Browsing by Subject "Forensic Genetics / methods"
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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 Alternative procedure for DNA recovery from cartridges and casings(2020-05) Bettex, Kelsey D.; Planz, John V.; Jones, Harlan P.; Warren, Joseph E.Both unfired and fired ammunition recovered from a crime scene are useful in connecting a crime to a specific firearm, however its use in identifying individuals is less common due to low-level or degraded DNA present. Traditionally, the standard procedure for DNA sampling of ammunition evidence is the use of a swabbing technique. The New York State Police (NYSP) Forensic Investigation Center conducted a study to test the efficacy of an alternative method for obtaining DNA from cartridges and casings, soaking the sample prior to extraction. This method presented a 186% increase in total DNA yield, 169% increase in male DNA yield, and a 44% increase in comparable profiles obtained using GlobalFiler(TM) and 240% using Yfiler(R) Plus. Results showed fewer 'no data' determinations using the soaking method, indicating more DNA obtained. Although a statistically significant difference was not observed between the two methods (p = 0.070), a qualitative significance was.Item Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel(MDPI, 2020-11-13) Cihlar, Jennifer Churchill; Amory, Christina; Lagace, Robert; Roth, Chantal; Parson, Walther; Budowle, BruceFor the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology-Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge-has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra- and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence.Item Evaluation of forensic DNA mixture evidence: protocol for evaluation, interpretation, and statistical calculations using the combined probability of inclusion(BioMed Central Ltd., 2016-08-31) Bieber, Frederick R.; Buckleton, John S.; Budowle, Bruce; Butler, John M.; Coble, Michael D.BACKGROUND: The evaluation and interpretation of forensic DNA mixture evidence faces greater interpretational challenges due to increasingly complex mixture evidence. Such challenges include: casework involving low quantity or degraded evidence leading to allele and locus dropout; allele sharing of contributors leading to allele stacking; and differentiation of PCR stutter artifacts from true alleles. There is variation in statistical approaches used to evaluate the strength of the evidence when inclusion of a specific known individual(s) is determined, and the approaches used must be supportable. There are concerns that methods utilized for interpretation of complex forensic DNA mixtures may not be implemented properly in some casework. Similar questions are being raised in a number of U.S. jurisdictions, leading to some confusion about mixture interpretation for current and previous casework. RESULTS: Key elements necessary for the interpretation and statistical evaluation of forensic DNA mixtures are described. Given the most common method for statistical evaluation of DNA mixtures in many parts of the world, including the USA, is the Combined Probability of Inclusion/Exclusion (CPI/CPE). Exposition and elucidation of this method and a protocol for use is the focus of this article. Formulae and other supporting materials are provided. CONCLUSIONS: Guidance and details of a DNA mixture interpretation protocol is provided for application of the CPI/CPE method in the analysis of more complex forensic DNA mixtures. This description, in turn, should help reduce the variability of interpretation with application of this methodology and thereby improve the quality of DNA mixture interpretation throughout the forensic community.Item How many familial relationship testing results could be wrong?(PLOS, 2020-08-13) Ge, Jianye; Budowle, BruceItem 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 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 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.Item Variation in Mitochondrial DNA Heteroplasmy from Blood, Buccal, and Hair Samples(2020-05) Colon, Natalie S.; Budowle, Bruce; Gwirtz, Patricia A.; Cihlar, Jennifer C.Mitochondrial DNA (mtDNA) sequence variation has important forensic implications. This research assesses five subjects for variation in point heteroplasmy (PHP) using blood, buccal, and head hair from five scalp sites. Five hairs from each site (25 total hairs) were sequenced for mtDNA using the Ion Chef and S5 technologies. Three hairs were used to study PHP variation along the hair shaft. Variance in hair PHP was more similar to blood (R2 = 0.3411) than buccal (R2 = 0.3059). Given sufficient signal, PHP was detected in all subjects, but was not concordant within or across all scalp sites. Along hair shafts, PHP was present in one distal hair portion. Future research is needed to elucidate PHP's impact on hair sample interpretation.