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Page 6 of 19 Wang et al. Microbiome Res Rep. 2025;4:23 https://dx.doi.org/10.20517/mrr.2024.94

Figure 1. Overview of the proposed DNA spike-in procedure, compared with other quantification methods.

maternal and infant fecal samples. Accurate documentation of the mother and infant sample weights and
elution buffer volumes during DNA extraction is crucial [Supplementary Table 1]. Following DNA
extraction, a specified quantity of spike-in DNA was added to each sample's DNA, after which library
preparation was undertaken. Given the predetermined number of rRNA copies added, this served as a back-
normalization factor for accurately determining the absolute abundance of all other organisms present
within the sample.

The spike-in bacteria correlate well with microbial loads
To validate the accuracy and reliability of the DNA spike-in method for estimating microbial loads, we
examined the correlation between the spike-in bacterial read counts and the total microbial loads across
different samples. Figure 2 illustrates the relationship between the read counts of the spike-in bacteria
Pseudoalteromonas and Planococcus and the total copy number obtained from the spike-in as calculated
from the weight of DNA added from the sample. The analysis shows an inverse correlation between the
read count assigned to a spike-in operational taxonomic unit (OTU) and the total copy number from the
spike-in method. For Planococcus, the correlation coefficient was r = -0.83, indicating a strong negative
correlation (P = 0.0008174). Similarly, for Pseudoalteromonas, the correlation coefficient was found to be r =