Page 124 - Read Online

Wang et al. Microbiome Res Rep. 2025;4:23 https://dx.doi.org/10.20517/mrr.2024.94 Page 9 of 19

To validate the reliability of the spike-in approach, genus-specific analysis using Bifidobacterium-specific
primers in qPCR was performed. The results revealed a similarly strong correlation (r = 0.96, P < 0.0001)
between Bifidobacterium copy numbers obtained via qPCR and spike-in quantification [Figure 3C]. Both
methods showed that infants tended to have a higher abundance of Bifidobacterium than mothers, though
this difference was not statistically significant [Figure 3C and D].

To compare bacterial loads in infant and mother fecal samples, we quantified the total DNA content. A
strong positive correlation was observed between total DNA content and the spike-in method (r = 0.94, P <
0.001) [Figure 3E], demonstrating the reliability of the spike-in approach for estimating microbial loads.
Consistent with findings from both the spike-in and qPCR methods, the total DNA content was
significantly higher in samples from mother than infants (P = 0.002) [Figure 3F].

Comparing relative and absolute abundance via spike-in

To evaluate the impact of spike-in on microbiome analysis outcomes, we examined its effect on phylum and
genus abundance profiles in the mother-infant pairs. In the top four phyla, significant differences were
observed in the microbial profiles of infants and mothers [Figure 4A and B]. Pseudomonadota showed a
significantly higher abundance in infants compared to mothers (relative: P = 0.0022; absolute: P = 0.0087),
while Bacillota was more abundant in mothers in both analyses (relative: P = 0.0087; absolute: P = 0.0022).
Actinomycetota and Bacteroidota did not display significant differences in either relative or absolute
abundance, though Bacteroidota showed a trend toward higher abundance in infants in the absolute
analysis (P = 0.0637).

To better compare the impact of the spike-in method on gut microbiota analysis, we selected the top 10
genera based on relative abundance and the top 10 genera obtained using the spike-in method. After using
the absolute quantitative analysis with spike-in, the rankings of several genera within the top 10 in relative
abundance changed significantly [Figure 4C and D]. For example, the rankings of the genera
Bifidobacterium, Escherichia-Shigella, and Bacteroides shifted notably. Additionally, some genera, such as
Veillonella, Streptococcus, and Enterobacter, that were within the top 10 in relative quantification are no
longer in the top 10 in absolute quantification. Conversely, some genera, such as Agathobacter,
Coprococcus, Subdoligranulum, and Anaerostipes, have entered the top 10 in absolute quantification.

Additionally, as observed in Figure 4D, the absolute quantification analysis revealed significant differences
in microbial loads between mothers and infants. This disparity affected the top 10 genera in the infant
group, which had lower microbial loads. To address this variation, we separately calculated the top 10
genera for the infant and mother groups. Upon independent calculation, we found that the top 10 rankings
for the mother group remained largely unchanged, with a few exceptions [Figure 4E]. Bifidobacterium
dropped from the second to the ninth position in abundance, and Escherichia-Shigella was no longer within
the top 10. Notably, these two genera were the first and second most abundant genera in the infant group.

Interestingly, when performing absolute quantification to independently compute the top 10 genera within
the infant group, the results show significant similarity to those obtained through relative quantification.
Veillonella, Erysipelatoclostridium, Streptococcus, and Enterobacter have reappeared among the top 10
genera. Moreover, in absolute quantification analysis, the independent calculation of the top 10 genera
within the mother group closely resembles the overall top 10 genera calculated across all samples. Key
differences in genus rankings between relative and absolute quantification, including group-specific shifts in
infants and mothers, are summarized in Supplementary Table 2, which facilitates clearer comparison across
quantification strategies.