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Page 2 of 15 Longhi et al. Microbiome Res Rep 2024;3:4 https://dx.doi.org/10.20517/mrr.2023.02

Methods: Here, we have investigated a saponin-based DNA isolation protocol commonly applied to different
biological matrices to deplete the released host DNA.

Results: The bacterial DNA obtained was used to assess the relative abundance of bacterial and human DNA,
revealing that the inclusion of 2.5% wt/vol saponin allowed the depletion of most of the host’s DNA in favor of
bacterial DNA enrichment. However, shotgun metagenomic sequencing showed inaccurate microbial profiles of
the DNA samples, highlighting an erroneous increase in Gram-positive DNA. Even the application of 0.0125%
wt/vol saponin altered the bacterial profile by depleting Gram-negative bacteria, resulting in an overall increase of
Gram-positive bacterial DNA.

Conclusion: The application of the saponin-based protocol drastically changes the detection of the microbial
composition of human-related biological specimens. In this context, we revealed that saponin targets not only host
cells but also specific bacterial cells, thus inducing a drastic reduction in the profiling of Gram-negative bacterial
DNA.
Keywords: Host DNA depletion, saponin, bacterial DNA enrichment, microbiome profiling

INTRODUCTION
Microbiome research, especially the detection of microorganisms by molecular techniques, has become a
fundamental tool for investigating host-associated bacteria, such as those harbored by veterinary or human
[1,2]
clinical samples . Next-generation sequencing (NGS) approaches now enable the identification of slow-
growing, non-cultivable, or non-viable bacteria contained in clinical specimens without relying on
conventional culture-based identification methods based on the isolation of microorganisms by in vitro
[3,4]
growth . Basic PCR amplicon systems were exploited for DNA sequencing cost reasons, amplifying a
specific gene for genus-level phylogenetic profiling purposes . The study of microbial composition is
[5]
rapidly progressing due to recent advances in DNA sequencing platforms employed for shotgun
metagenomics sequencing approaches, and concomitantly lowering of the costs of DNA sequencing, thus
guaranteeing the investigation of species-level taxonomic profiles along with genetic and functional
information of host-associated microbiomes .
[6]

However, despite using protocols specific for bacterial DNA extraction, the huge amount of host DNA in
many biological samples analyzed generates no reliable metagenomic data where the considerable amount
of eukaryotic DNA hinders the microbial sequences. High human-microbial DNA ratios have been
[7]
reported for skin swabs as well as sputum , saliva , oral swabs , vaginal samples , and human biopsies ,
[10]
[11]
[8]
[9]
making it difficult to investigate the resident microbial population. The human/microbial DNA ratio can
also be increased when samples belong to inflamed or infected sites due to an influx of immune cells, tissue
wounds, or necrosis .
[12]
Accordingly, the amount of bacterial DNA present in some biological samples can reach very low levels,
such as on skin samples due to the cutaneous low pH and the continuous secretion of antimicrobials or
[13]
[14]
biopsies where the bacterial content is mainly associated with tissue or mucosa . In this context, methods
have been developed to enrich bacterial DNA, but they proved to be partially ineffective and compatible
only with fresh specimens [15,16] . For this reason, the extraction of bacterial DNA from biological samples with
high contamination of host eukaryotic DNA is usually much more demanding in terms of target sequencing
depth and related costs to compensate for the lower fraction of microbial DNA. Thus, optimization of the
extraction and sequencing protocols is now mandatory .
[17]