Utility of Single-Stranded Library Preparation for Recovery of Complete Mitogenomes from Archaeological Skeletal Remains from Ajnala, India: A Forensic Anthropological Investigation

Authors

• Tanvi P. Honap, University of Oklahoma, University of Zürich,
• Shrushti Modi, University of Oklahoma, Wildlife Institute of India
• Cecil M. Lewis Jr, University of Oklahoma
• Mohammad N. Ahmad, Yenepoya Medical College
• Jagmahender S. Sehrawat, Panjab University

Document Type

Article

Anticipated Volume

96

Anticipated Issue

4

Abstract

Despite being a hotspot of cultural and genetic diversity, the Indian subcontinent has been under-represented in ancient genomics research. Poor biomolecular preservation in tropical and subtropical regions poses significant challenges for the recovery of degraded DNA from archaeological and forensic contexts. Recently developed DNA library preparation methods that incorporate natively single-stranded and heat-denatured double-stranded DNA molecules into sequencing libraries have been shown to outperform protocols that only incorporate double-stranded DNA molecules into libraries; however, these approaches are underutilized in ancient DNA and forensic anthropological research in India. Here, we report the recovery of complete mitochondrial genomes from 168-year-old fragmented and severely damaged commingled human skeletal remains (n=56) excavated from a historic well in Ajnala, Punjab, India. DNA was extracted from teeth, built into single- or double-stranded DNA libraries, enriched for the human mitochondrial genome using an in-solution hybridization capture, and sequenced using Illumina next-generation sequencing technology. A comparative analysis of 12 samples processed using both the double-stranded Blunt-Ended Single Tube (BEST) protocol and the single-stranded Santa Cruz Reaction (SCR) demonstrated that the SCR libraries exhibited significantly higher complexity and endogenous mitochondrial DNA content, resulting in a 98% success rate for mitogenome reconstruction. Near-complete mitochondrial genomes were recovered from 55 of 56 individuals, with macrohaplogroups H, M, R, T, U, and X being detected. Haplogroups U7a2 (10.9%) and U2b2 (7.2%) were found to be the most prevalent. Haplogroups rarely found in modern-day local Punjabi populations, such as M65a1, T2b34, and X2p, were also detected. These findings highlight the utility of single-stranded DNA library preparation for ancient genomics and forensic anthropological research in the Indian subcontinent.

Recommended Citation

Honap, Tanvi P.; Modi, Shrushti; Lewis, Cecil M. Jr; Ahmad, Mohammad N.; and Sehrawat, Jagmahender S., "Utility of Single-Stranded Library Preparation for Recovery of Complete Mitogenomes from Archaeological Skeletal Remains from Ajnala, India: A Forensic Anthropological Investigation" (2026). Human Biology Open Access Pre-Prints. 237.
https://digitalcommons.wayne.edu/humbiol_preprints/237