Investigative Genetics

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    Discovery and Characterization of Tetranucleotide Short Tandem Repeats in North American Bears (Ursids)
    (2015-03) Graham, Michelle; Planz, John; Allen, Michael; Shewale, Shantanu J.
    Background: Accurate individual identification is essential to wildlife crime investigation or associating individuals to source populations in conservation genetics. Current methodology utilized dinucleotide short tandem repeats (STRs) that can be difficult to type accurately and have high mutation rates. Tetranucleotide STRs, like those used in human forensics and population genetics, are more stable and can have a more diverse allele composition due to inherent motif substructure, making them more robust and informative for finer grained individualization. STRs are currently typed by PCR amplification followed by electrophoretic sizing that cannot identify polymorphisms in the repeat motif structure often observed at tetranucleotide loci. Hypothesis: The main objective of this study was to perform a preliminary identification of a suite of new tetranucleotide repeat STR loci, for each of the three primary bear species in North America: the American black bear, the brown (grizzly) bear, and the polar bear. These loci would be more informative than existing panels of dinucleotide STR loci reported in the literature and used in wildlife genetics. Methods: In this study, barcoded, adaptor-ligated genome libraries were prepared for four individuals from each bear species from enzymatically fragmented ursine DNA to be interrogated by massively parallel sequencing. Preliminary libraries for the set of samples were size selected and quantified to ensure sufficient concentrations for downstream processing. These preliminary libraries were enriched for tetranucleotide STR sequence regions prior to sequencing using biotinylated RNA baits designed to capture twelve sequence motifs common to mammals. The enriched libraries were sequenced using the Ion Torrent™ Personal Genome Machine™ (PGM) and the data analyzed using the NextGENe® software using a Floton assembly method for de novo alignment to synthetically reference sequences based on the nucleotide structure of the targeted STR motifs as reference genomes for the bear species examined in this study do not exist. Loci were sorted by species and then selected within a species based on the polymorphism observed among the individual samples. Results and Conclusions: Several tetranucleotide STR loci were identified within each bear species. Loci were characterized based upon common repeat sequence and motif structure, and individualized based on unique sequence composition in regions flanking the repeat region. . Further characterization of individual alleles observed at the loci within and between the bear species is underway, and additional individuals will be typed at the optimized loci to establish allelic frequency distributions, genetic stability, and establish their value as individualizing markers. All unambiguous sequences identified in this study will be submitted to Genbank.
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    Evaluation of the PowerSeq™ Auto System by Massively Parallel Sequencing
    (2015-03) Zeng, Xiangpei; King, Jonathan; Hermanson, Spencer; Patel, Jaynish; Storts, Doug; Budowle, Bruce
    Massively parallel sequencing (MPS) is potential technology for STR typing by forensic laboratories and that some of the CE-based limitations may be overcome by MPS. In this study, the PowerSeq Auto System (Promega) containing 23 STR loci and Amelogenin, was evaluated by MPS. The PCR products were size selected using the MinElute PCR Purification Kit (Qiagen). DNA libraries were normalized, pooled and sequenced on the MiSeq (Illumina; 2 x 250 bp). This multiplex STR system was tested for sensitivity of detection based on input DNA. The result showed that a broad range of the quantity of PCR products could be used for library preparation. In mixture study, the partial profile of minor contributor could be detected up to 19:1 mixture. These studies indicate that PowerPlex Fusion STR system and the Illumina MiSeq system can generate reliable DNA profiles with the types of samples and amounts of input DNA that are relevant to forensic analyses.
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    Detection of intra-allelic sequence variants within Short Tandem Repeats using the Illumina ForenSeq DNA Signature Prep Kit and MiSeq Desktop Sequencer
    (2015-03) Novroski, Nicole; Churchill, Jennifer D.; Budowle, Bruce; King, Jonathan
    Objective: The goal of this study was to identify and characterize intra-allelic sequence variants that exist inside the short tandem repeat (STR) sequences captured by the ForenSeq DNA Signature Prep Kit panel for a Hispanic sample population (n=150). Materials and methods: DNA from 150 Hispanic reference samples were used in this study. All samples were anonymized and collected according to IRB-approved protocols. The ForenSeq DNA Signature Prep Kit was used to barcode and generate sequence libraries so all 150 samples could be multiplexed. Sequencing was conducted on the MiSeq Desktop Sequencer, and data analysis was carried out using the ForenSeq Universal Analysis Software (UAS) and in-house Excel-based workbooks. Results: All 27 autosomal STRs, 24 Y-STRs and 7 X-STRs included in the ForenSeq DNA Signature Prep kit were evaluated. A total of 31 loci were found to have sequence variation in at least one nominal allele. Within that group, 21 loci exhibited variation at greater than or equal to 3 or more nominal alleles. Although most alleles had only two varying sequences per nominal allele, some loci demonstrated a larger amount of variation within their nominal alleles, increasing the diversity and discrimination power at those sites. Conclusions: These results demonstrated that intra-STR allele sequence variation can be detected and characterized for the STRs captured with the ForenSeq Signature DNA Prep kit. Furthermore, the characterization of these sequence variants in STRs offers great potential as a means of further individualizing DNA samples from one another, opportunities for improved mixture de-convolution, and kinship analyses, such as identifying victims from mass disasters.
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    Selection of an Ancestry-Informative Marker (AIM) Panel of INDELs
    (2015-03) Thompson, Lindsey M.; Zeng, Xiangpei; Sage, Kelly; Sturm, Sarah; LaRue, Bobby
    Purpose (a): In forensics, there are two main concerns with the marker systems currently used for human identification. First, the identification of the source of a biological sample found at a crime scene requires a comparison to a known reference sample. When no suspect is available for comparison, these genetic markers cannot provide any additional phenotypic information. In such cases, Ancestry-Informative Markers (AIMs) can be used as an investigative lead to law enforcement. A second issue commonly encountered is caused by sample degradation. When DNA is exposed to the elements, it can degrade to fragments of less than 200 base pairs (bps). The genetic markers currently used in forensic settings are called Short Tandem Repeats (STRs). The commercially available STR amplification kits generally yield amplified products that are 200-600 bps in length. Marker systems that yield short fragments. Methods (b): INDELs, insertions and deletions in the genetic sequence as compared to a consensus reference sequence, are a bi-allelic marker system that can be easily multiplexed and analyzed using the instrumentation currently available in forensic laboratories. In this project, publically available genome data was used to select a panel of INDELs that can distinguish between three major global population groups; Caucasian, African, and East Asian. The markers were selected based on specific criteria; length of INDEL, allele frequency divergence, population substructure, and genetic location. Results (c): "All INDELs were tested for departure from Hardy-Weinberg Equilibrium (HWE) and evidence of linkage disequilibrium (LD). Principal Component Analysis (PCA) of the markers indicate the AIMs ability to completely separate the three population groups. Additionally, analysis in STRUCTURE v2.3.4 gives statistical support for the presence of three separate population clusters. Conclusions (d): A robust panel of AIMs was chosen to distinguish between three major global population groups. By multiplexing these markers into a single reaction, the panel will provide a quick, reliable method for determining the ancestry origin of an unknown sample, which may provide significant benefits to forensic investigations.
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    Stated race/ethnicity is not a definitive indicator of patrilineal ancestry in males of major US populations.
    (2015-03) Nolan, Michael R.; Chakraborty, Ranajit
    Evolutionary analyses of Y-linked SNPs provide clustering of Y-haplotypes defining haplogroups, whose geographic origins have been studied at least at continental levels. Lack of recombination allows haplogroup prediction even with Y-STR haplotype data. In this research we examined variations of predicted patrilineal ancestry of individuals grouped by their self-described race/ethnicity. Y-STR haplotype data, encompassing 23 STR loci, on 936 unrelated males published by the US National Institute of Standards and Technology (NIST) were used to examine the Y-chromosomal diversity at the haplogroup level for the three self-described race/ethnicity groups, namely US Caucasians, African Americans, and US Hispanics. Of these samples, haplogroups of 814 Y-STR haplotypes were predicted with a Bayesian approach, from which haplogoup diversity was estimated for the three groups. The phenetic tree of the observed Y-STR haplotypes was drawn as a Median Joining Network (MJN) with the program Network, on which their haplogroup prediction and stated race/ethnicity information was superimposed to graphically demonstrate the differences of patrilineal ancestry among the three groups. These predictions were used to estimate the proportions of male ancestry from European, African, and Native American gene pools. Predominantly most (90.85%) US Caucasians had predicted male European ancestry, while the Hispanics and African-Americans had much lower levels of male European ancestry; 62.95% and 31.14%, respectively. Contributions of male African ancestry in Caucasians, Hispanics and African-Americans were approximately 4.27%, 15.23% and 67.47% , respectively. Lastly, all three groups exhibited variable amounts of male Native American ancestry; namely, 0.31%, 0.69%, and 14.72%, respectively for Caucasians, African-Americans, and Hispanics. At the haplogroup level, the coefficient of haplogroup diversity (GST) was 18.15%, suggesting that the three groups differed substantially with respect their male ancestral haplogroup lineages. In contrast, these three groups had 812 distinct 23-locus haplotypes; none of which were shared across the three groups. Only two haplotypes were observed twice each, one in African-Americans, and one in Hispanics. The 328 Caucasians had all distinct haplotypes. These resulted in a coefficient of haplotype diversity (GST) of 0.26% at the haplotype level. Together these results suggest that while there is virtually no haplotype sharing across these three groups, each of them exhibited considerable amount of haplogroup sharing, and hence prediction of male lineages is not definitive from the stated race/ethnicity of individuals with Y-STR haplotypes defined by the 23 loci of this study.
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    Effect of Dietary Methylmercury on the Gut Microbiome of Fathead Minnows (Pimephales Promelas)
    (2015-03) Lamar, Nicholas; Allen, Michael
    Purpose: The purpose of this study was to investigate the effect of dietary methylmercury on the gut microbiome of fathead minnows. We hypothesized that consumption of methylmercury would induce a shift in the composition of the gut microbiome. This shift could render the gut susceptible to takeover by opportunistic pathogens. This shift could also increase the concentration of organisms capable of the biotransformation of mercury. Methods: 66 gut samples were obtained from the lab of Dr. Aaron Roberts at the University of North Texas. Methylmercury concentrations ranged from 0.02 to 5.5 parts per million. Bacterial deoxyribonucleic acid (DNA) was extracted, and the sequences for the 16S ribosomal ribonucleic acid (rRNA) were amplified by polymerase chain reaction (PCR). Amplicons were optimized and sequenced using the Ion Torrent™ Personal Genome Machine (PGM™) Sequencer® (Life Technologies™, Carlsbad, CA) with barcoded primers to identify the samples. Sequence data was analyzed using mothur. Results: Data analysis revealed that the presence of dietary methylmercury induced a significant shift in the composition of the microbiome. Communities featured a decrease in order diversity, and an increased concentration of select taxa such as Fusobacteriales. Conclusions: The data supports our hypothesis that exposure to dietary methylmercury induced a shift in the composition of the fathead minnow gut microbiome. Further studies shall be devised to investigate the genome of those select taxa that flourished under these conditions.
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    Discovering the Optimal Hair Sections for Mitochondrial DNA Quantification Via a Multiplex Real-Time PCR Assay
    (2015-03) Nakhla, Meriam; Warren, Joseph
    Hair is among the frequently encountered evidence found in crime scenes. The average person loses approximately 100 hairs a day. Because these hairs are telogen strands, or at the end of their life-phase, there is very little tissue present to obtain nuclear DNA. Hair shafts, however, contain mitochondrial DNA that can be used for identification purposes. There are two areas of concern involving mtDNA analysis of hair shafts: 1) will there be enough mtDNA present to obtain a full profile, and 2) and has the integrity of mtDNA been compromised due to oxidative properties, and/or the keratinization of the hair. The purpose of this project is to elucidate whether the amount of mitochondrial DNA changes from the proximal to the distal end of the hair shaft. Three hair samples were obtained from three subjects and the hairs were dissected at every fourth centimeter. DNA was extracted from each hair section, and subjected to mitochondrial DNA quantification (via the control region of the genome), as well as assessed for any deletions seen within the coding region as a sign of damage that may have occurred, using an assay validated by the University of North Texas- Health Science Center (UNTHSC, Fort Worth, Texas). It was found that there was generally a gradual decrease in mitochondria copy number throughout the hair strands from the proximal to the distal end. Also, it was found that mitochondrial DNA is more susceptible to damage towards the distal end. Mitochondrial DNA sequencing was performed on specific samples to observe any relationship between the concentration of mitochondria and the stability of the sequence.