A comparison of five DNA extraction methods from degraded human skeletal remains

Extracting DNA from degraded human remains poses a challenge for any forensic genetics laboratory, as it requires efficient high-throughput methods. While little research has compared different techniques, silica in suspension has been identified in the literature as the best method for recovering small fragments, which are often present in these types of samples. In this study, we tested five DNA extraction protocols on 25 different degraded skeletal remains. Including the humerus, ulna, tibia, femur, and petrous bone. The five protocols were organic extraction by phenol/chloroform/isoamyl alcohol, silica in suspension, High Pure Nucleic Acid Large Volume silica columns (Roche), InnoXtract™ Bone (InnoGenomics), and PrepFiler™ BTA with AutoMate™ Express robot (ThermoFisher). We analysed five DNA quantification parameters (small human target quantity, large human target quantity, human male target quantity, degradation index, and internal PCR control threshold), and five DNA profile parameters (number of alleles with peak height higher than analytic and stochastic threshold, average relative fluorescence units (RFU), heterozygous balance, and number of reportable loci) were analysed. Our results suggest that organic extraction by phenol/chloroform/ isoamyl alcohol was the best performing method in terms of both quantification and DNA profile results. However, Roche silica columns were found to be the most efficient method

Extraction efficiency testing of degraded bone samples: Comparing four DNA extraction methods for downstream massively parallel sequencing applications

In recent years, investigative genetic genealogy (IGG), which involves the use of genealogical methods combined with DNA analysis to make potential familial matches, has become an important tool in solving cold and active cases. These cases can involve the identification of a perpetrator or the identification of missing persons. Estimates show that approximately 4,400 unidentified bodies are recovered each year, and up to one quarter of those individuals remain unidentified after one year. Traditionally, forensic DNA amplification methods have relied on the need to amplify 100-450 base-pair targets, specifically, short tandem repeats (STRs), for forensic profiles. With genetic genealogical approaches, smaller targets, such as single nucleotide polymorphisms (SNPs), have shown potential as tools for identification. Traditional extraction protocols for forensic DNA have focused on maximizing DNA recovery with the intent of amplifying larger STR targets. On the other hand, ancient DNA techniques have focused efforts on recovering smaller DNA fragments, like SNPs, and indeed have shown recovery of even highly degraded samples in excess of 400,000 years. This study aims to compare the extraction success and efficiency of one ancient DNA (aDNA) extraction technique from Rohland et al. (2018) and three forensic DNA extraction techniques, PrepFiler® BTA Forensic DNA Extraction Kit from Applied Biosystems, the Bone DNA Extraction Kit, Custom from Promega, and the InnoXtract™ from InnoGenomics, on compromised bone samples for the purposes of massively parallel sequencing (MPS). Quantitative PCR was used to compare the extraction performance of the protocols, while an MPS-based assay, the Ion AmpliSeq™ PhenoTrivium Panel, was used to assess informative characteristics, such as phenotype and biogeographic ancestry, for an investigation. The Rohland and modified PrepFiler protocols showed the most success in terms of DNA recovery and sequencing. These results show the utility of an ancient DNA extraction method in MPS research and the success of a widely used forensic method. The results of this study may add to the process of determining the most appropriate extraction method for massively parallel sequencing applications such as IGG in forensic contexts

InnoXtract vs PrepFiler BTA: Extracting DNA from Burned Skeletal Remains

The purpose of this study was to investigate the different levels of DNA extraction in thermally altered samples by comparing the InnoXtract and PrepFiler BTA extraction kits. PrepFiler BTA is a DNA extraction kit designed to use for bone samples which uses silica coated magnetic beads to extract DNA from powdered bone. InnoXtract is a specialized kit for bone samples which uses the same silica coated magnetic bead method for DNA extraction. Burned skeletal remains pose a challenge since high temperature exposure denatures the DNA and degrades DNA that can be extracted. For this experiment, porcine femurs were used as a human model. The porcine femurs were given one of three treatments, unburned, burned at 200 o C for 15 minutes, or burned at 200 o C for 30 minutes. Cuttings of each bone’s cross section were taken and powdered. The powder was used for extractions. Three extractions were done from each cross section per extraction kit. The extractions were analyzed by running real-time PCR quantitation on the samples. The data showed that the length of time the bones were burned was not statistically significant and that the extraction kit used was not statistically significant in relation to the quantity of DNA recovered

Testing the accuracy of osteometric pair-matched arm bones from the East Marshall Street Well with Insertion/Null (INNUL) genotyping

During the construction on the Virginia Commonwealth University medical campus in 1994, human remains and artifacts were discovered in what is now referred to as the East Marshall Street Well (EMSW). Artifacts found in the well can be dated back to the 19th century and human remains discovered were likely medical cadavers obtained illegally back in mid-19th century, when grave robbing was widely practiced. As a result, the East Marshall Street Well Project (EMSWP) was established in 2015, following the raising of public consciousness concerning the discovery. The aim of this study is to verify previously pair-matched arm element groups (performed by anthropologists at the Smithsonian using osteometric analysis) using Insertion/Null (INNULs) genotyping in order to assemble individuals for reburial purposes. Due to the bones recovered from EMSW being highly degraded, an optimization study was also performed prior to genotyping in order to determine which method was best suited to extract quality DNA from the remains. In total, three sampling methods (scalpel scraping, Dremel sanding, and cut bone sampling) in combination with three extraction methods (organic phenol-chloroform extraction, a modified organic phenol-chloroform extraction suggested by Loreille et al. (2007), and InnoXtract™️ Bone Extraction Kit) were tested on four femurs of donated individuals obtained for teaching purposes. Quantitation of DNA was performed using the InnoQuantÒ HY kit on QuantStudio™️ 6 Real-time PCR. Even though no statistical significance was found between the different combinations of sampling and extraction methods, the combination of the Dremel sampling method and InnoXtract™️ Bone Extraction Kit resulted in the second highest DNA yield and lowest degradation index. Therefore, the combination of the Dremel sampling and InnoXtract™️ Bone Extraction Kit was used to extract DNA from EMSW remains. The quantitation step was performed using the InnoQuantÒ HY kit and genotyping was performed using the InnoTyperⓇ 21 Human DNA Typing kit. Of 106 bone samples, 52 yielded full or partial profiles suitable for comparison, and 41 of them were included in the percent match calculation. Of the 41 bones, 28 (68.29%) bones were correctly matched within the group, 7 (17.07%) bones cannot be excluded as a match, and 6 (14.63%) bones were incorrectly matched. As suggested from the DNA extraction optimization portion of the study, Dremel sanding sampling method could be an alternative to the cutbone sampling method when there is a need to preserve the integrity of the bones. Overall, more than half of the bones were previously pair-matched correctly with some bones that were incorrectly matched, indicating that pair-matching could be a useful method for estimating bone groups but should ultimately be verified by DNA analysis

Testing the Accuracy of Osteometric Pair-matched Leg Bones from the East Marshall Street Well with Insertion/Null (INNULs) DNA Typing Method

In 1994, during the construction of one of Virginia Commonwealth University’s (VCU) medical science buildings, a well containing human skeletal remains was discovered. The uncovering of what is now referred to as the East Marshall Street Well (EMSW), exposed mid 19th-century medical practices on unlawfully obtained cadavers during a time when grave robbing was widely practiced. A seven – year community consultation concluded with the election of a Family Representative Council (FRC) to provide recommendations on research, commemoration, and dignified burials to honor the individuals whose remains resided in the EMSW. The aim of this study was to confirm the pair matching of leg bone elements from the ESMW that were previously examined by anthropologists at the Smithsonian, using an Insertion/Null (INNULs) DNA typing method. Human DNA recovered from bone often fails to generate profiles either because of low human DNA quality and quantity or because of presence of PCR inhibitors. For this research, an optimization of a DNA extraction method was performed prior to INNUL typing. In total, three bone sampling methods (scalpel scrapping, dremel sanding, and cut bone sampling) and three different DNA extraction methods (organic, Loreille et al., and InnoGenomics InnoXtractTM Bone Extraction Kit) were compared to test the recovery of DNA quality and quantity on alternative degraded femur bones. Human DNA quantitation was performed using the InnoQuant HY kit on the QuantStudioTM 6 Real-Time PCR. Considering the FRC’s wishes to preserve the intactness of the ancestral remains, the best least destructive method of DNA recovery of highly degraded bones was determined to be the dremel and InnoXtract methods. These methods were used to recovery DNA from the EMSW remains. The ancestral remains were genotyped using InnoTyper 21 Human DNA typing kit. Out of the total 115 lower limb elements (femora, fibulae, tibiae, innominates and sacrum) only 30 had developed partial profiles. Four bone elements that had been previously pair matched through osteometric methods, were also confidently matched through INNUL typing. Because of the limited results, testing the accuracy of osteometric pair matched leg elements using INNUL markers would need to be further investigated

Optimization of forensic DNA analysis of critical skeletal remains

El descubrimiento en 1986 de las aplicaciones forenses del análisis de ADN con fines de identificación supuso una revolución en el campo de las Ciencias forenses, consolidándose como una de las disciplinas más aceptadas por los tribunales de todo el mundo. Sin embargo, y pese a la multitud de avances que han enriquecido la disciplina como la PCR en un primer momento o la secuenciación masiva más recientemente, la Genética forense no ha estado exenta de problemas, como son los perfiles mezcla, las muestras mínimas, o la degradación del ADN. En contextos como el crimen organizado, el terrorismo, las grandes catástrofes, o las fosas comunes, el análisis de ADN en restos humanos suele constituir la única vía posible para la identificación, habida cuenta que huesos y dientes son las únicas muestras biológicas que permanecen con el paso del tiempo. Este tipo de muestras son especialmente desafiantes para los laboratorios de Genética forense, puesto que, además de tener poca cantidad de ADN, este suele encontrarse degradado. A este hecho se une la dificultad, en algunos contextos en los que el suceso y las actuaciones de identificación están muy separadas en el tiempo, de la relación familiar distante entre las muestras de restos humanos y los familiares de referencia con los que se harán las eventuales comparativas. La optimización y puesta a punto de los protocolos de análisis de restos humanos degradados para un máximo rendimiento en la obtención de un perfil genético resulta, por tanto, de especial interés para la disciplina. El Laboratorio de Identificación Genética de la Universidad de Granada, primero con el programa Fénix y más recientemente con el convenio con la Junta de Andalucía de identificación de las víctimas de la Guerra Civil y la Posguerra española, cuenta con una amplia experiencia en el análisis de restos humanos críticos. Es por ello por lo que se constituyó como el crisol para la realización de esta tesis doctoral, aportando tanto su experiencia previa y presente como la disponibilidad de muestras para los diferentes análisis realizados. El objetivo de la presente tesis doctoral es la optimización del análisis de ADN, en el contexto forense, de restos humanos críticos (huesos y dientes). Para ello se ha realizado una revisión de las diferentes etapas del análisis de ADN: la muestra de restos óseos/dientes, extracción, cuantificación, amplificación, secuenciación, visualización de resultados e informe, revisando también las pautas para asegurar la calidad del proceso en el marco de la norma ISO 17025. Para cada etapa analítica se ha tratado de hacer una comparación de las diferentes técnicas analíticas disponibles, buscando la que mejor rendimiento mostraba para cada una de ellas: En el Capítulo 1, sobre la muestra de restos óseos o dientes, se comparó el rendimiento del análisis de ADN en diferentes restos humanos del mismo individuo: fémur, tibia, dientes, y hueso petroso. Se observó que tanto los dientes como el hueso petroso, especialmente este último, son, en general, los mejores sustratos para obtener un perfil genético. En el Capítulo 2, sobre la extracción de ADN se compararon cinco protocolos de extracción distintos: extracción orgánica, sílice en suspensión, sílice en columna, y con los kits comerciales PrepFiler™ BTA e InnoXtract™. Se vio que el protocolo de extracción orgánica con fenol/cloroformo/alcohol isoamílico era el que mejor rendimiento tenía en términos de cuantificación y de perfil genético obtenido. En el Capítulo 3, sobre la cuantificación de ADN, se comparó la eficiencia de cuatro kits comerciales de qPCR: Quantifiler™ Trio, PowerQuant™, Quantiplex® Pro, e InnoQuant™ HY. Este último kit destacó en términos de sensibilidad a la hora de detectar ADN, así como en la correlación entre la cantidad de ADN observada en la qPCR y el perfil genético que se obtiene posteriormente. En el Capítulo 4, sobre la amplificación de ADN, se compararon kits de STRs autosómicos (Globalfiler™, PowerPlex® Fusion 6C, e Investigator® 24Plex QS), STRs del cromosoma Y (Yfiler™ Plus, PowerPlex® Y23, e Investigator® Argus Y-28), explorándose también la aplicación de un kit de INNULs (InnoTyper® 21) comparando su poder de discriminación con el de un kit de STRs autosómicos (Globalfiler™). Si bien no se observaron diferencias significativas entre los kits de STRs autosómicos, sí se vieron diferencias en términos de mayor número de marcadores obtenidos del kit PowerPlex® Y23 con respecto a los otros. El kit de INNULs obtuvo un mayor número de marcadores que el kit de STRs autosómicos, teniendo, no obstante, un menor poder de discriminación. En el Capítulo 5 se revisan las técnicas de visualización de los resultados, así como el análisis de los perfiles genéticos de restos humanos críticos, y los cálculos estadísticos en los resultados de identificación. En el Capítulo 6, sobre la secuenciación de ADN, se exploró la aplicación de las técnicas de análisis de ADN antiguo en muestras forenses, realizándose en muestras analizadas con STRs autosómicos las técnicas de preparación de librería, shotgun sequencing, enriquecimiento con captura de ADN con el kit comercial Twist Bioscience, y deep shotgun. Finalmente, en el Capítulo 7, se revisan los diferentes puntos contenidos en la norma ISO 17025, sobre los requisitos de competencia de los laboratorios de ensayo, haciendo especial incidencia en los aspectos a tener en cuenta en un laboratorio de Genética forense.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.Tesis Univ. Granada.Ayudas para la Formación de Profesorado Universitario (FPU) del Ministerio de Ciencia, Innovación y Universidades FPU 20/01967Ayudas complementarias de movilidad destinadas a beneficiarios del programa de Formación del Profesorado Universitario (FPU) EST 23/0011