Research Progress on the Application of Nanopore Long-Read Sequencing Combined with DNA Methylation Marking in Complex Forensic Evidence

Background In the analysis of complex forensic material evidence, conventional detection methods for highly degraded, trace, or mixed samples present several limitations, such as low typing success rates and limited information dimensions. Methods This study systematically examines the technical principles and compatibility of nanopore long-read sequencing combined with DNA methylation profiling, identifies optimization strategies to enhance sensitivity for trace materials, improves accuracy in methylation site interpretation, for instance, NanoFreeLunch, without raw signals and using base quality/error patterns, reaching 0.97–0.99 correlation with benchmark regional methylation levels, and streamlines library preparation workflows while evaluating its practical efficacy through domestic and international case studies. Results The research demonstrates that this technology enables simultaneous acquisition of genomic sequences and epigenetic information, facilitating multidimensional identification—including personal identification, age estimation, and body fluid identification—even in highly degraded samples, with significantly higher site detection rates compared to traditional methods. Conclusions Future efforts should focus on standardizing the technology and developing specialized databases to further refine its application frameworks for forensic investigations, thereby providing robust technical support for complex forensic evidence analysis.