Indiana University-Purdue University Indianapolis (IUPUI) / In the forensic science community, there is an immense need for tools to help assist investigations where conventional DNA profiling methods have been non-informative. Forensic DNA Phenotyping (FDP) aims to bridge that gap and aid investigations by providing physical appearance information when other investigative methods have been exhausted. To create a “biological eye witness”, it becomes necessary to constantly improve these methods in order to develop a complete and accurate image of the individual who left the sample. To add to our previous prediction systems IrisPlex and HIrisPlex, we have developed the HIrisPlex-S system for the all-in-one combined prediction of eye, hair, and skin color from DNA. The skin color prediction model uses 36 variants that were recently proposed for the accurate prediction of categorical skin color on a global scale, and the system is completed by the developmental validation of a 17-plex capillary electrophoresis (CE) genotyping assay that is run in conjunction with the HIrisPlex assay to generate these genotypes. The predicted skin color output includes Very Pale, Pale, Intermediate, Dark and Dark-to-Black categories in addition to categorical eye (Blue, Intermediate, and Brown) and hair (Black, Brown, Blond, and Red) color predictions. We demonstrate that the HIrisPlex-S assay performs in full agreement with guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM), achieving high sensitivity levels with a minimum 63pg DNA input. In addition to adding skin color to complete the pigmentation prediction system termed HIrisPlex-S, we successfully designed a Massively Parallel Sequencing (MPS) assay to complement the system and bring Next Generation Sequencing (NGS) to the forefront of forensic DNA analyses methods. Using Illumina’s MiSeq system enables the generation of HIrisPlex-S’s 41 variants using sequencing data that has the capacity to xiii better deconvolute mixtures and perform with even more sensitivity and accuracy. This transition opens the door for a plethora of new ways in which this physical appearance assay can grow as sequencing technology is not limited by variant number; therefore, in essence many more traits have the potential to be included in this one assay design. For now, the HIrisPlex-S design of 41 variants using MPS is being fully assessed according to SWGDAM validated guidelines; therefore, this design paves the way for Forensic DNA Phenotyping to be used in any forensic laboratory. This new and improved HIrisPlex-S system will have a profound impact on casework, missing persons cases, and anthropological cases, as it is relatively inexpensive to run, HIrisPlex-S is easy to use, developmentally validated and one of the largest systems freely available online for physical appearance prediction from DNA using the freely available online web tool found at https://hirisplex.erasmusmc.nl/. Lastly, moving forward in our aim to include additional traits for prediction from DNA, we contributed to a large-scale research collaboration to unearth variants associated with hair morphology. 1026 samples were successfully sequenced using an inhouse MPS design at 91 proposed hair morphological loci. From this reaction, we were able to contribute to the identification of significant correlations between the SNPs rs2219783, rs310642 and rs80293268 with categorical hair morphology: straight, wavy or curly.

Indiana University-Purdue University Indianapolis (IUPUI) / In the forensic science community, there is a vast need for tools to help assist investigations when standard DNA profiling methods are uninformative. Methods such as Forensic DNA Phenotyping (FDP) and proteomics aims to help this problem and provide aid in investigations when other methods have been exhausted. FDP is useful by providing physical appearance information, while proteomics allows for the examination of difficult samples, such as hair, to infer human identity and ancestry. To create a “biological eye witness” or develop informative probability of identity match statistics through proteomically inferred genetic profiles, it is necessary to constantly strive to improve these methods. Currently, two developmentally validated FDP prediction assays, ‘HIrisPlex’ and ‘HIrisplex-S’, are used on the capillary electrophoresis to develop a phenotypic prediction for eye, hair, and skin color based on 41 variants. Although highly useful, these assays are limited in their ability when used on the CE due to a 25 variant per assay cap. To overcome these limitations and expand the capacities of FDP, we successfully designed and validated a massive parallel sequencing (MPS) assay for use on both the ThermoFisher Scientific Ion Torrent and Illumina MiSeq systems that incorporates all HIrisPlex-S variants into one sensitive assay. With the migration of this assay to an MPS platform, we were able to create a semi-automated pipeline to extract SNP-specific sequencing data that can then be easily uploaded to the freely accessible online phenotypic prediction tool (found at https://hirisplex.erasmusmc.nl) and a mixture deconvolution tool with built-in read count thresholds. Based on sequencing reads counts, this tool can be used to assist in the separation of difficult two-person mixture samples and outline the confidence in each genotype call. In addition to FDP, proteomic methods, specifically in hair protein analysis, opens doors and possibilities for forensic investigations when standard DNA profiling methods come up short. Here, we analyzed 233 genetically variant peptides (GVPs) within hair-associated proteins and genes for 66 individuals. We assessed the proteomic methods ability to accurately infer and detect genotypes at each of the 233 SNPs and generated statistics for the probability of identity (PID). Of these markers, 32 passed all quality control and population genetics criteria and displayed an average PID of 3.58 x 10-4. A population genetics assessment was also conducted to identify any SNP that could be used to infer ancestry and/or identity. Providing this information is valuable for the future use of this set of markers for human identification in forensic science settings.

Indiana University-Purdue University Indianapolis (IUPUI) / In the past, the present and the future, Lebanon has been an important link between the East and the West. It was always known as the ‘Switzerland of the East’. Over the years, it was a hotspot for different civilizations that uniquely shaped the genomic backbone of the current Lebanese. It is also a good representation of genetically admixed individuals with diverse phenotype characteristics and unique features. Lebanon, quite like other Middle Eastern populations, lacks sufficient genetic studies that helps to better comprehend the complex genomic composition of different traits and diseases. The lack of good representation of the Middle East and North Africa (MENA) region in global studies has led to ambiguity in discovering special ancestry markers and patterns in the Lebanese genome. Yet, in this study, a thorough investigation into a Lebanese collection shows new patterns that potentially would be helpful in forensic and genealogical applications. The investigation into the autosomal and Y-STRs revealed unique alleles that would be valuable in future forensic investigation analysis. In addition, the assessment of phenotype prediction models to predict eye, hair and skin color showed promising results in terms of prediction performance. Those results encourage the future use of intelligence tools in the regions that in return would aid in serving justice and furthering science research. In fact, ancestry and genetic distance studies confirms the presence of admixture within Lebanon between Europe and North Africa. / 2029-06-01

<div><div>In the forensic science community, there is a vast need for tools to help assist investigations when standard DNA profiling methods are uninformative. Methods such as Forensic DNA Phenotyping (FDP) and proteomics aims to help this problem and provide aid in investigations when other methods have been exhausted. FDP is useful by providing physical appearance information, while proteomics allows for the examination of difficult samples, such as hair, to infer human identity and ancestry. To create a “biological eye witness” or develop informative probability of identity match statistics through proteomically inferred genetic profiles, it is necessary to constantly strive to improve these methods. </div><div><br></div><div>Currently, two developmentally validated FDP prediction assays, ‘HIrisPlex’ and ‘HIrisplex-S’, are used on the capillary electrophoresis to develop a phenotypic prediction for eye, hair, and skin color based on 41 variants. Although highly useful, these assays are limited in their ability when used on the CE due to a 25 variant per assay cap. To overcome these limitations and expand the capacities of FDP, we successfully designed and validated a massive parallel sequencing (MPS) assay for use on both the ThermoFisher Scientific Ion Torrent and Illumina MiSeq systems that incorporates all HIrisPlex-S variants into one sensitive assay. With the migration of this assay to an MPS platform, we were able to create a semi-automated pipeline to extract SNP-specific sequencing data that can then be easily uploaded to the freely accessible online phenotypic prediction tool (found at https://hirisplex.erasmusmc.nl) and a mixture deconvolution tool with built-in read count thresholds. Based on sequencing reads counts, this tool can be used to assist in the separation of difficult two-person mixture samples and outline the confidence in each genotype call.<br></div><div><br></div><div>In addition to FDP, proteomic methods, specifically in hair protein analysis, opens doors and possibilities for forensic investigations when standard DNA profiling methods come up short. Here, we analyzed 233 genetically variant peptides (GVPs) within hair-associated proteins and genes for 66 individuals. We assessed the proteomic methods ability to accurately infer and detect genotypes at each of the 233 SNPs and generated statistics for the probability of identity (PID). Of these markers, 32 passed all quality control and population genetics criteria and displayed an average PID of 3.58 x 10-4. A population genetics assessment was also conducted to identify any SNP that could be used to infer ancestry and/or identity. Providing this information is valuable for the future use of this set of markers for human identification in forensic science settings. </div></div><div><br></div>