Browsing by Subject "Polymorphism, Single Nucleotide"
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Item A Continuous Statistical Phasing Framework for the Analysis of Forensic Mitochondrial DNA Mixtures(MDPI, 2021-01-20) Smart, Utpal; Cihlar, Jennifer Churchill; Mandape, Sammed N.; Muenzler, Melissa; King, Jonathan L.; Budowle, Bruce; Woerner, August E.Despite the benefits of quantitative data generated by massively parallel sequencing, resolving mitotypes from mixtures occurring in certain ratios remains challenging. In this study, a bioinformatic mixture deconvolution method centered on population-based phasing was developed and validated. The method was first tested on 270 in silico two-person mixtures varying in mixture proportions. An assortment of external reference panels containing information on haplotypic variation (from similar and different haplogroups) was leveraged to assess the effect of panel composition on phasing accuracy. Building on these simulations, mitochondrial genomes from the Human Mitochondrial DataBase were sourced to populate the panels and key parameter values were identified by deconvolving an additional 7290 in silico two-person mixtures. Finally, employing an optimized reference panel and phasing parameters, the approach was validated with in vitro two-person mixtures with differing proportions. Deconvolution was most accurate when the haplotypes in the mixture were similar to haplotypes present in the reference panel and when the mixture ratios were neither highly imbalanced nor subequal (e.g., 4:1). Overall, errors in haplotype estimation were largely bounded by the accuracy of the mixture's genotype results. The proposed framework is the first available approach that automates the reconstruction of complete individual mitotypes from mixtures, even in ratios that have traditionally been considered problematic.Item Ancestry Informative Markers Tailored to Hispanic Populations(2020-05) Setser, Casandra H.; Cross, Deanna S.; Planz, John V.; Barber, Robert C.; Phillips, Nicole R.; Krishnamoorthy, Raghu R.Hispanic populations are highly heterogeneous despite being grouped together as a conglomerate population; this makes an accurate panel of ancestry informative markers (AIMs) especially important for human identification. In Chapter 2, the Genomic Origins and Admixture in Latinos (GOAL) dataset containing 494,886 SNPs was used for SNP ascertainment. Utilizing a country attributable variant of Wright's FST, 234 SNPs were selected for biogeographic ancestry (BGA) determination by tailoring each SNP to genetic differentiation of specific populations. Accuracy of BGA prediction was tested using multinomial logistic regression (MLR) and as few as 55 SNPs were robust to 90% for all populations studied. The panel of 234 SNPs was compressed by 65.8% to 80 SNPs by decreasing the influence of Honduras and the Dominican Republic SNPs with high country attributable mean FST values in favor of additional SNPs for Colombia, Cuba, and Puerto Rico; this balanced small panel size with classification accuracy. In Chapter 3, the Setser80 Hispanic AIMs panel was tested against the panels of 128 SNPs developed by the Seldin group and 55 SNPs developed by the Kidd group using STRUCTURE, PCA, a naive Bayesian classifier and MLR. In STRUCTURE, the Setser80 was able to distinguish Honduras, the Dominican Republic, and Colombia at K=4, where the Seldin and Kidd panels were optimized at K=3 and distinguished only Honduras and the Dominican Republic; similar results were obtained by PCA. The GOAL dataset was combined with the Admixed American super-population from the 1000 Genomes Project to test the panel on an expanded dataset of seven populations. Overall, the Setser80 had superior results to the Seldin and Kidd panels with 91.5% accuracy by naive Bayesian classifier and 93.2% by MLR. As an indication of its portability, the Setser80 had accuracies of >98% for Peru and >80% for Mexicans living in Los Angeles, which were not involved in SNP ascertainment. Given its accuracy and lack of overlap, the Setser80 may supplement existing panels for more granular Hispanic BGA determination. In Chapter 4, the application of allele frequencies to forensic genetics, genealogy, and clinical genetics are discussed as well as future directions and ethical considerations.Item Circulating mitochondrial DNA: New indices of type 2 diabetes-related cognitive impairment in Mexican Americans(PLoS, 2019-03-12) Silzer, Talisa K.; Barber, Robert C.; Sun, Jie; Pathak, Gita A.; Johnson, Leigh A.; O'Bryant, Sid E.; Phillips, NicoleMitochondrial function has been implicated and studied in numerous complex age-related diseases. Understanding the potential role of mitochondria in disease pathophysiology is of importance due to the rise in prevalence of complex age-related diseases, such as type 2 diabetes (T2D) and Alzheimer's disease (AD). These two diseases specifically share common pathophysiological characteristics which potentially point to a common root cause or factors for disease exacerbation. Studying the shared phenomena in Mexican Americans is of particular importance due to the disproportionate prevalence of both T2D and AD in this population. Here, we assessed the potential role of mitochondria in T2D and cognitive impairment (CI) in a Mexican American cohort by analyzing blood-based indices of mitochondrial DNA copy number (mtDNACN) and cell-free mitochondrial DNA (CFmtDNA). These mitochondrial metrics were also analyzed for correlation with relevant neuropsychological variables and physiological data collected as indicators of disease and/or disease progression. We found mtDNACN to be significantly decreased in individuals with CI, while CFmtDNA was significantly elevated in T2D; further, CFmtDNA elevation was significantly exacerbated in individuals with both diseases. MtDNACN was found to negatively correlate with age and fatty acid binding protein concentration, while positively correlating with CFmtDNA as well as CERAD total recall score. Candidate gene SNP-set analysis was performed on genes previously implicated in maintenance and control of mitochondrial dynamics to determine if nuclear variants may account for variability in mtDNACN. The results point to a single significant locus, in the LRRK2/MUC19 region, encoding leucine rich repeat kinase 2 and mucin 19. This locus has been previously implicated in Parkinson's disease, among others; rs7302859 was the driver SNP. These combined findings further indicate that mitochondrial dysfunction (as assessed by proxy via mtDNACN) is intimately linked to both T2D and CI phenotypes as well as aging.Item Improving Human Identification Using the Human Skin Microbiome(2021-12) Sherier, Allison J.; Budowle, Bruce; Leudtke, Robert; Phillips, Nicole R.There are times when biological evidence has too low of quality or quantity of human DNA to provide enough information for human identification (HID). However, nucleic acids from the human skin microbiome are sources of genetic material that may be useful for HID. The studies in this dissertation test the hypothesis that specific single nucleotide polymorphisms (SNPs) of selected human skin microorganisms can be used to attribute an unknown microbiome sample to an individual. The first study investigated how Wright's fixation index (FST) can be used to select potentially informative SNPs for HID. SNPs with high estimated FST were ascertained in three different ways to examine three distinct hypotheses. The hypotheses focused on testing whether a high FST, increased taxonomic abundance, and/or using a predetermined panel would be the most effective for HID. Classification accuracies ranged from 88 – 95%, and the method using the most taxa possible performed the best. Results from the study support that using genetic distance to select informative markers from the human skin microbiome for HID was viable. The predetermined panel only achieved an 88% accuracy, although it would be the most applicable of the tested method for forensic case work. The second study focused on using FST estimations to select SNPs abundant in 51 individuals sampled at three body sites in triplicate for HID. The most common SNPs (present in ≥ 75% of the samples) which had FST estimates ≥ 0.1 were used with least absolute shrinkage and selection operator (LASSO) to select a list of informative SNPs for HID. The final list (i.e., hidSkinPlex+) contains 365 SNPs and achieved a 95% classification accuracy on 459 samples. The hidSkinPlex+ lays the foundation for a targeted sequencing panel that can be used to further study the stability and specificity of human skin microorganism SNPs for HID applications.Item Patient genetics is linked to chronic wound microbiome composition and healing(PLOS, 2020-06-18) Tipton, Craig D.; Wolcott, Randall D.; Sanford, Nicholas E.; Miller, Clint; Pathak, Gita A.; Silzer, Talisa K.; Sun, Jie; Fleming, Derek; Rumbaugh, Kendra P.; Little, Todd D.; Phillips, Nicole; Phillips, Caleb D.The clinical importance of microbiomes to the chronicity of wounds is widely appreciated, yet little is understood about patient-specific processes shaping wound microbiome composition. Here, a two-cohort microbiome-genome wide association study is presented through which patient genomic loci associated with chronic wound microbiome diversity were identified. Further investigation revealed that alternative TLN2 and ZNF521 genotypes explained significant inter-patient variation in relative abundance of two key pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis. Wound diversity was lowest in Pseudomonas aeruginosa infected wounds, and decreasing wound diversity had a significant negative linear relationship with healing rate. In addition to microbiome characteristics, age, diabetic status, and genetic ancestry all significantly influenced healing. Using structural equation modeling to identify common variance among SNPs, six loci were sufficient to explain 53% of variation in wound microbiome diversity, which was a 10% increase over traditional multiple regression. Focusing on TLN2, genotype at rs8031916 explained expression differences of alternative transcripts that differ in inclusion of important focal adhesion binding domains. Such differences are hypothesized to relate to wound microbiomes and healing through effects on bacterial exploitation of focal adhesions and/or cellular migration. Related, other associated loci were functionally enriched, often with roles in cytoskeletal dynamics. This study, being the first to identify patient genetic determinants for wound microbiomes and healing, implicates genetic variation determining cellular adhesion phenotypes as important drivers of infection type. The identification of predictive biomarkers for chronic wound microbiomes may serve as risk factors and guide treatment by informing patient-specific tendencies of infection.Item skater: an R package for SNP-based kinship analysis, testing, and evaluation(F1000 Research Ltd., 2022-01-07) Turner, Stephen D.; Nagraj, V. P.; Scholz, Matthew; Jessa, Shakeel; Acevedo, Carlos; Ge, Jianye; Woerner, August E.; Budowle, BruceMotivation: SNP-based kinship analysis with genome-wide relationship estimation and IBD segment analysis methods produces results that often require further downstream process- ing and manipulation. A dedicated software package that consistently and intuitively imple- ments this analysis functionality is needed. Results: Here we present the skater R package for SNP-based kinship analysis, testing, and evaluation with R. The skater package contains a suite of well-documented tools for importing, parsing, and analyzing pedigree data, performing relationship degree inference, benchmarking relationship degree classification, and summarizing IBD segment data. Availability: The skater package is implemented as an R package and is released under the MIT license at https://github.com/signaturescience/skater. Documentation is available at https://signaturescience.github.io/skater.Item SNP Genotyping of Native DNA using Oxford Nanopore MinION Sequencing(2018-05) Ludwick, Whitney N.; Planz, John V.; Zascavage, Roxanne R.; Phillips, Nicole R.; Yang, ShaohuaShort tandem repeats (STRs) are the primary system of genetic variation used for human identity testing in forensics; however, STR typing relies on the use of time- consuming polymerase chain reaction and expensive laboratory equipment. The use of single nucleotide polymorphisms (SNPs) in forensics have several advantages over STRs. In this study, a panel of Identity SNPs were interrogated and typed from native genomic DNA sequencing libraries using the Oxford Nanopore Technologies (ONT) MinION sequencer. We determined that SNPs could be effectively captured using existing software. Four different methods of alignment were investigated, and we found that aligning sequence data to the human genomic sequence (hg19) provided partial profiles, while aligning data to a merged reference profile resulted in more complete profiles. As ONT?s platform continues to improve, SNP genotyping using the MinION may be used to generate complete SNP profiles with the sufficient depth of coverage for reliable genotype determination.Item Typing Highly Degraded DNA Using Target Enrichment(2020-05) Kieser, Rachel E.; Budowle, Bruce; Phillips, Nicole R.; Coble, Michael D.; Berg, Rance E.; Salvatore, MichaelForensic genetic profiling is the process of targeting unique positions within the human genome for identity testing of biological DNA evidence. Forensic profiling of highly degraded DNA samples is one of the primary challenges faced by forensic analysts. These compromised biological samples are difficult to genetically profile, due to the highly fragmented nature of the target molecules, using traditional methods which centers around the detection of short tandem repeats (STRs). For STR typing to be successful, DNA must be relatively intact in order to amplify by PCR. Molecular biology approaches have been developed that may be applied to severely degraded samples to increase the capability of DNA profiling. Targeting single nucleotide polymorphisms (SNPs) holds potential as their amplicons can be designed to be substantially smaller than those for STRs, making these markers a viable alternative for typing degraded (fragmented) DNA. Additionally, rolling circle amplification (RCA) can be exploited as a tool as it has the capacity to amplify all genomic DNA in a circular template present in a sample. A circular molecule essentially creates an infinitely long template for amplification. RCA generates linear tandem copies of the circular template sequence. However, nuclear DNA is not circular and thus RCA cannot be used to its full potential. CircLigase II is an enzyme that circularizes single-stranded DNA. Thus, it may be possible to generate circular DNA from the highly degraded fragments of challenged samples. Molecular inversion probes (MIPs) are an alternative circle-based enrichment approach. A MIP is a single-stranded oligonucleotide that contains two target-specific arms flanking a SNP of interest (capture) and internal PCR primer binding sites for controlled amplification. The two target-specific arms hybridize to the target DNA, the gap is filled resulting in the complementary state of the SNP of interest, the MIP dissociates from the target, and the target site is amplified employing the internally incorporated primer binding sites. Coupled to massively parallel sequencing (MPS), both circle-based approaches were attempted with limited to no success. Reverse Complement PCR (RC-PCR) was pursued to address the same problem of analyzing degraded DNA. RC-PCR is an innovative, one-step PCR target enrichment technology adapted for the amplification of highly degraded (fragmented) DNA. It provides simultaneous amplification and tagging of a targeted sequence construct in a single, closed-tube assay. A human identification (HID) RC-PCR panel was designed targeting 27 identity SNPs generating targets only 50 base pairs in length. In a single reaction, the complete sequencing construct is produced which is essential for MPS library preparation. The RC-PCR approach produced reliable and concordant genotyping results as well as demonstrated a sensitivity of detection of a majority of alleles down to 60 pg of input DNA. In addition, RC-PCR showed robustness tolerating known PCR inhibitors, especially calcium and collagen. The RC-PCR system may be an effective alternative to current forensic genetic methods in the analysis of highly degraded DNA.