Enhanced SNP genotype recovery from low-template DNA using primary template-directed amplification and hybrid capture-based MPS

Abstract Forensic DNA analysis often faces challenges with low-template DNA (LT DNA) due to stochastic effects that compromise genotyping reliability. Although whole genome amplification (WGA) has previously been investigated as a strategy to amplify trace amounts of DNA, its forensic application remains limited by amplification bias and artifacts. Primary template-directed amplification (PTA), a recently developed low-bias WGA method, offers improved coverage uniformity and genome reproducibility. However, its applicability in forensic contexts has not yet been explored. In this study, we developed a workflow integrating PTA with hybrid capture-based massively parallel sequencing (MPS) targeting 1,225 identity-informative single nucleotide polymorphisms (SNPs). Serially diluted genomic DNA (gDNA) samples (1 ng to 6.25 pg) were amplified using PTA, followed by library preparation and hybrid capture-based sequencing. To evaluate the applicability of PTA for SNP genotyping of LT DNA, coverage uniformity, library complexity, SNP genotype recovery, and false-positive rates were investigated. The improvements in coverage uniformity and library complexity achieved by PTA led to a marked enhancement in SNP genotype recovery, reaching 96.0% at 100 pg and 39.7% (more than 400 autosomal SNPs) at a single-cell-equivalent input (6.25 pg). Furthermore, PTA maintained false-positive rates consistently below 1% regardless of input amounts, indicating high reliability of the developed PTA-based workflow. These findings demonstrate the utility of PTA in enhancing SNP genotype recovery for LT DNA analysis, highlighting its potential for forensic applications.