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Page 12 of 15 Longhi et al. Microbiome Res Rep 2024;3:4 https://dx.doi.org/10.20517/mrr.2023.02

in the 2% wt/vol treated saponin sample with a relative abundance of 8% and decreased to 5.2% due to 0.05%
wt/vol saponin treatment while an abundance of 2.4% was observed in the control. Moreover, several Gram-
negative taxa, such as other members of the Prevotella and Veillonella genera, as well as unknown species of
the Lancefieldella and Schalia genera, decreased with the increasing amount of saponin, while the Gram-
positive Actinomyces and Streptococcus spp. increased in relative abundance, reinforcing the previous
findings regarding the depletion protocol’s impact on the taxonomic profile [Supplementary Table 5 and
Supplementary Figure 3].

These results confirmed that saponin treatment has an impact on the Gram-positive/Gram-negative ratio.
Gram-negative percentages decreased with the increasing concentration of saponin applied, thus inducing a
concomitant increase in the relative abundance of Gram-positive bacteria in the overall taxonomic profile.
Indeed, Gram-negatives ranged from an average of 42.3% in the 0.0125% wt/vol saponin-treated samples to
an average of 35% and 24.3% in the 0.5% and 2% wt/vol saponin treated samples, respectively, compared to
the starting 82.3% in the untreated saliva sample [Supplementary Table 6 and Figure 2B].

Remarkably, despite the fact that even low saponin amounts are able to modulate the retrieved bacterial
DNA in terms of Gram-negative taxonomic representation, saponin could be efficiently employed for the
depletion of human DNA when targeting the detection of microbial populations dominated by Gram-
positive bacteria or when targeting specific microbial taxa such as pathogens in clinical contexts. Thus, data
retrieved in this study indicate the need for preliminary tests on the biological matrices under investigation
to assess the feasibility and effects of the saponin protocol use. In this context, in the case of studies
employing long-read sequencing, a further novel approach that can be employed to overcome contaminants
derived from high amounts of host DNA, as an alternative to saponin, could be represented by the
SQK-RPB004 kit combined with the SQK-LSK109 kit proposed by Oxford Nanopore Technologies. This
consists of the specific enrichment or depletion of target DNA directly during sequencing by simply
[36]
providing reference fasta sequence . Nevertheless, it should be considered that this approach may
considerably impact the amount of sequencing data retrieved.

In conclusion, in the framework of this study, we investigated the performances of the saponin-based
eukaryotic DNA depletion approach on different biological matrices and the impact on the correlated
microbial taxonomic profiles retrieved after DNA sequencing and data analysis.

Overall, this depletion method successfully reduced the amount of human DNA but drastically changed the
detected microbial composition harbored by the specimens analyzed, inducing a drastic reduction of Gram-
negative bacteria DNA.

However, although the saponin-based approach is one of the most used methods, other protocols pursue
the same aim [18,19,37] . In these cases, it will also be necessary to carry out a very careful evaluation of protocol
limitations to avoid alteration of the retrieved microbial DNA profile. Our findings highlight a serious issue
that might affect the reliability of the microbial profiles and the biological meaning of the associated
metagenomic studies that use these specific DNA extraction protocols involving the use of saponin.

Based on the results obtained in our study, we highlighted that the use of saponin should be avoided if
specific evaluations of the microbial/host DNA ratio and complexity of the targeted microbial population
are not known. For this reason, we suggest that for each study intending to apply saponin treatment, it is
necessary to introduce detailed preliminary tests on the biological matrices investigated to assess the
feasibility and efficacy of the saponin-based host DNA depletion approach.