Whole-genome sequencing (WGS) enables systematic acquisition of high-density genetic variation from a single dataset, providing rich information for forensic identification, kinship analysis, and comprehensive characterization of trace biological materials. However, in challenging samples with low DNA input or damaged molecules, conventional double-stranded DNA (dsDNA) library preparation often fails to efficiently convert available DNA into sequenceable libraries, limiting the potential of WGS for forensic applications.

Single-stranded DNA (ssDNA) WGS library preparation overcomes this limitation by denaturing DNA and ligating adapters directly to single-stranded templates. This approach enhances conversion efficiency for damaged and short fragments, preserves strand-specific information, and recovers molecules that are otherwise inaccessible to dsDNA workflows.

In this study, we evaluated ssWGS libraries across picogram-level DNA inputs (66–264 pg) and representative forensic trace samples. Libraries consistently yielded sequenceable data, with Q30 values of 0.77–0.86 and on-target rates of 96.84%–98.91%. Median insert sizes were substantially shorter than in dsDNA libraries (93–104 bp vs. 336–411 bp), reflecting improved retention of short fragments. Across sample types, library efficiency exceeded 96%, average mapping rate was 94.45%, and Q30 values ranged from 87.24% to 96.01%, demonstrating robustness despite sample variability.

Moreover, ssWGS libraries supported downstream targeted panel enrichment, with genotype concordance to reference profiles remaining stable (79.90%–82.73%), indicating that genome-wide libraries can be flexibly converted into targeted genotyping outputs. In highly compromised samples, fragment peaks of 50–80 bp and partial genomic coverage (up to ~2.15%) enabled successful identification in selected cases.

In summary, ssWGS library preparation addresses the conversion inefficiency of dsDNA workflows under challenging conditions, maximizing DNA utilization and recovering molecules otherwise lost. This strategy provides a robust and flexible framework for forensic genomic analyses, supporting both genome-wide and targeted applications from trace and low-quality samples.

Authors

• Anqi Chen (Institute of Forensic Science, Fudan University; School of Forensic Medicine and Science, Fudan University, China)
• Sitong Liu (Institute of Forensic Science, Fudan University; School of Forensic Medicine and Science, Fudan University, China)
• Yujia Xuan (Institute of Forensic Science, Fudan University; School of Forensic Medicine and Science, Fudan University, China)
• Jinyuan Zhao (Institute of Forensic Science, Fudan University; School of Forensic Medicine and Science, Fudan University, China)
• Yu Xing (Institute of Forensic Science, Fudan University; School of Forensic Medicine and Science, Fudan University, China)
• Chengtao Li (Institute of Forensic Science, Fudan University; School of Forensic Medicine and Science, Fudan University, China)
• Suhua Zhang (Institute of Forensic Science, Fudan University; School of Forensic Medicine and Science, Fudan University, China)