From degraded to deciphered: Applying ATAC-seq to forensic molecular diagnosis under degradation conditions

Current molecular diagnostic techniques in forensic science are significantly hindered by DNA degradation, underscoring the urgent need for robust molecular tools with resistance to degradation. In our previous study using a murine traumatic brain injury (TBI) model, we were the first to propose the feasibility of using ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) to identify trauma-related chromatin changes under early postmortem degradation conditions (2 h). However, its applicability to human forensic samples remains insufficiently evaluated. This study aims to systematically assess the utility of ATAC-seq in identifying chromatin accessibility changes induced by TBI in human samples, with a particular focus on its stability and reliability under late-stage postmortem degradation. Cortical brain tissues from three human TBI cases and three matched controls were collected and subjected to sampling at 0 and 24 h postmortem. Standard ATAC-seq protocols were used for library construction and sequencing. Downstream analyses included quality control, differential accessibility analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment, and transcription factor motif analysis. All 12 samples exhibited high-quality sequencing metrics. Numerous differentially accessible chromatin regions (peaks) were identified between TBI and control groups, demonstrating the sensitivity of ATAC-seq in detecting trauma-associated chromatin alterations. Notably, no significant differences were observed between samples degraded for 0 and 24 h, indicating the method's robustness to degradation. Enrichment analyses revealed that differential peaks were significantly associated with biological processes and pathways relevant to TBI pathology, including axon development, synaptic signaling, and neurotrophin signaling pathways. The SOX transcription factor family emerged as a dominant motif, consistent with known mechanisms of TBI pathophysiology. This study provides compelling evidence that trauma-induced chromatin accessibility changes remain detectable and relatively stable in degraded forensic brain tissues, including those subject to late postmortem intervals. These results, obtained in a small proof-of-concept cohort, support the feasibility of applying ATAC-seq to human postmortem brain samples and motivate validation in larger, systematically collected forensic case series.