Browsing by Subject "Reproducibility of Results"
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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 An assessment of qPCR assays for DNA concentration and degradation(2019-05) Cropper, Emily R.; Coble, Michael D.; Warren, Joseph E.; Phillips, Nicole R.Forensically challenged samples are often composed of degraded, damaged, or low template mitochondrial DNA (mtDNA). A real-time quantitative polymerase chain reaction (qPCR) assay can help determine if there is sufficient quantity and robust quality of mtDNA to move forward with downstream sequencing and analysis. The fundamental issue with qPCR is that the nominal quantity of the DNA calibrated along the commercial standard used for quantification can vary depending on the supplier and lot numbers. The National Institute of Standards and Technology (NIST) has developed a commercially available human DNA standard, Standard Reference Material (SRM) 2372a, which consists of nuclear DNA (nDNA) and mtDNA data on three wellcharacterized human genomic DNA preparations. The SRM 2372a was used to compare three qPCR assays: a non-commercial triplex assay, for mtDNA quantification, and two commercial assays, Quantifiler Trio (QFTrio) for nDNA quantification, and NovaQUANT for nDNA quantification and determination of the mtDNA/nDNA ratio. Quantification of the SRM uniformly across these three qPCR assays allowed for the conclusion that a robust, reproducible, accurate, and efficient qPCR assay is dependent on (1) the quality and reliability of the DNA standard, (2) the specificity of the qPCR chemistry, and (3) sound primers and probes, to name a few. The findings indicate that commercially available qPCR assays do not necessarily perform as marketed and should be re-verified by a validated DNA SRM.Item How many familial relationship testing results could be wrong?(PLOS, 2020-08-13) Ge, Jianye; Budowle, BruceItem Transfer function analysis of dynamic cerebral autoregulation: A CARNet white paper 2022 update(International Society for Cerebral Blood Flow and Metabolism, 2022-08-14) Panerai, Ronney; Brassard, Patrice; Burma, Joel S.; Castro, Pedro; Claassen, Jurgen AHR; van Lieshout, Johannes J.; Liu, Jia; Lucas, Samuel JE; Minhas, Jatinder S.; Mitsis, Georgios D.; Nogueira, Ricardo C.; Ogoh, Shigehiko; Payne, Stephen J.; Rickards, Caroline A.; Robertson, Andrew D.; Rodrigues, Gabriel D.; Smirl, Jonathan D.; Simpson, David M.Cerebral autoregulation (CA) refers to the control of cerebral tissue blood flow (CBF) in response to changes in perfusion pressure. Due to the challenges of measuring intracranial pressure, CA is often described as the relationship between mean arterial pressure (MAP) and CBF. Dynamic CA (dCA) can be assessed using multiple techniques, with transfer function analysis (TFA) being the most common. A 2016 white paper by members of an international Cerebrovascular Research Network (CARNet) that is focused on CA strove to improve TFA standardization by way of introducing data acquisition, analysis, and reporting guidelines. Since then, additional evidence has allowed for the improvement and refinement of the original recommendations, as well as for the inclusion of new guidelines to reflect recent advances in the field. This second edition of the white paper contains more robust, evidence-based recommendations, which have been expanded to address current streams of inquiry, including optimizing MAP variability, acquiring CBF estimates from alternative methods, estimating alternative dCA metrics, and incorporating dCA quantification into clinical trials. Implementation of these new and revised recommendations is important to improve the reliability and reproducibility of dCA studies, and to facilitate inter-institutional collaboration and the comparison of results between studies.