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Updated: Sep 29, 2025
Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Impact of Early Feeding: Metagenomics Analysis of the Infant Gut Microbiome
Matthew D Di Guglielmo1, Karl R Franke2, Alan Robbins2
1Division of General Academic Pediatrics, Department of Pediatrics, Nemours Children's Health, Wilmington, DE, United States and Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, United States.
Background:
Different feeding regimens in infancy alter the gastrointestinal (gut) microbial environment. The fecal microbiota in turn influences gastrointestinal homeostasis including metabolism, immune function, and extra-/intra-intestinal signaling. Advances in next generation sequencing (NGS) have enhanced our ability to study the gut microbiome of breast-fed (BF) and formula-fed (FF) infants with a data-driven hypothesis approach.
Methods:
Next generation sequencing libraries were constructed from fecal samples of BF (n=24) and FF (n=10) infants and sequenced on an Illumina HiSeq 2500. Taxonomic classification of the NGS data was performed using the Sunbeam/Kraken pipeline and a functional analysis at the gene level was performed using publicly available algorithms, including BLAST, and custom scripts. Differentially represented genera, genes, and NCBI Clusters of Orthologous Genes (COG) were determined between cohorts using count data and R (statistical packages edgeR and DESeq2).
Results:
Thirty-nine genera were found to be differentially represented between the BF and FF cohorts (FDR ≤ 0.01) including Parabacteroides, Enterococcus, Haemophilus, Gardnerella, and Staphylococcus. A Welch t-test of the Shannon diversity index for BF and FF samples approached significance (p=0.061). Bray-Curtis and Jaccard distance analyses demonstrated clustering and overlap in each analysis. Sixty COGs were significantly overrepresented and those most significantly represented in BF vs. FF samples showed dichotomy of categories representing gene functions. Over 1,700 genes were found to be differentially represented (abundance) between the BF and FF cohorts.
Conclusions:
Fecal samples analyzed from BF and FF infants demonstrated differences in microbiota genera. The BF cohort includes greater presence of beneficial genus Bifidobacterium. Several genes were identified as present at different abundances between cohorts indicating differences in functional pathways such as cellular defense mechanisms and carbohydrate metabolism influenced by feeding. Confirmation of gene level NGS data via PCR and electrophoresis analysis revealed distinct differences in gene abundances associated with important biologic pathways.
