Assessment of protocols for characterization of the human skin microbiome using shotgun metagenomics and comparative analysis with 16S metabarcoding

The skin microbiome includes bacteria, fungi, and viruses, with composition varying significantly across body sites. Although 16S rRNA gene sequencing is common, it excludes non-prokaryotic taxa and offers limited functional data. Shotgun metagenomics provides broader taxonomic and functional insights but is challenging for low-biomass skin samples due to limited microbial DNA and high host contamination. In this study, we characterized the microbiome of the forehead and armpits in healthy individuals using shotgun metagenomics and assessed the strategies to improve sequencing success. We compared collection kits, DNA extraction protocols, and tested multiple displacement amplification (MDA). We found that sampling with D-Squame discs followed by an in-house DNA extraction protocol was the most effective combination to maximize DNA yields. MDA introduced significant compositional biases and is not recommended. Shotgun sequencing, without MDA, produced microbial compositions and diversity indices broadly consistent with 16S rRNA metabarcoding, although it showed discrepancies in the relative abundance of some genera. Consistent with prior studies, the armpit microbiome was dominated by <i>Staphylococcus spp</i>., whereas the forehead microbiome was dominated by <i>Cutibacterium spp</i>. Critically, shotgun sequencing provided additional insights into viral and eukaryotic microorganisms and revealed the functional potential of microbial communities, demonstrating its clear advantages over 16S rRNA metabarcoding for comprehensive skin microbiome research.IMPORTANCEWith growing evidence of the role of microorganisms in maintaining healthy skin, accurately characterizing the skin microbiome remains a significant challenge. In this study, we demonstrate that shotgun sequencing, carried out with adapted wet lab protocols, provides deep insights into the microbiome composition of specific areas, such as the forehead or the armpits. Notably, it enables the characterization of fungi and viruses while offering direct functional insights into microbial communities, providing a clear advantage over 16S ribosomal RNA gene sequencing. Our findings highlight the potential of shotgun metagenomics as a powerful tool for comprehensive skin microbiome analysis. They emphasize the importance of tailored protocols for low-biomass samples, improving the reliability of shotgun sequencing and paving the way for more robust clinical studies focused on the skin microbiome.