Browsing by Subject "Microsatellite Repeats"
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Item A Bone and Buccal Sensitivity Study Comparison and Stability Study using the PowerPlex[R] Fusion 6C System(2018-05) McDaniel, Ethan L.; Warren, Joseph E.; Planz, John V.; Krishnamoorthy, Raghu R.; Gaydosh-Combs, LauraA validation study, a bone sensitivity study and a stability study were performed using the PowerPlex[R] Fusion 6C System. These studies were performed on a 7500 Real-Time PCR System, 9700 GeneAmp Thermocycler and 3500xL Genetic Analyzer. Buccal DNA was used to develop a method to analyze the DNA profiles gathered during the bone sensitivity study and stability study. DNA profiles for specific concentrations of DNA in solution were obtained during the bone sensitivity study. The stability study showed profiles exhibiting the effects of metal PCR inhibitors being introduced to the DNA extract solutions. Full profiles were obtained for calcium concentrations less than 7.35 mM, while the instrument was fully inhibited for copper concentrations between 0mM and 7.35 mM. Based on the limited data, the PowerPlex Fusion 6C System cannot tolerate copper when present in DNA solutions; whereas, calcium may be tolerated as an inhibitor up to 7.35mM.Item ParaDNA: A Novel Instrument for Presumptive DNA Analysis(2018-05) Moussa, Ola; Warren, Joseph E.; Allen, Michael S.; Staub, Rick W.; Millar, J. CameronCurrent methods for human identification are time consuming and can take weeks to complete, leading to a backlog of evidence needing to be processed and a slowdown in investigations. The ParaDNA[R] Instrument is designed to address this issue using the Screening and Intelligence Systems which can detect the relative amount of DNA in an evidence item, and analyze 5 short tandem repeats (STRs) and Amelogenin, respectively [3]. The Instrument uses HyBeacons[R] which target specific STRs to identify the presence of DNA, and detect STR alleles. This is a validation of the ParaDNA[R] Screening and Intelligence Systems using saliva and blood samples to assess the sensitivity and reliability of the instrument. The data collected using the ParaDNA[R] Instrument show that it can reliably identify the relative amount of DNA in a sample, and display useful STR profiles that are 99% concordant with Qiagen Investigator 24Plex QS STR Kits.Item Precision DNA Mixture Interpretation with Single-Cell Profiling(MDPI, 2021-10-20) Ge, Jianye; King, Jonathan L.; Smuts, Amy; Budowle, BruceWet-lab based studies have exploited emerging single-cell technologies to address the challenges of interpreting forensic mixture evidence. However, little effort has been dedicated to developing a systematic approach to interpreting the single-cell profiles derived from the mixtures. This study is the first attempt to develop a comprehensive interpretation workflow in which single-cell profiles from mixtures are interpreted individually and holistically. In this approach, the genotypes from each cell are assessed, the number of contributors (NOC) of the single-cell profiles is estimated, followed by developing a consensus profile of each contributor, and finally the consensus profile(s) can be used for a DNA database search or comparing with known profiles to determine their potential sources. The potential of this single-cell interpretation workflow was assessed by simulation with various mixture scenarios and empirical allele drop-out and drop-in rates, the accuracies of estimating the NOC, the accuracies of recovering the true alleles by consensus, and the capabilities of deconvolving mixtures with related contributors. The results support that the single-cell based mixture interpretation can provide a precision that cannot beachieved with current standard CE-STR analyses. A new paradigm for mixture interpretation is available to enhance the interpretation of forensic genetic casework.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 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.Item USAT: a bioinformatic toolkit to facilitate interpretation and comparative visualization of tandem repeat sequences(BioMed Central Ltd., 2022-11-20) Wang, Xuewen; Budowle, Bruce; Ge, JianyeBACKGROUND: Tandem repeats (TR), highly variable genomic variants, are widely used in individual identification, disease diagnostics, and evolutionary studies. The recent advances in sequencing technologies and bioinformatic tools facilitate calling TR haplotypes genome widely. Both length-based and sequence-based TR alleles are used in different applications. However, sequence-based TR alleles could provide the highest precision in characterizing TR haplotypes. The need to identify the differences at the single nucleotide level between or among TR haplotypes with an easy-use bioinformatic tool is essential. RESULTS: In this study, we developed a Universal STR Allele Toolkit (USAT) for TR haplotype analysis, which takes TR haplotype output from existing tools to perform allele size conversion, sequence comparison of haplotypes, figure plotting, comparison for allele distribution, and interactive visualization. An exemplary application of USAT for analysis of the CODIS core STR loci for DNA forensics with benchmarking human individuals demonstrated the capabilities of USAT. USAT has user-friendly graphic interfaces and runs fast in major computing operating systems with parallel computing enabled. CONCLUSION: USAT is a user-friendly bioinformatics software for interpretation, visualization, and comparisons of TRs.