Mitochondrial DNA control region typing from highly degraded skeletal remains by single‐multiplex next‐generation sequencing

Abstract

Poor nuclear DNA preservation from highly degraded skeletal remains is the most limiting factor for the genetic identification of individuals. Mitochondrial DNA (mtDNA) typing, and especially of the control region (CR), using next‐generation sequencing (NGS), enables retrieval of valuable genetic information in forensic contexts where highly degraded human skeletal remains are the only source of genetic material. Currently, NGS commercial kits can type all mtDNA‐CR in fewer steps than the conventional Sanger technique. The PowerSeq CRM Nested System kit (Promega Corporation) employs a nested multiplex‐polymerase chain reaction (PCR) strategy to amplify and index all mtDNA‐CR in a single reaction. Our study analyzes the success of mtDNA‐CR typing of highly degraded human skeletons using the PowerSeq CRM Nested System kit. We used samples from 41 individuals from different time periods to test three protocols (M1, M2, and M3) based on modifications of PCR conditions. To analyze the detected variants, two bioinformatic procedures were compared: an in‐house pipeline and the GeneMarker HTS software. The results showed that many samples were not analyzed when the standard protocol (M1) was used. In contrast, the M3 protocol, which includes 35 PCR cycles and longer denaturation and extension steps, successfully recovered the mtDNA‐CR from highly degraded skeletal samples. Mixed base profiles and the percentage of damaged reads were both indicators of possible contamination and can provide better results if used together. Furthermore, our freely available in‐house pipeline can provide variants concordant with the forensic software.

The Fascinating World of Forensic Sciences: Multidisciplinary Approaches in Human Remains and the Role of Forensic Pathology in Calabrian Experience

“…Forensic anthropology is a discipline that uses methods of physical anthropology to assist law enforcement in the investigation of crimes and other legal cases. These include sex and age estimation methods to identify individuals as well as techniques to identify trauma and taphonomic markers [5,6]. In particular, in our cases reported, these forensic activities were carried out for human remains found outdoors that were partially or totally decomposed or skeletonized [7].…”

A Customized Human Mitochondrial DNA Database (hMITO DB v1.0) for Rapid Sequence Analysis, Haplotyping and Geo-Mapping

“…The non-coding region, or control region (CR), plays a crucial role in mitochondrial DNA replication and transcription. The hypervariable regions (HV-I, HV-II, and HV-III) within the CR have a higher mutation rate than coding regions [18][19][20]. Consequently, the discriminative power of HV-polymorphisms has been exploited for maternal lineage tracing, population studies, and forensics [18][19][20].…”

“…Today, next-generation sequencing (NGS) has revolutionized the field of mtDNA haplotyping. NGS enables rapid and cost-effective sequencing of the mitogenome, allowing for the identification of single-nucleotide polymorphisms (SNP), insertions, deletions, and structural variants, providing a comprehensive view of mtDNA diversity [18][19][20]. Over the last decade, the number of bioinformatics tools developed for mtDNA haplotyping has also increased [23].…”