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Amplification of STRs using Polymerase Chain Reaction (PCR) is the foundation of current forensic DNA analysis methods throughout the world. While it remains the gold standard, an inevitable drawback of this process is the production of stutter artifacts due to strand slippage during PCR. Stutter appears as smaller peaks typically positioned one or two repeat units away from an allele peak on an electropherogram and can cause issues in interpretation or mixture deconvolution if not accounted for effectively. Current DNA analysis software attempts to recognize the expected occurrence of stutter and allow analysts to remove it from the allelic data. These stutter models have evolved over time from locus-specific thresholds to allelespecific filters based on a linear relationship between stutter peak height ratio and allele number.1 While this allele-specific stutter modeling offers improvements over the locus-specific thresholds in more accurately predicting the occurrence of stutter in simple loci, loci with more complex repeat units tend to show major deviation from the linear model, suggesting that additional factors may still need to be considered. The advancement and growing adoption of NGS technology offers exciting new possibilities for stutter modeling. NGS still allows laboratories to assess STR marker length but provides the additional ability to view their sequences, which can reveal differences in STR alleles that would have appeared identical on CE. These differences could potentially be used to develop a more accurate model for stutter filtering based not only on allele, but also on characteristics of the sequence. This presentation details part of a study that sequenced batches of 96 and 32 single-source samples for autosomal and Y-chromosomal Short Tandem Repeats (Y-STRs) using the QIAGEN® ForenSeq™ MainstAY Kit. These sequence data were used to develop a model for sequence-specific stutter to explore its potential effectiveness in comparison to locus- or allele-based stutter filter model. While preliminary data suggests that additional sequence characteristics may still need to be considered, this presentation will demonstrate stutter ratio plots showing improved R2 measures of linearity at complex loci when considering the Longest Uninterrupted Stretch (LUS) in the repeat unit of the STR sequence rather than allele number.2 Others have looked at various characteristics of NGS stutter before; however, this stutter model is the first to also incorporate the simplicity of the Sequence Identifier (SID) nomenclature first described by Young et al.3-5 These NGS stutter models will ultimately need to be incorporated into analysis and PG software to take full advantage of the potential interpretation and deconvolution improvements. While stutter data from many more samples will be needed, along with validation tools and downstream software, this presentation will serve as an introduction of sequence-specific stutter to the forensic community. References: 1. Kalafut T, Schuerman C, Sutton J, Faris T, Armogida L, Bright JA, et al. Implementation and validation of an improved allele specific stutter filtering method for electropherogram interpretation. Forensic Sci Int Genet. 2018;35:50-6. 10.1016/j.fsigen.2018.03.016. 2. Bright JA, Taylor D, Curran JM, Buckleton JS. Developing allelic and stutter peak height models for a continuous method of DNA interpretation. Forensic Sci Int Genet. 2013;7(2):296-304. 10.1016/j.fsigen.2012.11.013. 3. D’Angelo O, Vandepoele ACW, Adelman J, Marciano MA. Assessing non-LUS stutter in DNA sequence data. Forensic Sci Int Genet. 2022;59:102706. 10.1016/j.fsigen.2022.102706. 4. Agudo MM, Aanes H, Roseth A, Albert M, Gill P, Bleka O. A comprehensive characterization of MPS-STR stutter artefacts. Forensic Sci Int Genet. 2022;60:102728. 10.1016/j.fsigen.2022.102728. 5. Young B, Faris T, Armogida L, Meles V. The sequence identifier (SID): Nomenclature for mixed-DNA casework using MPS. Forensic Science International: Genetics Supplement Series. 2019;7. 10.1016/j.fsigss.2019.10.125. 

Engineering risk assessment has traditionally focused on direct impacts to individual assets or systems. However, as society's most notable risks increasingly stem from complex, interdependent systems, conventional methods fail to capture the cascading consequences and deepening uncertainty. Addressing this gap requires developing or extending assessment methods. To guide such development and align research efforts, this paper introduces a taxonomy of risk assessment methods. The taxonomy classifies methods by their capability to assess different types of consequences and systemic behaviors. Tier 1 methods can assess direct, localized impacts; Tier 2 methods also capture cascading consequences in interconnected systems; Tier 3 methods also address emergent, transformative, and uncertain dynamics. We identify key system and knowledge characteristics that challenge existing methods and outline research priorities necessary to advance systemic risk assessment: Analyzing consequences, structuring uncertainty, evaluating trade‐offs, strengthening causal inference, ensuring defensibility, and communicating results. This agenda aims to guide future research towards risk assessment methods suitable for the systemic risk challenges society increasingly faces. 

The growing threat of antibiotic-resistant bacteria necessitates the development of alternative antimicrobial agents. Gallium oxyhydroxide (GaOOH) is a promising candidate, though its direct antibacterial efficacy is unexplored. This study provides the first direct evidence of GaOOH microparticles exhibiting cytotoxic effects against both Gram-positive Staphylococcus aureus (S.aureus) and Gram-negative Escherichia coli (E. coli). Orthorhombic GaOOH particles were synthesized hydrothermally, with their morphology influenced by the pH of the synthesis process, as confirmed by scanning electron microscopy and x-ray diffraction analysis. Antibacterial assays revealed that cytotoxicity against E. coli increases with a higher synthesis pH, a trend we demonstrate to be associated with the enhanced defect density in particles, as supported by photoluminescence spectra and FTIR analysis. The study underscores the significant influence of synthesis conditions on the morphology and crystallinity of the resulting GaOOH microparticles, highlighting the influence of surface characteristics on antibacterial agents. 

Next Generation Sequencing (NGS) has been widely considered to be the future of forensic casework, with increasing numbers of labs beginning to implement it into their workflows alongside, or even in place of, traditional Capillary Electrophoresis (CE). There are numerous advantages to NGS such as shorter amplicons, increased sensitivity, the potential detection of isoalleles, and the massive number of markers and marker types that can be run simultaneously. One main limiting factor, though, is plexity, the number of samples that can be run at once. Plexity is affected by the physical capacity of the flow cell, which facilitates the formation of clusters that each represent a distinct PCR target (DPT). Since only so many DPTs can fit on the flow cell, the read coverage at each locus in an NGS run is highly dependent on the flow cell’s capacity, the number of markers in the kit, and the number of DPTs generated during the PCR process. The total number of DPTs includes things like forward and back stutter as well as the alleles. More loci and higher numbers of contributors result in higher DPTs. Verogen’s™ recently released ForenSeq™ MainstAY kit is specifically aimed at a forensically relevant set of 27 autosomal STR and 25 Y-STR markers. It uses the MiSeq® FGx Reagent Micro Kit, with a flow cell capable of five million reads. This combination of markers and flow cell capacity should allow full coverage of a run of 96 single-source samples. However, forensic casework samples are rarely this simple. Labs regularly process samples that contain a mixture of two or more contributors, often with low-level trace donors. Mixed samples can multiply the number of DPTs present, causing a 96-sample run that should fall within the capacity of the flow cell to exceed it, reducing the effective coverage of each locus. In addition, the increased sensitivity promised by NGS has not yet been fully explored for mixed samples, especially for batched samples such as sexual assault swabs, which commonly have very high female-to-male mixture ratios. Such samples, if included in a full run of 96 libraries, must compete for space on the flow cell for detection. The study presented here aimed to test the limits of MainstAY’s plexity by simultaneously running a variety of sample types and mixtures that would be typically encountered in forensic casework. The presentation will explore the trade-off of more rare genotypes due to isoalleles against detection challenges due to extreme plexity and competition for a spot on the flow cell. Additionally, properties unique to NGS such as sequence-specific stutter models will be explored. Last, the presentation will provide recommendations for optimal batching of samples for an efficient NGS case processing workflow. 

Consumer-mediated movement can couple food webs in distinct habitats and facilitate energy flow between them. In New England saltmarshes, mummichogs (Fundulus heteroclitus) connect the vegetated marsh and creek food webs by opportunistically foraging on the invertebrate communities of the marsh surface when access is permitted by tidal flooding and marsh-edge geomorphology. Via their movements, mummichog represent a critical food web node, as they can potentially transport energy from the marsh surface food web to creek food web and exert top-down control on the communities of the vegetated marsh surface. Here, I use gut content analysis, calorimetric analysis, and field surveys to demonstrate that access to the marsh surface (afforded by marsh-edge geomorphology) impacts the trophic relay of marsh production to creek food webs. Fish populations in creeks with greater connectivity had a higher total biomass of terrestrial invertebrates in their guts. However, bomb calorimetry showed no difference in the average caloric content of mummichog individuals from creeks with different creek edge geomorphology. Access also did not impact mummichog distribution across the marsh platform and exhibited no evidence of top-down control on their invertebrate prey. Thus, mummichogs function as initial nodes in the trophic relay, unidirectionally moving energy from the vegetated marsh to the creek food web. Reduced marsh surface access via altered marsh-edge geomorphology results in a 50 % to 66 % reduction in total energy available to aquatic predators via this route. Estuarine systems are intimately connected to coastal and offshore systems via consumer mediated flows of energy; thus, disruptions to the trophic relay from the marsh surface at the tidal creek scale can have far reaching impacts on secondary productivity in multiple disparate systems and must be accounted for in considerations of impacts to future food-web function. 

Gram-positive bacteria are some of the earliest known life forms, diverging from gram-negative bacteria 2 billion years ago. These organisms utilize sortase enzymes to attach proteins to their peptidoglycan cell wall, a structural feature that distinguishes the two types of bacteria. The transpeptidase activity of sortases make them an important tool in protein engineering applications, e.g., in sortase-mediated ligations or sortagging. However, due to relatively low catalytic efficiency, there are ongoing efforts to create better sortase variants for these uses. Here, we use bioinformatics tools, principal component analysis and ancestral sequence reconstruction, in combination with protein biochemistry, to analyze natural sequence variation in these enzymes. Principal component analysis on the sortase superfamily distinguishes previously described classes and identifies regions of relatively high sequence variation in structurally-conserved loops within each sortase family, including those near the active site. Using ancestral sequence reconstruction, we determined sequences of ancestral Staphylococcus and Streptococcus Class A sortase proteins. Enzyme assays revealed that the ancestral Streptococcus enzyme is relatively active and shares similar sequence variation with other Class A Streptococcus sortases. Taken together, we highlight how natural sequence variation can be utilized to investigate this important protein family, arguing that these and similar techniques may be used to discover or design sortases with increased catalytic efficiency and/or selectivity for sortase-mediated ligation experiments.