Page 131 - Read Online

Page 16 of 19 Wang et al. Microbiome Res Rep. 2025;4:23 https://dx.doi.org/10.20517/mrr.2024.94

while reducing distortion from low-abundance taxa.

However, the use of marine-derived spike-in strains presents a limitation in broader microbiome research.
While these strains are ideal for human gut studies due to their absence in mammalian microbiomes, they
may be less suitable for environmental or indoor microbiomes, where trace marine DNA is more likely to
be present. In such contexts, the spike-in signal may be confounded by endogenous microbial DNA,
reducing quantification accuracy. Alternative strategies, such as qPCR, have been more commonly adopted
for absolute quantification in these settings [58,59] . Future adaptations of this method may include selecting
habitat-specific spike-ins to improve applicability across different ecosystems.

Our data suggest that the spike-in method offers a strong balance between practicality and accuracy,
particularly in 16S rRNA-based studies involving low-biomass or compositionally variable samples. While
not a universal standard, it is especially well suited for microbiome comparisons in mother-infant cohorts,
Authors’ contributions
intervention studies, and archived samples with limited input. In contrast, for environmental microbiomes,
adapting spike-in strains may be necessary. Overall, the method provides a scalable and accessible approach
when total microbial load is a key variable of interest.

In conclusion, by spiking exogenous bacterial DNA from Pseudoalteromonas sp. APC 3896 and Planococcus
sp. APC 3900 into extracted fecal DNA, we demonstrated that DNA spike-in can reliably normalize
microbial load differences and enable accurate comparisons of community profiles. Even under fluctuating
microbial biomass, this approach effectively stabilized quantification across samples. The application of
spike-in-based absolute quantification revealed notable shifts in the rankings of predominant genera,
underscoring its importance in uncovering biologically meaningful variation otherwise masked in relative
abundance analyses.

This method also captured clearer distinctions in β-diversity between mother and infant microbiomes,
supporting its utility for detecting biologically relevant community differences. Overall, the spike-in strategy
offers a practical, scalable, and reproducible alternative to traditional relative abundance measures -
improving accuracy in quantifying microbial dynamics and advancing our understanding of microbiome
ecology in health and disease.

DECLARATIONS
Acknowledgments
The authors would like to thank the MIMIC study participants for sample donations.

Conducting laboratory experiments: Wang S, Uniacke-Lowe S, Kamilari E, Kozak IM
Performing bioinformatic and statistical analysis: Wang S, Patangia D
Figure generation: Wang S
Writing Manuscript: Wang S, Yang B, Dempsey EM, Stanton C, Ross RP
Participant recruitment and sample collection: Healy D, Dempsey EM
Study conception: Yang B, Dempsey EM, Stanton C, Ross RP
Study design: Yang B, Dempsey EM, Stanton C, Ross RP
Funding acquisition: Yang B, Dempsey EM, Stanton C, Ross RP
Interpretation of the results: Yang B, Dempsey EM, Stanton C, Ross RP
All authors read and approved of the final manuscript.