Evaluation of AutoMate and EZ2 extraction methods with surface cleaning pretreatments for ancient DNA recovery from pre-Hispanic individual using STR and SNP analyses.

The recovery of ancient DNA (aDNA) from skeletal remains continues to be a critical challenge in forensic genetics and paleogenomics due to its high fragmentation, low quantity, and susceptibility to exogenous contamination. This study assessed the combined effect of four surface cleaning pretreatments (PT) and two automated extraction methods, AutoMate Express (AM) and EZ2 Connect Fx (EZ), on aDNA recovery from a pre-Hispanic individual dated to the Postclassic period (950-1521 CE). The pretreatments included chlorine (C), chlorine with ultraviolet irradiation (CUV), chlorine with bone fragment demineralization (CDB), chlorine with bone powder demineralization (CDP), and a non-treated control (NT). DNA quantity and quality were assessed using spectrophotometry, fluorometry, and quantitative PCR (qPCR), while genetic profiling was evaluated using autosomal short tandem repeat (A-STR) analysis and next-generation sequencing (NGS) of STR and SNP markers. The EZ method consistently generated higher DNA concentrations and greater amounts of amplifiable human DNA than the AM method across all pretreatments. However, differences in input mass limit direct comparisons (200 mg for EZ vs. 80 mg for AM); after normalization, EZ maintained a general performance advantage. STR analysis revealed more informative partial profiles with EZ, particularly under CDB and CUV pretreatments, while NGS results also favored EZ, showing improved locus recovery and higher read depth. Extraction methodology was the primary determinant of DNA recovery, with PT providing additional profiling. This study provides an optimized workflow for degraded human DNA, with direct relevance to forensic genetics and paleogenomics.