Chasing the code : Advancing Precision Diagnostics through Next Generation Sequencing

Next generation sequencing (NGS) has revolutionized precision diagnostics by enabling high-throughput analysis of nucleic acids. This thesis combines technical validations with innovative applications of NGS across five studies. Papers I–II focus on molecular autopsies, demonstrating that hybridisation-based whole-exome sequencing can be successfully applied to formalin-fixed paraffin-embedded (FFPE) tissue, even in severely fragmented samples. Using matched blood and FFPE samples, our complete workflow for variant detection achieved a sensitivity of 97% and a positive predictive value of 98%. Applied to clinical cases, 23 of 35 FFPE samples were successfully sequenced, and relevant variants were detected in previously unresolved cases of sudden unexplained death. Paper III expanded forensic analysis on blood using the same hybridisation-based NGS-technology. Papers IV–V explore liquid biopsy for pan-cancer detection. Using enzymatic conversion and targeted methylation sequencing of plasma circulating cell-free DNA (cfDNA), we identified 162 differentially methylated regions (DMRs) and developed a classifier for pan-cancer detection with sensitivity and specificity of 83.8%. Fragmentomics analysis revealed cancer-associated patterns in cfDNA fragment length and end motifs: cancer samples exhibited shorter median fragment lengths and alterations in fragment end motifs. These findings highlight fragmentomics as a promising biomarker for cancer detection. Together, these studies illustrate the versatility of NGS for precision diagnostics—from post-mortem genetic analysis to minimally invasive cancer screening—and underscore the importance of rigorous validation to bridge research and clinical implementation.