Browsing by Subject "STR"
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Item ALLELE CHARACTERIZATION OF FIFTEEN SHORT TANDEM REPEAT LOCI OF NORTH AMERICAN GOLDEN (AQUILA CHYSAETOS) AND BALD (HALIAEETUS LEUCOCEPHALUS) EAGLES USING NEXT-GENERATION SEQUENCING(2014-03) Howard, Taylor E.; Kreutzer, McKensie; Curtis, Mary; Allen, Michael; Planz, JohnPurpose (a): The purpose of this study was to characterize by deep sequencing fifteen short tandem repeat (STR) loci in North American bald and golden eagles currently utilized by the US Fish and Wildlife Service. These STR loci are used for eagle identification in North America, however they were all developed from related species of European eagles. We hypothesized that using next-generation sequencing techniques we would be able to not only show concordance between allele calls using capillary electrophoresis (CE) methods and our new sequencing method, but also have an increased discrimination power of individuals by using this technique. Methods (b): Five samples of both bald and golden eagles were provided by the US Fish and Wildlife Service and deoxyribonucleic acid (DNA) was extracted from each using organic extraction. The current method of analysis through polymerase chain reaction (PCR) using fluorescent CE size based detection was adapted to create a protocol with increased specificity for the 15 sets of primers. Once the amplicon pool was developed, the amplicons were combined into a library using Library Prep Set for Ion Torrent™ kit for sequencing on the Ion Torrent™ Personal Genome Machine (PGM™) Sequencer® (Life Technologies™, Carlsbad, CA) with individual samples identified through barcodes embedded in the sequencing adaptors of the kit. After sequencing, the alleles were analyzed to identify the allele classification and determine the repeat structure of the STR motifs. The sequence data was used to determine if the sequences of North American population differed from existing reference sequences in GenBank®. Data was evaluated with the NextGENe® software using a virtual allelic ladder developed for each locus and contigs were assembled by anchoring the reads with the primer sequence. Results (c): Variants of alleles and allele distributions were recorded. The data revealed that the presence of sequence motif variation can increase the power of discrimination when using sequence analysis as compared to CE. Concordance was observed between the allele calls made using CE and with the sequence determined alleles. Conclusions (d): The method developed can be used to identify individual eagles with a higher discrimination power, which will be helpful in determining identity of individuals when needed for law enforcement investigations of harmed protected species such as bald and golden eagles.Item Allele Characterization of Fifteen Short Tandem Repeat Loci of North American Golden (Aquila Chysaetos) and Bald (Haliaeetus leucocephalus) Eagles using Next-Generation Sequencing(2014-05-01) Howard, Taylor E.; John PlanzBald and golden eagles are species of conservation concern in North America, and are protected under the Bald and Golden Eagle Protection Act (BGEPA; 16 U.S.C. 668-668d). Wildlife forensics utilizes short tandem repeat (STR) loci for identification purposes, however the loci currently used for bald and golden eagle in North America were developed from related species of European eagles. In this study, STR loci were sequenced using the Ion Torrent™ Personal Genome Machine (PGM™) Sequencer® (Life Technologies™, Carlsbad, CA) to characterize the alleles (e.g. the repeat motifs, presence of SNPs and indels). These methods were used to evaluate the discriminatory power of the loci for individualization and for species differentiation.Item An Initial Comparison of Applied Biosystems Quantifiler Duo and Promega Plexor HY Real-time PCR DNA Quantification Systems(2008-05-01) Cole, Sarah Kathleen; Arthur Eisenberg; John Planz; Joseph WarrenObjective 1: Sensitive Study: This study was designed to determine the quantity of template DNA below which amplification is not expected to yield a DNA profile. Dilution series of male and female stock DNA ranging from 0.003 ng/μl will independently be run with both Quantifiler Duo and Plexor HY. These samples will be run in duplicate per plate, with duplicate plates being run. We want to determine if the published lowest detection thresholds (0.023 ng/μl for Duo; 0.0032 ng/μl for HY) are concordant with the data obtained. Objective 2: Mixture Study: The purpose of this study is to obtain quantification results for mixtures of male and female DNA, which should allow for calculations of autosomal:Y ratios that can be helpful in determining what type of genetic analysis to pursue (autosomal STR, Y-STR, or both). Mixtures of female and male DNA ranging from 1:1 to 1024:1 (female: male) will be run in duplicate per plate, with duplicate plates being run. We want to find out how minor of a contributor the male can be in an excess of female DNA and still be detected. This is especially important in sexual assault cases where the major contributor is usually female or when the offender is a vasectomized male. Objective 3: Concordance Study: The purpose of this study is to compare quantification results from Quantifiler Duo and Plexor HY with those from Quantifiler Human, specifically in cases when samples are degraded. The majority of these samples originate from unidentified human remains. Patterns of overestimation or underestimation of DNA concentration can help determine which system will be most beneficial in these cases. This is where the new amplicons size featured in Quantifiler Duo is important in comparing the values with previous results for Quantifiler Human. Sample choice will be at the discretion of the laboratory technical leader and Unidentified Human Remains section analysts. These samples will be the ones that are known to be degraded and have previously yielded overestimated results from the Quantifiler Human quantification system, resulting in poor STR data.Item Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assault Kit Swabs(2016-05-01) Brignac, Francine J.; Joseph E. WarrenSexual assault kits make up 40-50% of a typical Forensic Laboratory caseload. The traditional method to process these samples is time-consuming and requires the use of hazardous chemicals such as Phenol:Chloroform:Isoamyl Alcohol (PCIA). This study compares another manual differential extraction method and two automated methods to the traditional standard differential extraction. Results indicate that as sperm sample concentration decreases, automated methods produce superior results both in DNA quantity obtained and in quality of STR profiles produced. Automated methods reduce hands-on time, facilitate higher through-put of samples, and reduce analyst contact with hazardous chemicals such as PCIA, making it an excellent choice for labs.Item Development of a Comprehensive Massively Parallel Sequencing Panel of Single Nucleotide Polymorphism and Short Tandem Repeat Markers for Human Identification(2015-08-01) Warshauer, David H.; Budowle, Bruce; Chakraborty, Ranajit; LaRue, Bobby L.Massively parallel sequencing (MPS) technologies allow for the detection of an unparalleled amount of genetic information with unprecedented speed and relative ease. These qualities make the technology desirable for generating DNA profiles that may be uploaded into forensic offender, arrestee, and family reference database files. This doctoral dissertation research was conducted under the hypothesis that MPS, with its exquisitely high throughput, can provide a system whereby reference samples can be typed for a large battery of markers, providing more discrimination power for forensic DNA typing and offering increased opportunities to develop investigative leads. The design and implementation of large marker panels for the typing of reference samples will reduce debates on the best core markers for forensic utility, generate innovation because focus will not be solely on a core set of autosomal STRs, promote the development of better systems that can analyze more challenging samples, and enable sharing of data across laboratories worldwide. The primary goal of this project was to develop the capability of typing reference samples for a large battery of markers: 84 autosomal, Y-chromosome, and X-chromosome short tandem repeats (STRs), Amelogenin, and 275 human identity single nucleotide polymorphisms (SNPs), in a single multiplex analysis. To that end, a bioinformatic software package, STRait Razor, was developed to detect STR alleles in raw MPS data. A proof-of-concept study was performed to evaluate the efficacy of using MPS to type forensically relevant markers, using a PCR multiplex-based SNP assay. The proposed comprehensive capture-based MPS panel then was designed and extensively tested. Finally, the benefits of the additional genetic data afforded by MPS, as opposed to traditional methods, were illustrated through the characterization of intra-repeat nucleotide variation within Y-chromosome STR alleles. The results of this dissertation research indicate that MPS is capable of providing robust genetic data from a wide variety of forensically-relevant STR and SNP loci in a single analysis. To date, the comprehensive MPS panel developed during the course of these studies is the most potentially informative assay for reference sample testing for human identification.Item Effect of Collagenase Type 2 and Proteinase K Digestion on DNA Yield from Bone Samples Purified on the EZ1 Advanced XL(2015-08-01) Barrett, Lisa C.; Arthur J. Eisenberg; Joseph E. Warren; Raghu R. KrishnamoorthyGenetic results from bone samples often yield low quantities of DNA and poor quality of genetic data. Proteinase K is a proteinase that is commonly used in DNA extraction methods, however the target proteins of Proteinase K do not closely align with the makeup of bone. Collagenase Type 2 is a protease that is more specific to the breakdown of collagen, which bone is comprised of. This study looked at the potential effects of Collagenase Type 2 digestion on bone samples compared to the effects of Proteinase K on quantity and quality of genetic typing. This study also incorporates the EZ1 Advanced XL purification platform and the AmpFLSTR Globalfiler Amplification Kit.Item Effect of Detergent Selection on Quantity of DNA Obtained and on STR Profile Developed from Bone-Derived DNA(2016-05-01) Proctor, Frances N.; Warren, Joseph E.; LaRue, Bobby L.; Krishnamoorthy, Raghu R.Bone is sometimes the only source of DNA in cases of unidentified persons, missing persons, and mass fatality incidents, but bone characteristically provides lower quantities of DNA and lower quality short tandem repeat (STR) profiles than that of other sample types. Bone composition is very different from that of blood and soft tissue, therefore using a method designed for these other sample types is less effective for bone. A bone-specific extraction method is needed in order to improve these results. This study investigated the effects of detergent selection on both the quantity of DNA obtained and on the STR profiles produced from bone-derived DNA, as a part of developing a bone-specific DNA extraction protocol.Item Effects of pH on Binding of DNA Using the PrepFiler BTA Forensic DNA Extraction Kit on the AutoMate Express Forensic DNA Extraction System(2014-05-01) Ferguson, Erin E.; Arthur EisenbergThe extraction method for the recovery of DNA from bone samples at the UNT Center for Human Identification (UNTCHI) Missing Persons Laboratory is both time consuming and laborious. The results of this study show that the AutoMate Express™ can yield both DNA quantity and STR profiles comparable or greater to that of the standard organic extraction method. The incorporation of this automated technology could significantly reduce the time and streamline the process while increasing the amount of genetic information obtained. Based on the results of this study, the use of the AutoMate Express™ for the extraction of DNA from skeletal remains could be very advantageous to UNTCHI DNA analysts.Item Extraction and STR Amplification of HAEIII Restriction Cut, Membrane Bound Human DNA(2005-07-01) Andrews, John S.; Joseph Warren; Arthur Eisenberg; John PlanzThe use of DNA in forensics has become widely accepted since its introduction into the field in 1985 with Restriction Fragment Length Polymorphisms (RFLP) by Alec Jeffreys (1). RFLP techniques were utilized in the forensic DNA community until the mid 1990s when less labor-intensive PCR-STR techniques became available. During the transition from RFLP technology to PCR based STR technology a method for comparing RFLP profiles to that of STR profiles was not developed. Currently there have been no published studies where STR profiles have been obtained from membrane-bound, restriction cut human DNA. The only way to compare RFLP profiles to STR profiles would be to obtain STR profiles from the bound restriction cut DNA left on the nylon membranes. Since the shift in technology from RFLP to PCR-STR most labs, including the FBI, have stopped RFLP analysis as of the year 2000 (4). Today many unsolved cases exist that utilized RFLP technology. Due to the nature of RFLP analysis many times all of the biological sample must be consumed in order to obtain an RFLP profile. When this occurs, there is no longer biological sample left for future testing. In these instances the only DNA left from the case is restriction cut and bound to nylon RFLP membranes. The only chance of determining the STR profile of the source of the biological sample found at the crime scene would be to remove the membrane bound DNA and obtain an STR profile. The experimental hypothesis of this study is that DNA can be recovered from nylon membranes and interpretable STR results can be obtained. The use of multiple STRs are highly discriminatory being able to generate rare DNA profiles possessing a discriminatory power of in many times that of the earth’s population. Due to this discrimination power, profiles are able to individualize the source of a biological sample and aid in criminal investigations. If STR profiles could be obtained from old RFLP membranes numerous cold cases could be reopened and reinvestigated. The STR profiles obtained from the RFLP membranes could be placed into the Combined DNA Indexing System (CODIS). CODIS blends forensic science and computing software into a tool for solving violent crimes. Through CODIS, STR profiles can be entered into the database and searched against possible suspects at the local, state, and national level. Obtaining STR profiles from RFLP membranes would allow for the comparison of these profiles to those found in CODIS for a possible suspect. This project will employ methods to try and obtain an STR profile from Hae III restriction cut DNA bound to Magna Graph membranes. Attempts will be made to obtain STR profiles through direct amplification off of the membrane with PowerPlex 16 and separation on the Avant 3100 equipped with GeneMapper ID. Methods will also be utilized to remove the bound DNA from the membrane prior to amplification and separation. Removal of the bound DNA from the membrane will involve physical means, as well as, the use of various extraction chemicals. If a technique is found successful at removing DNA from Magna Graph membranes, then the technique will be applied to true RFLP membranes donated by the UNTHSC.Item Sequencing Long Amplicon Microsatellite Loci Using the Oxford Nanopore Technologies MinION[TM] Device(2019-05) Hall, Courtney L.; Planz, John V.; Zascavage, Roxanne R.; Phillips, Nicole R.; Menegaz, Rachel A.Forensic DNA typing utilizes highly variable short tandem repeat (STR) markers to differentiate individuals. Despite the power and reliability of current techniques, sequence-level variations are masked in the length-based profiles generated. Nanopore sequencing has the ability to provide long-read data, allowing for accurate alignment and identification of single nucleotide polymorphisms (SNPs) within and around microsatellite loci. To evaluate the applicability of nanopore sequencing to forensically-relevant autosomal and Y chromosome markers, selected STRs and their flanking regions (~800 bp) were amplified using custom primer sets, barcoded by sample, and sequenced on the MinION[TM] device. High quality sequencing data were obtained for all 24 samples at the 45 STRs interrogated using a customized data analysis pipeline. This project sets the foundation for future development of STRs for potential forensic applications as well as biomedically-relevant regions.Item STRspy-ing hidden variation in forensic DNA profiles using the Oxford Nanopore Technologies MinION device(2022-12) Hall, Courtney L.; Phillips, Nicole R.Forensic DNA examinations harness the high degree of repeat length variation characteristic of short tandem repeats (STRs) for human identification. Conventional approaches to STR profiling consist of PCR amplification followed by length-based separation and detection via capillary electrophoresis (CE). These well-established methods are used in forensic laboratories throughout the world to generate robust and reliable profiles that can discriminate between individuals based on differences in STR repeat length alone. The power of discrimination achieved with length-based allele designations across established panels of autosomal and YSTRs is often sufficient for routine DNA examinations. However, nucleotide-level variation within and around STRs has been shown to increase resolution and facilitate interpretation in more challenging casework scenarios such as those involving partial and mixed DNA profiles. The MinION is a DNA sequencer from Oxford Nanopore Technologies (ONT) that is small in both size and price tag. This portable device could provide an alternative for STR sequencing in forensic laboratories that cannot afford the initial investment or commitment of common next-generation sequencing (NGS) platforms. Despite this potential, the relatively high error rate and lack of STR analysis software have precluded accurate forensic profiling with nanopore sequencing in previous studies. This project aims to determine whether STRs amplified with a commercial kit can be sequenced and profiled on the ONT MinION device. To achieve our overall objective, we developed and tested a novel bioinformatic method known as STRspy that is designed to produce forensic STR profiles from third-generation sequencing data. The results presented herein demonstrate that STRspy can predict the correct sequence- and length-based allele designations across an entire panel of autosomal and Y-STRs using error-prone ONT reads as well as detect variation in the flanking regions with a high level of accuracy. Moreover, these data provide novel insight into how PCR-induced stutter and sample multiplexing impact STR profiling on the MinION. Ultimately, this work increases the feasibility of nanopore sequencing in forensic investigations and provides the foundation for future efforts that aim to harness the big potential of the small MinION device.Item Testing Minimum Ultraviolet Light Exposures to Effectively Remove Contaminating DNA for Use in Forensics(2005-08-01) Kanaly, Angela Catherine; John Planz; Joseph Warren; Arthur EisenbergThis study introduces a solar lamp UV light source, for the purpose of removing contaminating DNA in direct relation to forensic testing. The study attempts to demonstrate what level of decontamination occurs from sun lamp exposure at given time intervals of exposure, set at distances from the lamp, and for different types of biological samples. A FS-40 solar lamp was used to irradiate samples of amplified DNA and cellular samples at distances of 5 cm, 10 cm, and 60 cm from the source, with varied exposure times of 15 min, 30 min, 3 hrs, 6 hrs, 12 hrs and 24 hrs. Common forensic DNA typing concerns include contamination by previously amplified DNA products or from transfer of cellular material onto testing materials. Samples exposed included dried PCR products amplified by AmpFlSTR COfiler kit, dried whole blood, and dried saliva. An organic extraction of the blood and saliva samples isolated any remaining genomic DNA. Control blood and saliva samples were quantitated for accurate DNA concentration. All samples were then amplified by AmpFlSTR COfiler kit and analyzed on an ABI 310 Genetic Analyzer, along with an unexposed control PCR product, blood, and saliva samples, reagent blank run alongside each PCR product, blood, and saliva series, and positive and negative PCR controls. Fragment analysis data was analyzed by GeneScan and Genotyper software to obtain any detectable genetic profile from the samples. This experimental design mimics a true forensic casework scenario by following a routine chain of procedures used widely throughout the field. The current standard in forensic DNA testing measures short tandem repeats (STRs), which vary significantly in length between individuals. There are thirteen loci used by the Combined DNA Index System (CODIS), the national DNA index managed by the FBI Laboratory. All thirteen loci are typed in a typical DNA test, with the AmpFlSTR COfiler kit amplifying seven of these loci. For the purposes of this study, successfully decontaminated PCR products, blood, and saliva samples would show no detectable genotype at any of the seven loci. Other DNA testing, such as mitochondrial DNA analysis from hair, bone or teeth, or very low copy number DNA from a small number of cells, require extreme caution to avoid contamination, as these tests have increased sensitivity over standard STR testing. The level of decontamination detected through UV exposure in this study would not provide sufficient information for application to the more sensitive techniques.Item The Validation of the RapidHIT ID System for Human Identification(2020-05) Campos, Gemma R.; Budowle, Bruce; Cihlar, Jennifer C.; Gwirtz, Patricia A.Forensic scientists routinely employ short tandem repeat (STR) typing as a means to identify or exclude individuals as the donors of biological evidence at crime scenes. Despite developments in automation of STR typing workflow, this time-consuming work requires the expertise and resources of a dedicated forensic genetics laboratory. Rapid DNA technology, a fully automated, turnkey system, offers a simple, 90-minute swab-to-profile approach to generate STR profiles, requiring minimal training and expertise to operate the system. As Rapid DNA continues to be developed and implemented, validation studies are necessary to ensure the quality of analyses performed by Rapid DNA instruments. A series of studies in accordance with SWGDAM guidelines were designed to test different performance measures of the RapidHIT ID System for Human Identification (Thermo Fisher Scientific). The results from this study provide opportunities for protocol and user guide revisions, software improvements, and highlight parts of instrumentation that can be improved for future models.