Methodological advances and computational frameworks in ancient DNA research: a narrative review

Abstract

Ancient DNA research has progressed from a limited technical field to a genome-scale discipline capable of reconstructing evolutionary history, past environments, and host-pathogen interactions over long periods. Early studies faced challenges such as severe molecular fragmentation, chemical damage, low endogenous DNA content, and a high risk of modern contamination. Improvements in laboratory techniques and computational methods have enhanced the accuracy and scope of analyses involving degraded DNA. This review synthesizes recent methodological and computational advances across the complete ancient DNA workflow. We discuss how environmental factors, including temperature, pH, and burial context, influence molecular preservation and inform substrate selection, with an emphasis on mineralized tissues and sediments. Key laboratory advances include optimized cleanroom practices, demineralization and preprocessing strategies, silica-based and single-stranded extraction methods for ultrashort fragments, and damage-aware library preparation that balances sequencing efficiency and authenticity. We outline the core authentication criteria, including fragment length distribution, nucleotide misincorporation patterns, endogenous DNA assessment, and contamination monitoring via controls and replication. In addition, we summarize current sequencing strategies and bioinformatic pipelines tailored for short, error-prone reads, highlighting approaches for mapping, contamination estimation, reference bias mitigation, and probabilistic genotype calling in low-coverage datasets. The review further highlights how integrated laboratory and computational frameworks collectively improve the recovery, authentication, and interpretation of ancient genetic material. By integrating methodological rigor with technological innovation, ancient DNA research now enables robust reconstruction of degraded genomes. Continued refinement of laboratory standards and computational frameworks is essential to ensure accuracy, reproducibility, and responsible application in evolutionary, archaeological, and forensic investigations. Future advances in sequencing technologies, authentication strategies, and bioinformatic methodologies are expected to further expand the analytical power and reliability of paleogenomic research. • Ancient DNA research has evolved from PCR-based analysis of short fragments to genome-scale paleogenomic investigations enabled by high-throughput sequencing and specialized laboratory workflows. • Successful recovery of ancient DNA depends on strategic sample selection, with mineralized tissues such as petrous bones and teeth generally providing higher endogenous DNA yields than most other substrates. • Rigorous contamination control, including dedicated cleanroom facilities, demineralization procedures, optimized extraction methods, and damage-aware library preparation, is essential for generating authentic ancient DNA datasets. • Authentication of ancient DNA relies on characteristic molecular signatures, including short fragment lengths, cytosine deamination patterns, endogenous DNA assessment, contamination monitoring, and independent replication. • High-throughput sequencing platforms, particularly Illumina-based technologies combined with target enrichment approaches, have substantially improved the recovery of degraded genomes from low-abundance ancient DNA samples. • Modern bioinformatic frameworks incorporating damage assessment, contamination estimation, reference-bias mitigation, probabilistic genotype calling, and genome reconstruction have become indispensable for accurate interpretation of ancient DNA data.