Optimización del análisis de ADN de restos óseos críticos

Abstract

The discovery in 1986 of the forensic applications of DNA analysis for identification purposes marked a revolution in the field of forensic sciences, establishing itself as one of the most accepted disciplines by courts worldwide. However, despite numerous advances that have enriched the discipline, such as PCR initially and more recently massively parallel sequencing, forensic genetics has not been without its challenges. These include DNA mixture analysis, low copy number DNA, or DNA degradation. In contexts such as organized crime, terrorism, disaster victim identification, or mass graves, DNA analysis on human remains often constitutes the only possible method for identification, given that bones and teeth are the only biological remain that endure over time. These types of samples pose challenges for forensic genetics laboratories due to their low DNA quantity and DNA degradation. Additionally, the distant familial relationship between human remains and reference family members for eventual comparisons can complicate identification efforts, especially in situations in which the incident and identification actions are widely separated in time. Therefore, optimizing and fine-tuning protocols for the analysis of degraded human remains to achieve maximum efficiency in obtaining a genetic profile is of special interest to the discipline. The Laboratory of Genetic Identification at the University of Granada, initially with the Phoenix program and more recently through an agreement with the Andalusian government for the identification of victims from the Spanish Civil War and post-war period, possesses extensive experience in analysing critical human remains. This laboratory served as the crucible for conducting this doctoral thesis, contributing both its prior and current experience, as well as the availability of samples for the various analyses conducted. The objective of this doctoral thesis is the optimization of DNA analysis, in the forensic context, for critical human remains (bones and teeth). To achieve this, a review of the different stages of DNA analysis was conducted: sampling of bone/teeth remains, extraction, quantification, amplification, sequencing, results visualization, and reporting. The review also considered guidelines to ensure process quality within the framework of ISO 17025 standards. For each analytical stage, a comparison of different available analytical techniques was attempted, seeking the one that showed the best performance for each stage: In Chapter 1, concerning bone or teeth samples, the performance of DNA analysis was compared in different human remains from the same individual: femur, tibia, teeth, and petrous bone. It was observed that both teeth and the petrous bone, especially the latter, are generally the best substrates for obtaining a genetic profile. In Chapter 2, regarding DNA extraction, five different extraction protocols were compared: organic extraction, silica in suspension, silica on a column, and with commercial kits PrepFiler™ BTA and InnoXtract™. It was found that the organic extraction protocol with phenol/chloroform/isoamyl alcohol had the best performance in terms of quantification and obtained genetic profile. In Chapter 3, about DNA quantification, the efficiency of four commercial qPCR kits was compared: Quantifiler™ Trio, PowerQuant™, Quantiplex® Pro, and InnoQuant™ HY. The latter kit stood out in terms of sensitivity in detecting DNA, as well as the correlation between the amount of DNA observed in qPCR and the genetic profile obtained subsequently. In Chapter 4, concerning DNA amplification, autosomal STRs commercial kits (Globalfiler™, PowerPlex® Fusion 6C, and Investigator® 24Plex QS), Y-STRs commercial kits (Yfiler™ Plus, PowerPlex® Y23, and Investigator® Argus Y-28), and the application of an INNULs kit (InnoTyper® 21) were compared, also comparing its discrimination power with an autosomal STR kit (Globalfiler™). While no significant differences were observed between autosomal STR kits, differences were seen in terms of a higher number of markers obtained from the PowerPlex® Y23 kit compared to the others. The INNULs kit obtained a higher number of markers than the autosomal STR kit, albeit with lower discrimination power. Chapter 5 reviews the different visualization techniques, genetic profile analysis of critical human remains, and statistical calculations in identification results. In Chapter 6, on DNA sequencing, the application of ancient DNA analysis techniques on forensic samples was explored. Techniques such as library preparation, shotgun sequencing, DNA capture enrichment with the commercial kit Twist Bioscience, and deep shotgun were performed on samples analyzed with autosomal STRs. Finally, in Chapter 7, the different recommendations contained in ISO 17025 standards regarding the competence requirements of testing laboratories are reviewed, with a particular emphasis on aspects to consider in a forensic genetics laboratory.