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Page 2 of 19 Wang et al. Microbiome Res Rep. 2025;4:23 https://dx.doi.org/10.20517/mrr.2024.94

total DNA quantification. We observed that mothers exhibited higher total bacterial loads than infants by
approximately half a log, while the abundance of Bifidobacterium was comparable in both groups. The spike-in
method revealed significant differences in taxonomic composition, highlighting the impact of absolute
quantification on microbiome analysis outcomes. Importantly, the method did not alter alpha diversity measures
but slightly affected beta diversity analysis, reflecting more precise inter-group differences.

Conclusion: Marine-sourced bacterial DNA spike-in offers a reliable, scalable, and accurate approach for absolute
microbiome quantification. This method enhances microbiome analysis by addressing biases inherent in relative
abundance measures, providing a deeper understanding of microbial dynamics in mother-infant gut microbiomes.

Keywords: Spike-in, absolute quantification, marine-sourced bacteria

INTRODUCTION

Relative abundance measurements rely on normalizing sequencing data to account for variations in
[1]
sequencing depth, sample composition, and other technical factors . However, normalization methods may
introduce biases and inaccuracies, particularly when dealing with complex microbial communities or
[2,3]
samples with low biomass . When examining relative abundance data, an increase in the abundance of
one taxon results in a corresponding decrease in the abundance of other taxa. Consequently, assessing the
relative abundance of a taxon depends on the abundance of all other taxa, potentially leading to elevated
false-positive rates in differential taxon analyses and negative correlation biases in correlation-based
[4,5]
analyses .
Absolute Quantification methods offer a more accurate approach by directly measuring the absolute
abundance of microbial taxa within a sample. Techniques such as flow cytometry , qPCR , machine-
[7-9]
[6]
learning , and total DNA [11,12] quantification have been utilized for absolute quantification but may face
[10]
limitations, including issues with specificity, sensitivity, and scalability. For instance, flow cytometry-based
techniques necessitate the sample’s dissociation into individual bacterial cells, often involving intricate
sample preparation . In addition, bacterial suspensions must be diluted to optimal concentrations
[13]
5
[14]
(typically 10 -10 cells/mL) to avoid coincidence artifacts and ensure accurate event detection . Achieving
7
this range can be technically challenging in low-biomass or small-volume samples, such as infant feces,
potentially compromising measurement accuracy. Total DNA-based approaches are confounded by the
presence of host DNA, particularly in low-biomass samples such as infant feces. qPCR provides taxonomic
specificity but is subject to primer-dependent amplification bias, which may disproportionately affect the
quantification of dominant taxa such as Bifidobacterium in infant gut samples . These limitations are
[15]
especially consequential in mother-infant microbiome studies, where sample volumes are typically small,
host DNA contamination is common, and microbial composition differs markedly from adults.

The spike-in method has also been employed in microbial absolute quantification, mainly in two forms:
either by directly introducing exogenous cells into fecal samples [16,17] or by adding synthetic DNA to the
sample DNA [18,19] . Corresponding reagent kits have already been developed and are commercially
available [19,20] . However, to date, no published studies have explored the use of marine bacterial DNA as a
spike-in for absolute quantification in sample DNA.

To address these challenges, reliable methods are needed that offer substantial reproducibility and
accurately provide the overall absolute abundance across both high- and low-density samples. Additionally,
the method should be easily applicable to high throughput workflows and be cost-effective for large-scale
studies.