Gunmeet Kaur Bali* | 10x Genomics Mary Wisner | Texas Department of Public Safety Sandy Calloway and Henry Erlich | Children’s Hospital Oakland Research Institute Steven J. Mack | University of California San Francisco Abstract: The clonal property of next generation sequencing (NGS), also known as massively parallel sequencing, facilitates the de-convolution of mixtures, a common and challenging category of crime scene evidence. Using a probe capture strategy for target enrichment enables the analysis of the short and degraded DNA fragments common in many forensic specimens because the alternative strategy of polymerase chain reaction (PCR) enrichment requires the presence of two intact priming sites. Moreover, a single shotgun library prepared from a forensic sample can, in principle, be analyzed with probe panels for mitochondrial DNA (mtDNA), single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs), maximizing the genetic information that can be extracted from quality- and quantity- compromised forensic samples. The presenters have developed NimbleGen probe panels for the entire mtDNA genome,1 for 496 SNPs,2 and for 44 STR genetic markers. The captured libraries are sequenced on the Illumina® MiSeq™ platform. When sequencing the entire mitochondrial DNA genome, a haploid lineage marker allows de-convolution of mixtures by counting individual clonal sequence reads and assigning them to one of the contributors, aided by mixemt, a phylogenetic- based software.3 This software can estimate the number of contributors and their proportions in the mixture; this information can be helpful in interpreting the SNP and STR analyses of the same mixtures. The presenters have recently reported the analysis of several contrived forensic type mixtures (e.g., hair, blood) using the mtDNA probe panel and mixemt.4 Recently, the presenters have developed a probe panel for 44 different STR loci that includes 30 autosomal STRs, five X-STRs, eight Y-STRs, and the amelogenin marker. The probes target unique flanking sequences, and the sequence files are analyzed with the web-based software toaSTR,5 developed by LABCON-OWL, for genotyping NGS STRs. The toaSTR analysis of STR markers enables the identification of STR alleles that are identical by size but different by sequence and can distinguish biological (iso-)alleles from stutter and other artifacts, which is useful in mixture interpretation. The SNP analysis is valuable in highly degraded forensic samples where the STR analysis may be compromised. The custom SNP probe panel consists of a set of 496 nuclear SNPs (367 SNPs and 129 microhaplotype markers). The presence of tri-allelic, tetra-allelic, and microhaplotype markers in the SNP probe panel facilitates possible mixture detection and analysis. The presenter’s experience with contrived mixtures indicates that counting sequence reads for mtDNA markers can be used to estimate proportions but estimating proportions for SNP and STR markers by counting reads is less reliable at DNA inputs <1 ng. This study presents the results obtained from a single shotgun DNA library made from contrived mixtures of hair and blood target enriched using the STR, SNP, and mtDNA probe capture panels, demonstrating proof of concept for application to forensically relevant degraded and mixed samples. References 1. Shih, Shelly Y., Nikhil Bose, Anna Beatriz R. Gonçalves, Henry A. Erlich, and Cassandra D. Calloway. "Applications of Probe Capture Enrichment Next Generation Sequencing for Whole Mitochondrial Genome and 426 Nuclear SNPs for Forensically Challenging Samples." Genes 9, no. 1 (2018): 49. http://doi.org/10.3390/genes9010049. 2. Bose, Nikhil, Katie Carlberg, George Sensabaugh, Henry Erlich, and Cassandra Calloway. “Target Capture Enrichment of Nuclear SNP Markers for Massively Parallel Sequencing of Degraded and Mixed Samples.” Forensic Science International: Genetics 34 (2018): 186–196. https://doi.org/10.1016/j.fsigen.2018.01.010. 3. Vohr, Samuel H., Rachel Gordon, Jordan M. Eizenga, Henry A. Erlich, Cassandra D. Calloway, and Richard E. Green. “A Phylogenetic Approach for Haplotype Analysis of Sequence Data from Complex Mitochondrial Mixtures.” Forensic Science International: Genetics 30 (2017): 93–105. https://doi.org/10.1016/j.fsigen.2017.05.007. 4. Wisner, Mary, Henry Erlich, Shelly Shih, and Cassandra Calloway. “Resolution of mitochondrial DNA mixtures using a probe capture next generation sequencing system and phylogenetic-based software.” Forensic Science International Genetics 53 (2021). https://doi.org/10.1016/j.fsigen.2021.102531. 5. Ganschow, Sebastian, Janine Silvery, Jörn Kalinowski, and Carsten Tiemann. “toaSTR: A Web Application for Forensic STR Genotyping by Massively Parallel Sequencing. Forensic Science International: Genetics 37 (2018): 21–28. https://doi.org/10.1016/j.fsigen.2018.07.006.