Biological Data

Revisiting the Sterile Tissue Paradigm: Systematic Re-analysis of Microbial Signals in Germ-Free Mouse Tissues

Document Type : Original Article

Author

Biology Department, Faculty of Sciences, Malayer University First of Arak Road

Abstract

The paradigm of sterile internal tissues is challenged by high-sensitivity sequencing, creating an analytical dilemma for low-biomass samples. Distinguishing genuine endogenous signals from pervasive contamination requires stringent computational frameworks. Here, we developed and applied a systematic bioinformatic pipeline to re-analyze public 16S rRNA gene sequencing data from germ-free and conventional mouse tissues. Our approach integrated rigorous contamination control, diversity analysis, and differential abundance testing. We found no significant difference in microbial community structure (PERMANOVA: R²=0.47, p=0.15) or alpha diversity (Shannon Index, p=0.63) between germ-free and conventional groups, failing to support the hypothesis of a structured endogenous microbiome in germ-free tissues. While we observed tissue-correlated profiles, these patterns could not be definitively attributed to biological colonization versus technical artifacts. The most parsimonious interpretation is that the observed signals are consistent with contamination. This study provides a replicable analytical framework and underscores the critical importance of robust bioinformatic decontamination for validating claims of microbiomes in low-biomass environments.

Keywords

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Biological Data
Volume 1, Issue 1 - Serial Number 1
June 2026
Pages 86-103
  • Receive Date: 22 April 2026
  • Revise Date: 22 May 2026
  • Accept Date: 11 June 2026
  • First Publish Date: 13 June 2026