Presentation number: FG 11
DETECTION OF 13 HYPERVARIABLE REGION 1 (HV1) SNPS USING SINGLE-BASE EXTENSION (SBE) PRIMERS IN PARALLEL WITH SANGER SEQUENCING
Rijad Konjhodžić1, Lana Salihefendić1,2, Ivana Čeko1,2, Enis Kandić1, Adna Ašić2, Milovan Kubat3
1Alea Genetic Center, Sarajevo, Bosnia and Herzegovina, 2International Burch University, Department of Genetics and Bioengineering, Sarajevo, Bosnia and Herzegovina, 3University of Zagreb, Faculty of Medicine, Institute for Forensic Medicine, Zagreb, Croatia
The aim of this study was to determine whether single-base extension (SBE) chemistry can be applied to the forensic practice of testing the target single nucleotide polymorphisms (SNPs) of the mitochondrial DNA (mtDNA) Hypervariable Region 1 (HV1). Despite its rather weak discrimination power compared to the short tandem repeat (STR) markers, high copy number of mtDNA per cell and its stability against degradation still guarantee mtDNA testing a place in modern forensic genetics. Buccal swab samples were obtained from 294 unrelated individuals from Bosnia and Herzegovina, following signing of the informed consent form by all participants. After DNA isolation, full sequencing of HV1 was done using chain-termination Sanger sequencing method. SBE reactions were then performed by targeting 13 SNPs that were identified to be the most frequent in the study population. Uniplex SBE reactions for each individual SNP, as well as two multiplex reactions were prepared for both pure and mixed samples, and results thus obtained were compared with those obtained by Sanger sequencing. The results showed complete agreement of the Sanger sequencing results with SBE reactions for both uniplex and multiplex reactions. No significant differences in signal intensity between reactions with forward and reverse SBE primers were observed. The results obtained with SBE were encouraging in regard to multiplexing and processing of the mixed samples, since the allele of minor contributor to the sample was observed in SBE electropherogram in all prepared mixtures. SBE method is limited by the fact that only target SNPs of interest will be analyzed, meaning that they must be carefully selected and curated for each population. However, typing with SBE protocol is accurate, as compared to the golden standard of Sanger sequencing, but was more time- and labor-efficient and simpler to analyze.
Key words: hypervariable region 1 (HV1), mtDNA, mtDNA control region, single-base extension (SBE), single nucleotide polymorphism (SNP)
Presentation number: FG 12
SINGLE CELL TRANSCRIPTOME AND GENOME SEQUENCING FOR GENETICALLY SEPARATING, CHARACTERIZING AND IDENTIFYING INDIVIDUALS FROM BIOLOGICAL MIXTURES
Lucie Kulhankova1, Diego Montiel Gonzales1, Eric Bindels2, Daniel Kling4, Eskeatnaf Mulugeta1,3, Manfred Kayser1
1Erasmus University Medical Center, Department of Genetic Identification, Rotterdam, The Netherlands, 2Erasmus University Medical Center, Department of Hematology, Rotterdam, The Netherlands, 3Erasmus University Medical Center, Department of Cell Biology, Rotterdam, The Netherlands, 4National Board of Forensic Medicine, Department of Forensic Genetics and Toxicology, Linkoping, Sweden
Separating individuals who contributed to biological mixtures and their genetic identification are crucial in forensic investigations where mixed crime scene traces are often encountered, but remains largely unsolved despite several attempts. Here, we present a single cell transcriptome sequencing approach with a novel bioinformatics pipeline aiming to solve this long-standing, societally relevant problem. Our pipeline extracts different sets of single nucleotide polymorphisms (SNPs) from single cell RNA sequencing (scRNA-seq) data we obtained from biological mixtures and uses them for the different purposes. Our approach also allows determining the tissue(s) of origin of the cells present in the mixture. We validated our approach using de novo generated scRNA-seq datasets from multi-person blood mixtures and in-silico mixtures generated from individual scRNA-seq datasets involving different numbers and bio-geographic ancestries of contributors and different ratios. For all up to 9-person balanced and imbalanced mixtures with ratios up to 1:60, we achieved a clear single cell cluster separation. Sex, bio-geographic ancestry of the maternal, paternal and bi-parental sides as well as individual identification were genetically determined correctly for all separated contributors. To further increase the number of captured SNPs, thereby increasing the ability of deconvoluting more complex mixtures including minor contributions based on less cells, we additionally investigated single cell genome sequencing by applying the single cell chromatin accessibility assay (scATAC-seq) to various biological mixtures. Our single cell omics approach has the potential to solve forensic mixture deconvolution for genetically separating, characterizing, and individually identifying perpetrators from multi-person biological mixtures found at crime scenes and can also be applied for detecting and resolving contamination in cell cultures or to separate cancer cells from normal ones.
Key words: mixtures, single cell, transcriptomics, SNPs
Presentation number: FG 13
EVALUATING THE USE OF ANCIENT DNA LABORATORY PROTOCOLS IN THE DOWNSTREAM DNA IDENTIFICATION OF BURNED FORENSICALLY-DERIVED SAMPLES
Cody Parker1, Mathew V. Emery1,2,3, Katelyn L. Bolhofner4,5, Suhail Ghafoor2, Erin Rawls6, Stevie Winingear1, Robert Oldt6, Sreetharan Kanthaswamy4, Jane E. Buikstra1,5, Giovanna Vidoli7, Joanne Devlin7, Laura C. Fulginiti8, Anne C. Stone1,2,5
1School of Human Evolution and Social Change, Arizona State University, Tempe, AZ, USA, 2Center for Evolution and Medicine, ASU, Tempe, AZ, USA, 3Department of Anthropology, Binghamton University, Binghamton, NY, USA, 4School of Math and Natural Sciences, ASU, Glendale, AZ, USA, 5Center for Bioarchaeological Research, ASU, Tempe, AZ, USA, 6School of Life Sciences, ASU, Tempe, AZ, USA, 7Department of Anthropology UT, Knoxville, TN, USA, 8Maricopa County Office of the Medical Examiner, Phoenix, AZ, USA
DNA analysis is a pivotal tool in the identification of human remains recovered from forensic contexts. Under ideal conditions, DNA is sufficiently preserved for DNA identification, including short tandem repeat (STR) profiling. However, natural exogenous factors can limit the amount of recoverable DNA from skeletal material. Where skeletal tissues are exposed to more severe insults, such as fire, obtaining adequate quantities of DNA for downstream analysis has proven challenging. As ancient DNA (aDNA) research is already heavily invested in optimizing the recovery of DNA from challenging samples under similar contexts (e.g., low yields of highly fragmented/degraded DNA), we evaluate DNA yield, quality, usability in STR analyses, NGS library preparation, mitochondrial DNA (mtDNA) recovery, and targeted single nucleotide polymorphism (SNP) assays using protocols adapted from aDNA and forensic analyses. Initial STR and NGS analyses showed that the aDNA extraction protocol recovered higher quantities of shorter DNA fragments at temperatures >350°C. Additionally, our results suggest that there may be an acute point of DNA degradation at temperatures >350°C resulting in a drop in observed STR allele recovery, mtDNA genome read counts and depth of coverage, and SNP calling efficiency. We continue this research using samples obtained via controlled burning of ~10 donor cadavers to evaluate DNA recovery and useability across multiple skeletal elements by comparing methodologies in samples from the same sampling locations across individuals. Preliminary results from this second study show that aDNA extraction protocols appear to provide adequate DNA yields more consistently for initial STR analyses, especially at higher levels of thermal exposure. Our results demonstrate that by optimizing our laboratory protocols to recover DNA efficiently from highly degraded bone samples, we greatly enhance individual identification possibilities in such challenging forensic contexts.
Key words: forensic DNA, burned bone, genomes
Presentation number: FG 14
EFFECT OF OZONE DISINFECTION ON FORENSIC SAMPLES
Vivien Fejes, Dominika Szűcs, Katalin Sipos, Gábor Simon, Viktor Soma Poór
University of Pécs, Medical School, Department of Forensic Medicine, Pécs, Hungary
In certain situations, disinfection of forensic case sample may be required, e.g., in the early stages of COVID-19 pandemic, or in case of objects which might have been related to biological warfare or terrorism. Our aim was to test the applicability of ozone disinfection technique on forensic samples. We examined the effect of ozone on mock casework samples. We collected samples from worn surgical masks. We investigated blood, saliva, and semen stains. Immunoassays were applied to detect special antigens in blood, saliva and semen samples. As supplementary test, luminol, Phadebas test and microscopy was applied, respectively, in addition to examination of body fluid specific RNA markers. In surgical masks, the detected allele counts between control and ozone-treated samples showed no significant difference (p = 0.513). However, the two tested sampling sites, the elastic earloop and the middle part of the nosepiece, showed significant difference in detected allele numbers (p = 0.011). Comparison revealed a statistically significant difference between the contributors (p = 0.001). Immunoassays were able to identify the sample type after the ozone-treatment. Phadebas showed that ozone-treated samples showed no or only very low enzyme activity. By RT-PCR, we could detect the specific markers in both ozone-treated and control samples. The STR profiles from the masks showed that sampling site and contributor had greater effects on profiling that the ozone treatment itself. Ozone does not damage the structure of hemoglobin, amylase and prostate specific antigen; however, it decreases the enzymatic function of salivary amylase. Detection with luminol was successful. Microscopic observation of sperm cells also showed no alteration between the ozone-treated and the control samples. RT-PCR was successful in all cases; therefore this disinfection method does not hamper the RNA-based biological fluid identification. According to our pilot study, ozone treatment does not encumber the routine forensic sample processing, so ozone treatment could become an accepted method to disinfect crime scene samples.
Key words: STR, ozone, biological fluid, RNA biomarkers
Presentation number: FG 15
AN ASSESSMENT OF PROBABILISTIC APPROACHES TO MTDNA MIXTURE INTERPRETATION
Jennifer McElhoe1, Alyssa Addesso1, Brian Young2, Jeff Smith2, Mitchell Holland1
1The Pennsylvania State University, Forensic Science Program, Department of Biochemistry and Molecular Biology, University Park, PA, USA, 2NicheVision, LLC, Akron, OH, USA
Mitochondrial (mt) DNA plays an important role in the fields of forensic and clinical genetics, molecular anthropology, and population genetics, with mixture interpretation being of particular interest in medical and forensic genetics. In forensics, mixture deconvolution generally relies on genotyping of STRs, but this approach struggles to resolve samples with similar contributor proportions and degraded samples. The high copy number, haploid state (single haplotype contributed per individual), high mutation rate, and well-known phylogeny of mtDNA, makes it an attractive marker for mixture deconvolution in damaged and low quantity samples of all types. Given the desire to deconvolute mtDNA mixtures, the goals of this study are to 1) combine and assess two existing software tools, MixtureAce™ and Mixemt (1), to deconvolute mtDNA mixtures 2) create a dataset of in-silico MPS mixtures from whole mitogenome haplotypes representing a diverse set of population groups, and consisting of two and three contributors at different dilution ratios to test the combined tools, and 3) since amplicon targeted sequencing is desirable, and is a commonly used approach is forensic laboratories, create biological mixture data associated with two amplification kits: PowerSeq™ Whole Genome Mito (Promega™) and Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific by AB™) to further validate the software for use in forensic laboratories. Findings will include qualitative measures and statistical evaluation of forensic evidence and will significantly enhance the value of mtDNA testing in forensic laboratories through the assessment of software tools and best practices regarding the deconvolution of mtDNA mixtures. Overall, exact contributors were detected in 17.7% (42/237) of 2 and 3-person in-silico mixtures of small amplicon targeted MPS data, and increased to 70% when closely related haplogroups were included. Spurious haplogroups, most likely due to private mutations, were detected in addition to the contributing haplogroups in approximately 30% of the mixtures. Inclusion of a randomly selected, known haplotype in the analysis reduced the number of samples with spurious haplogroups from 32 (56.1%) to 15 (26.4%). Biological samples were also considered. 1. Samuel H. Vohr, et al. 2017. FSIG 30; 93-105.
Key words: mtDNA, mixtures, MPS