The human microbiome is increasingly explored as a complementary source of forensic intelligence due to its relative resilience to environmental stressors and its potential to provide information when nuclear DNA is limited or degraded. However, methodological variability introduced during sample handling and laboratory processing remains a critical limitation. Therefore, we evaluated the extent to which storage conditions and DNA extraction methods influence saliva-derived microbiome profiles. Saliva samples from five individuals were collected using the Copan® eNAT® system and subjected to three storage conditions: immediate processing, short-term storage (6-10 h at 4°C) followed by long-term storage at -20°C, and short-term storage followed by long-term storage at -80°C. DNA was extracted using two widely applied protocols: the QIAGEN® DNeasy® PowerSoil® Pro Kit (chemical and mechanical lysis) and the QIAGEN® QIAamp® DNA Mini Kit (chemical lysis). Full-length 16S rRNA sequencing was performed on the PacBio® Revio® platform, and microbial community composition was assessed using alpha and beta diversity metrics. Across multiple diversity metrics, DNA extraction method consistently showed a statistically significant effect on microbiome composition, whereas storage condition did not. Moreover, the DNeasy® PowerSoil® Pro Kit yielded more amplicon sequence variants (ASVs), with Venn diagram analysis indicating a higher proportion of ASVs shared among storage conditions. These findings indicate that variation introduced during DNA extraction outweighs variation associated with storage conditions. Accordingly, storage at -80°C is recommended as the preferred condition for long-term preservation, along with DNA extraction using the DNeasy® PowerSoil® Pro Kit due to its superior performance and widespread use in microbiome research. This pilot study contributes to the establishment of robust methodological standards for microbiome-based forensic applications and provides a foundation for our subsequent large-scale citizen science study investigating biological sources of microbiome variation. By analysing saliva-derived microbiomes from cohorts of related individuals, including monozygotic and dizygotic twin brothers as well as more distant paternal relatives, we aim to explore whether microbiome variation reflects paternal lineage, as defined by Y-chromosomal haplotypes and haplogroups. This controlled relatedness framework will enable us to disentangle genetic and environmental contributions and to quantify whether Y-chromosomal lineage explains a detectable and consistent component of microbiome variation, thereby evaluating the potential of microbiome profiling for enhanced donor characterisation. Results from this study are expected in the coming months.

Authors

• Heleen Coreelman (KU Leuven Kulak, Belgium)
• Ronny Decorte (KU Leuven, Belgium)
• Sofie Claerhout (KU Leuven Kulak, Belgium)
• Karen Bisschop (KU Leuven Kulak, Belgium)
• Ellen Decaestecker (KU Leuven Kulak, Belgium)