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Longhi et al. Microbiome Res Rep 2024;3:4 https://dx.doi.org/10.20517/mrr.2023.02 Page 7 of 15

[20]
eukaryotic DNA removal . Subsequently, the DNA extracts were treated with DNase to cleave the
eukaryotic content released before proceeding with standard microbial DNA extraction. For the bacterial
DNA extraction from different biological matrices, distinct commercially available kits were used, as
described in the Experimental Procedures section. The amount of bacterial and eukaryotic DNA in the
nucleic acid extracts was evaluated by qPCR using specific primers to discriminate bacterial 16S rRNA and
β-globulin genes.

Data collected revealed that saponin treatment induced a depletion of eukaryotic DNA in all the samples
analyzed [Figure 1C]. Notably, after the saponin treatment, host DNA content decreased by 17.5% in
vaginal, 11.7% in oral, and 22.6% in nasopharyngeal swab samples with respect to the same untreated
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samples [Figure 1C]. In contrast, bacterial content seemed to slightly increase from 10 to 10 gene copy
number/mL in the nasopharyngeal swab as well as in the oral and the nasopharyngeal samples after
saponin-based depletion, suggesting that lower host’s DNA abundance may support the recovery of a higher
amount of bacterial DNA in some specific biological matrices in contrast to what observed for saliva
[Figure 1D]. However, confirming the previous findings, the saponin depletion protocol seemed to impact
also on bacterial cells by promoting their lysis in biological samples such as vaginal swabs. In this case,
probably due to the large amount of bacterial DNA, bacterial content decreased from 10 to 10 gene copy
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number/mL in the saponin-treated vaginal swab [Figure 1D].
Remarkably, the results confirmed what was observed for the saliva samples, i.e., saponin targets mainly
human DNA, showing great performances in removing eukaryotic DNA from the biological samples while
sometimes impacting the bacterial DNA content. The observed differences in bacterial population reduction
can be considered biological matrix dependent. Thus, we suggest preliminary testing of each biological
matrix included in a study to identify the best depletion approach in terms of saponin concentration
according to the study purpose.

Validation of the saponin DNA extraction protocol by shallow shotgun metagenomics of a broader
range of biological matrices
The shotgun metagenomics approach allows to randomly sequence all the DNA fragments obtained from
DNA extraction protocols, thus allowing the quantification of the prokaryotic/eukaryotic DNA ratio
through specific bioinformatics applications . To validate the outcome of our qPCR analyses, we
[29]
performed shallow shotgun metagenomic sequencing , a specific low-depth shotgun metagenomics
[30]
[29]
technique that provides a reliable bacterial profile at the species level . DNA samples from different
biological matrices rich in eukaryotic DNA were submitted to DNA sequencing after being processed with
the extraction protocol involving 2.5% wt/vol saponin and DNase, followed by bead-beating and kit
extraction steps. To provide a more comprehensive overview of the human-associated microbiota, we
included samples rich in eukaryotic DNA, such as a vaginal swab, saliva, skin, sputum, and nasopharyngeal
swab samples [Table 1]. Our analysis also included a molecular biology-grade water sample undergoing the
same depletion protocol. Notably, it did not provide enough sequencing data to be analyzed as it contained
no eukaryotic or bacterial DNA contaminations.

Sequencing output resulted in an average of circa 144,585 pair-end reads per sample [Table 1], thus allowing
an accurate assessment of the microbial profile associated with each specimen . Each biological matrix
[30]
treated according to the depletion approach was compared to its untreated counterpart to investigate the
impact of the saponin method on the bacterial profile of each sample [Table 1].