Differential DNA Preservation of Thermally Altered Tissue and Bone

Description
Recovering high-quality DNA from thermally altered human remains poses a significant challenge for research and law enforcement agencies due to high levels of DNA degradation resulting from exposure to extremely high temperatures (e.g., fire). The current standard practice for the…

Recovering high-quality DNA from thermally altered human remains poses a significant challenge for research and law enforcement agencies due to high levels of DNA degradation resulting from exposure to extremely high temperatures (e.g., fire). The current standard practice for the DNA identification of badly burned skeletal remains is to extract DNA from dense cortical bone collected from recovered skeletal elements. Some of the problems associated with this method are that it requires specialized equipment and training, is highly invasive (involving the physical destruction of sample material), time-consuming, and does not reliably guarantee the successful identification of the remains in question. At low-medium levels of thermal exposure, charred tissue is often adhered to these skeletal remains and typically discarded. In cases where burned/charred tissue is recoverable, it has the potential to be a more efficient alternative to the sampling of cortical bone. However, little has been done to test the viability of thermally altered soft tissue in terms of DNA identification to date. Burned/charred tissue was collected from skeletal samples provided by the University of Tennessee Forensic Anthropology Center, as a part of a controlled burn from donor individuals, for downstream laboratory processing and DNA analysis as part of the Stone Lab (Arizona State University, School of Human Evolution and Social Change). DNA from this charred tissue was extracted using the Qiagen DNeasy Blood and Tissue Kit, and resulting yields were quantified via fluorometry using the Qubit Fluorometer 2.0 and Agilent TapeStation 4200 High-Sensitivity D5000 assay. It was found that between the temperatures of ~200-300 ℃ (burn category 2) and ~300-350 ℃ (burn category 3), tissue was the most efficient extraction type, especially from tissue taken from the surface of the ilium and the rib. As for bone, both the Dabney and the Loreille protocol performed similarly, so choice in extraction type comes down to personal preference, type of equipment on hand, and training. Although, for samples with low input material, the Dabney protocol is optimal.
Date Created
2023
Agent

Mitochondrial D-loop Phylogeography of the Northwest Atlantic Porbeagles (Lamna nasus) in Comparison to the Southwest Pacific porbeagles

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Description
Porbeagles (Lamna nasus) are a highly commercially important shark species that is threatened with extinction due to overfishing. Mitochondrial DNA (mtDNA) displacement loop (D-loop) sequence data from 18 Northwest (NW) Atlantic and 30 Southwest (SW) Pacific porbeagles reveal that these…

Porbeagles (Lamna nasus) are a highly commercially important shark species that is threatened with extinction due to overfishing. Mitochondrial DNA (mtDNA) displacement loop (D-loop) sequence data from 18 Northwest (NW) Atlantic and 30 Southwest (SW) Pacific porbeagles reveal that these regional populations have been genetically separated between 1.39 and 1.25 million years ago (MYA), a time frame which correlates with the end of the earth’s last cooling period. There is far greater genetic differentiation (FST = 0.835) between the NW and SW populations than among sharks within each population supporting a very high level of divergence. A lack of gene flow probably stemming from their limited distribution to cold water temperatures (-1oC to 15oC) has led to their genetic divergence. The NW Atlantic population exhibited fewer haplotypes than the SW Pacific population (2 vs 4). The mean nucleotide diversity value of the NW Atlantic population was also 50% lower (0.00143 vs. 0.00228). Male and female NW Atlantic individuals reflected virtually identical mean population diversity values (0.00393 vs 0.00399); however, females were prevalent near shorelines while the males were more often found in open waters. Of the three age groups within the NW Atlantic population, the immature individuals exhibited the greatest mean nucleotide diversity (0.00452), followed by the sub-adult group (0.00293) and the mature group (0.00288), suggesting that dispersion starts earlier in their life cycle and reduces as they get older. The porbeagle population biology, as revealed by D-loop sequence information, may have significant implications for the conservation efforts of this species. As differences in age-based and sex-based dispersion exist, it is important to understand the relative contributions of gene flow by adults of both sexes in order to implement more effective conservation strategies.
Date Created
2022
Agent

Analyzing SNPs Derived From High-Throughout RadSeq Method to Optimize Genetic Management of Captive Macaques

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Description

The study of macaque monkeys harbors advancements in the field of biomedical research. It is imperative to understand the genetic composition of different species of macaques to assess their accuracy as non-human primate (NHP) models for disease detection and treatment…

The study of macaque monkeys harbors advancements in the field of biomedical research. It is imperative to understand the genetic composition of different species of macaques to assess their accuracy as non-human primate (NHP) models for disease detection and treatment assessments. We sought to characterize the hybridization and admixture of the Southeast Asian macaques using single nucleotide polymorphism markers and analyzing the populations on the mainland and the island. Using AMOVA tests and STRUCTURE analysis, we determined that there are three distinct populations: Macaca mulatta, M. fascicularis fascicularis, and M. f. aurea. Furthermore, the island species holds an isolated population of M. f. aurea that demonstrate high inbreeding and genetic uniqueness compared to the mainland species. Findings from this study confirm that NHP models may need to be modified or updated according to changing allelic frequencies and genetic drift.

Date Created
2022-05
Agent

From Report to Prosecution: A Comprehensive Approach to Sexual Assault Response in Arizona

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Description
Every minute and a half, an American is sexually assaulted (Department of Justice, 2017). After an instance of sexual assault, some victims are given the choice of having a sexual assault evidence kit (SAK) collected. These kits are designed…

Every minute and a half, an American is sexually assaulted (Department of Justice, 2017). After an instance of sexual assault, some victims are given the choice of having a sexual assault evidence kit (SAK) collected. These kits are designed to collect DNA evidence that will, in the best case scenario, result in the identification of the perpetrator. If the perpetrator cannot be located, the DNA profile can still be submitted to the FBI’s CODIS databank, which houses hundreds of thousands of DNA profiles from criminal cases, and may still lead to apprehension of the rapist. Unfortunately, some SAKs experience long delays, decades even, before being tested. To date, there are hundreds of thousands of untested SAKs that remain in police custody awaiting to be submitted for forensic profiling across the country. Here, we completed a holistic investigation of sexual assault response and SAK processing in Arizona. It is important to notice that the focus of our study not only includes SAK processing and the backlog but sexual assault prevention and improving victim reporting in an effort to understand the SAK “pipeline,” from assault to prosecution.
We identified problems in three major categories that negatively impact the SAK pipeline: historical inertia, legislative and institutional limitations, and community awareness. We found that a large number of SAKs in Arizona have remained untested due insufficient funding and staffing for public crime labs making it difficult for state labs to alleviate the SAK backlog while simultaneously responding to incoming cases (“Why the Backlog Exists,” n.d.). However, surveys of ASU undergraduate students revealed a significant interest in campus assault and the SAK backlog. Based on our findings, we suggest harnessing the interest of undergraduate students and recruiting them to specialized SAK-oriented forensic technician and sexual assault nurse examiner (SANE) training at ASU with the goal of creating a workforce that will alleviate the absence of trained professionals within the country. We also explore the possibility of the creation of a private crime laboratory at ASU devoted the processing of SAKs in Arizona as a measure of alleviating the demand on local public laboratories and providing a more economic alternative to commercial laboratories. The creation of an SAK laboratory at ASU would provide undergraduates the opportunity to learn more about real forensic analysis on campus, provide a pipeline for students to become technicians themselves, and help reduce and prevent a future SAK backlog in Arizona.
Date Created
2019-05
Agent

A protocol for Resolving Indeterminate Blood Phenotypes in Rhesus (Macaca mulatta) and Cynomolgus Macaques (M. fascicularis)

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Description
Rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaques are the most commonly used nonhuman primate models in biomedical research. It is therefore critical to correctly infer each study animal's ABO blood group phenotype to prevent fatal transfusion- and transplantation-induced immune…

Rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) macaques are the most commonly used nonhuman primate models in biomedical research. It is therefore critical to correctly infer each study animal's ABO blood group phenotype to prevent fatal transfusion- and transplantation-induced immune responses. While most macaques can be efficiently and accurately phenotyped using a DNA-based assay, we have identified some animals that are unable to be classified as type A, B, or AB and therefore exhibit an indeterminate phenotype. The purpose of this study was to develop a protocol for resolving indeterminate blood group phenotypes and consequently determine if these animals do indeed belong to an O blood phenotype. We attempted both direct and cloning-based sequencing of 21 animals phenotyped as A, B, AB, or indeterminate in order to assess variation at the functional mutation site in exon 7 of the macaque ABO gene. Although direct-from-PCR Sanger sequencing was unable to generate reliable sequence results, our cloned plasmid protocol yielded high quality sequences consistent with known blood group-specific alleles and as such can be used to identify informative polymorphisms at this locus.
Date Created
2018-05
Agent

Ancestry Estimation in Forensic Anthropology: Osteological vs. Molecular Methods and Social Implications

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Description
Ancestry estimation in forensic anthropology has been one of the most complex determinations to make from the human skeleton. There is a long history in biological anthropology using different morphological characteristics from the skull and other areas of the skeleton…

Ancestry estimation in forensic anthropology has been one of the most complex determinations to make from the human skeleton. There is a long history in biological anthropology using different morphological characteristics from the skull and other areas of the skeleton but it remains a difficult estimation that always has some variability. Currently, more studies have been conducted in morphological and metric methods from the skull of ancestry estimation to better the accuracy of the determination. Since most forensic cases are not in the best condition, there also must be other estimation methods from other bones from the remains such as the cervical vertebrae and the femur. These methods have some degree of accuracy but are not as commonly used in forensic cases as the skull is. It seems that the best method for ancestry estimation is to use a combination of multiple methods, having multiple lines of evidence. With the advent of DNA, many researchers have started to study the use of DNA in ancestry estimation. Genetics can be used in ancestry estimation as certain populations have allele frequencies that can be quantified. Using ancestry informative markers (AIMS), DNA can be used to estimate the ancestry of an individual as well as the amount of admixture in the individual. Many different methods have been tested in genetic evaluation of ancestry and have been supported with good accuracy. However, DNA analysis is expensive and time consuming, putting more reliance on osteological methods. Social implications have had a tremendous impact on the fate of ancestry estimation in forensic anthropology. Anthropology has generally rejected the notion of races but it is still used in forensics due to how much it is inculcated into everyday society. Also, the overarching theme of admixture is becoming more prevalent in society. This causes the estimations in forensic anthropology to be extremely difficult. If more research into ancestry estimation does not continue, the determination will almost be impossible to be made.
Date Created
2016-12
Agent

Identifying Rhesus Macaque Gene Orthologs Using Heterospecific Human CNV Probes

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Description

We used the Affymetrix® Genome-Wide Human SNP Array 6.0 to identify heterospecific markers and compare copy number and structural genomic variation between humans and rhesus macaques. Over 200,000 human copy number variation (CNV) probes were mapped to a Chinese and…

We used the Affymetrix® Genome-Wide Human SNP Array 6.0 to identify heterospecific markers and compare copy number and structural genomic variation between humans and rhesus macaques. Over 200,000 human copy number variation (CNV) probes were mapped to a Chinese and an Indian rhesus macaque sample. Observed genomic rearrangements and synteny were in agreement with the results of a previously published genomic comparison between humans and rhesus macaques. Comparisons between each of the two rhesus macaques and humans yielded 206 regions with copy numbers that differed by at least two fold in the Indian rhesus macaque and human, 32 in the Chinese rhesus macaque and human, and 147 in both rhesus macaques. The detailed genomic map and preliminary CNV data are useful for better understanding genetic variation in rhesus macaques, identifying derived changes in human CNVs that may have evolved by selection, and determining the suitability of rhesus macaques as human models for particular biomedical studies.

Date Created
2015-09-16
Agent

The Population Genomics of Rhesus Macaques (Macaca Mulatta) Based on Whole Genome Sequences

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Description

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate in biomedical research, have the largest natural geographic distribution of any nonhuman primate, and have been the focus of much evolutionary and behavioral investigation. Consequently, rhesus macaques are one…

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate in biomedical research, have the largest natural geographic distribution of any nonhuman primate, and have been the focus of much evolutionary and behavioral investigation. Consequently, rhesus macaques are one of the most thoroughly studied nonhuman primate species. However, little is known about genome-wide genetic variation in this species. A detailed understanding of extant genomic variation among rhesus macaques has implications for the use of this species as a model for studies of human health and disease, as well as for evolutionary population genomics. Whole genome sequencing analysis of 133 rhesus macaques revealed >43.7 million single nucleotide variants, including thousands predicted to alter protein sequences, transcript splicing and transcription factor binding sites. Rhesus macaques exhibit 2.5-fold higher overall nucleotide diversity and slightly elevated putative functional variation compared with humans. This functional variation in macaques provides opportunities for analyses of coding and non-coding variation, and its cellular consequences. Despite modestly higher levels of non-synonymous variation in the macaques, the estimated distribution of fitness effects and the ratio of non-synonymous to synonymous variants suggest that purifying selection has had stronger effects in rhesus macaques than in humans. Demographic reconstructions indicate this species has experienced a consistently large but fluctuating population size. Overall, the results presented here provide new insights into the population genomics of nonhuman primates and expand genomic information directly relevant to primate models of human disease.

Date Created
2016-10-17
Agent
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