area of Science:
- Microbiology
- Genomics
- Bioinformatics
Background:
- The gut microbiota plays crucial roles in human health and disease pathogenesis.
- Accurate identification of intestinal bacterial species is essential for understanding these roles.
- Existing methods may lack the capacity to comprehensively profile the diverse gut microbial community.
Purpose of the Study:
- To develop a custom microarray for identifying hundreds of intestinal bacterial species.
- To create a high-throughput tool for analyzing gut microbiota composition.
- To investigate potential differences in gut microbiota between children and adults.
Main Methods:
- Compiled a dataset of bacterial 16S rRNA gene sequences from the Entrez nucleotide database.
- Clustered sequences into phylospecies and selected representative sequences.
- Designed a microbiota microarray on the Affymetrix GeneChip platform with probes for 775 bacterial phylospecies.
- Validated the array's accuracy and sensitivity using known bacterial species and DNA amounts.
Main Results:
- The custom microarray successfully identified genomic DNA from 15 bacterial species in validation experiments.
- The array demonstrated a detection sensitivity of at least 1 pg of genomic DNA.
- Analysis of fecal samples revealed 227-232 species in children and 191-208 species in adults.
- The majority of identified species belonged to Clostridia and Bacteroidetes classes.
- Putative differences were observed in the gut microbiota of healthy children versus adults, with adults having more Clostridia and less Bacteroidetes and Proteobacteria.
Conclusions:
- The developed microbiota microarray is a sensitive and accurate tool for identifying a large number of intestinal bacterial species.
- The study identified distinct gut microbiota profiles in healthy children and adults.
- These findings highlight the utility of custom microarrays for gut microbiome research and potential disease association studies.
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