CyberSec Research

Sequencing of PCR amplicons generated using degenerate primers (typically targeting a region of the 16S ribosomal gene) is widely used in metagenomics to profile the taxonomic composition of complex microbial samples. To reduce taxonomic biases in primer selection it is important to conduct in silico PCR analyses of the primers against large collections of up to millions of bacterial genomes. However, existing in silico PCR tools have impractical running time for analyses of this scale. In this paper we introduce AmpliconHunter, a highly scalable in silico PCR package distributed as open-source command-line tool and publicly available through a user-friendly web interface at https://ah1.engr.uconn.edu/. AmpliconHunter implements an accurate nearest-neighbor model for melting temperature calculations, allowing for primer-template hybridization with mismatches, along with three complementary methods for estimating off-target amplification. By taking advantage of multi-core parallelism and SIMD operations available on modern CPUs, the AmpliconHunter web server can complete in silico PCR analyses of commonly used degenerate primer pairs against the 2.4M genomes in the latest AllTheBacteria collection in as few as 6-7 hours.

Summary: Uchimata is a toolkit for visualization of 3D structures of genomes. It consists of two packages: a Javascript library facilitating the rendering of 3D models of genomes, and a Python widget for visualization in Jupyter Notebooks. Main features include an expressive way to specify visual encodings, and filtering of 3D genome structures based on genomic semantics and spatial aspects. Uchimata is designed to be highly integratable with biological tooling available in Python. Availability and Implementation: Uchimata is released under the MIT License. The Javascript library is available on NPM, while the widget is available as a Python package hosted on PyPI. The source code for both is available publicly on Github (https://github.com/hms-dbmi/uchimata and https://github.com/hms-dbmi/uchimata-py). The documentation with examples is hosted at https://hms-dbmi.github.io/uchimata/ Contact: david_kouril@hms.harvard.edu or nils@hms.harvard.edu.

Summary: Microbiome HiFi Amplicon Sequence Simulator (MHASS) creates realistic synthetic PacBio HiFi amplicon sequencing datasets for microbiome studies, by integrating genome-aware abundance modeling, realistic dual-barcoding strategies, and empirically derived pass-number distributions from actual sequencing runs. MHASS generates datasets tailored for rigorous benchmarking and validation of long-read microbiome analysis workflows, including ASV clustering and taxonomic assignment. Availability and Implementation: Implemented in Python with automated dependency management, the source code for MHASS is freely available at https://github.com/rhowardstone/MHASS along with installation instructions. Contact: rye.howard-stone@uconn.edu or ion.mandoiu@uconn.edu Supplementary information: Supplementary data are available online at https://github.com/rhowardstone/MHASS_evaluation.

TRPM4 is overexpressed in prostate cancer (PCa) associated with metastasis or recurrence. There is paucity of information pertaining to TRPM4 characterization and functions at single-cell level in PCa. In this study, generalized additive model (GAM) was utilized to model the relationship between TRPM4 and genes shortlisted using Spearman-Kendall dual-filter in aggressive PCa and benign prostate (BP) control cells derived from scRNA-seq dataset. Seven ribosomal genes (RPL10, RPL27, RPL28, RPS2, RPS8, RPS12, and RPS26; averaged into Ribo as the gene set), passed the dual-filter specifically in PCa cells. GAM modeling of TRPM4-Ribo significantly outperformed TRPM4 modeling with alternative cancer gene sets (GSK-3B, mTOR, NF-KB, PI3K/AKT, and Wnt). Cell explanatory power (CEP) classification was devised and verified by cross-validation to identify individual PCa cells most well-predicted by the model. CEP classification binarized PCa cells into top-ranked explanatory power (TREP; more well-predicted by the model) and non-TREP cells. In TRPM4-Ribo GAM plots, distribution pattern of TREP cells shifted at an inflection point (IP) i.e., the specific TRPM4 expression value that further binarized the plot into pre-IP (TRPM4 values below IP) and post-IP (TRPM4 values above IP) regions, producing a quadrant of TREP versus non-TREP cells for each PCa patient. Gene Ontology (GO) enrichment analysis showed that pre-IP TREP cells enriched for immune-related GOs, while post-IP TREP cells enriched for ribosomal, translation, and cell adhesion GOs. In conclusion, the CEP-IP framework based on pairwise genes produces quadrants of cancer cell subpopulations, enabling the identification of distinctive biology with potential therapeutic implications.

We introduce Genome-Factory, an integrated Python library for tuning, deploying, and interpreting genomic models. Our core contribution is to simplify and unify the workflow for genomic model development: data collection, model tuning, inference, benchmarking, and interpretability. For data collection, Genome-Factory offers an automated pipeline to download genomic sequences and preprocess them. It also includes quality control, such as GC content normalization. For model tuning, Genome-Factory supports three approaches: full-parameter, low-rank adaptation, and adapter-based fine-tuning. It is compatible with a wide range of genomic models. For inference, Genome-Factory enables both embedding extraction and DNA sequence generation. For benchmarking, we include two existing benchmarks and provide a flexible interface for users to incorporate additional benchmarks. For interpretability, Genome-Factory introduces the first open-source biological interpreter based on a sparse auto-encoder. This module disentangles embeddings into sparse, near-monosemantic latent units and links them to interpretable genomic features by regressing on external readouts. To improve accessibility, Genome-Factory features both a zero-code command-line interface and a user-friendly web interface. We validate the utility of Genome-Factory across three dimensions: (i) Compatibility with diverse models and fine-tuning methods; (ii) Benchmarking downstream performance using two open-source benchmarks; (iii) Biological interpretation of learned representations with DNABERT-2. These results highlight its end-to-end usability and practical value for real-world genomic analysis.

Differentiating between the two main subtypes of Inflammatory Bowel Disease (IBD): Crohns disease (CD) and ulcerative colitis (UC) is a persistent clinical challenge due to overlapping presentations. This study introduces a novel computational framework that employs spatial transcriptomics (ST) to create an explainable machine learning model for IBD classification. We analyzed ST data from the colonic mucosa of healthy controls (HC), UC, and CD patients. Using Non-negative Matrix Factorization (NMF), we first identified four recurring cellular niches, representing distinct functional microenvironments within the tissue. From these niches, we systematically engineered 44 features capturing three key aspects of tissue pathology: niche composition, neighborhood enrichment, and niche-gene signals. A multilayer perceptron (MLP) classifier trained on these features achieved an accuracy of 0.774 +/- 0.161 for the more challenging three-class problem (HC, UC, and CD) and 0.916 +/- 0.118 in the two-class problem of distinguishing IBD from healthy tissue. Crucially, model explainability analysis revealed that disruptions in the spatial organization of niches were the strongest predictors of general inflammation, while the classification between UC and CD relied on specific niche-gene expression signatures. This work provides a robust, proof-of-concept pipeline that transforms descriptive spatial data into an accurate and explainable predictive tool, offering not only a potential new diagnostic paradigm but also deeper insights into the distinct biological mechanisms that drive IBD subtypes.

The gene set analysis (GSA) is a foundational approach for uncovering the molecular functions associated with a group of genes. Recently, LLM-powered methods have emerged to annotate gene sets with biological functions together with coherent explanatory insights. However, existing studies primarily focus on proprietary models, which have been shown to outperform their open-source counterparts despite concerns over cost and data privacy. Furthermore, no research has investigated the application of advanced reasoning strategies to the GSA task. To address this gap, we introduce Gene-R1, a data-augmented learning framework that equips lightweight and open-source LLMs with step-by-step reasoning capabilities tailored to GSA. Experiments on 1,508 in-distribution gene sets demonstrate that Gene-R1 achieves substantial performance gains, matching commercial LLMs. On 106 out-of-distribution gene sets, Gene-R1 performs comparably to both commercial and large-scale LLMs, exhibiting robust generalizability across diverse gene sources.

Motivation: Low-complexity (LC) DNA sequences are compositionally repetitive sequences that are often associated with increased variant density and variant calling artifacts. While algorithms for identifying LC sequences exist, they either lack rigorous mathematical foundation or are inefficient with long context windows. Results: Longdust is a new algorithm that efficiently identifies long LC sequences including centromeric satellite and tandem repeats with moderately long motifs. It defines string complexity by statistically modeling the k-mer count distribution with the parameters: the k-mer length, the context window size and a threshold on complexity. Longdust exhibits high performance on real data and high consistency with existing methods. Availability and implementation: https://github.com/lh3/longdust

As synthetic genomics scales toward the construction of increasingly larger genomes, computational strategies are needed to address technical feasibility. We introduce an algorithmic framework for the Minimum-Cost Synthetic Genome Planning problem, aiming to identify the most cost-effective strategy to assemble a target genome from a source genome through a combination of reuse, synthesis, and join operations. By comparing dynamic programming and greedy heuristic strategies under diverse cost regimes, we demonstrate how algorithmic choices influence the cost-efficiency of large-scale genome construction. In parallel, solving the Minimum-Cost Synthetic Genome Planning problem can help us better understand genome architecture and evolution. We applied our framework in case studies on viral genomes, including SARS-CoV-2, to examine how source-target genome similarity shapes construction costs. Our analyses revealed that conserved regions such as ORF1ab can be reconstructed cost-effectively from related templates, while highly variable regions such as the S (spike) gene are more reliant on DNA synthesis, highlighting the biological and economic trade-offs of genome design.

The frequency distributions of DNA k-mers are shaped by fundamental biological processes and offer a window into genome structure and evolution. Inspired by analogies to natural language, prior studies have attempted to model genomic k-mer usage using Zipf's law, a rank-frequency law originally formulated for words in human language. However, the extent to which this law accurately captures the distribution of k-mers across diverse species remains unclear. Here, we systematically analyze k-mer frequency spectra across more than 225,000 genome assemblies spanning all three domains of life and viruses. We demonstrate that Zipf's law consistently underperforms in modeling k-mer distributions. In contrast, we propose the truncated power law and Zipf-Mandelbrot distributions, which provide substantially improved fits across taxonomic groups. We show that genome size and GC content influence model performance, with larger and GC-content imbalanced genomes yielding better adherence. Additionally, we perform an extensive analysis on vocabulary expansion and exhaustion across the same organisms using Heaps' law. We apply our modeling framework to evaluate simulated genomes generated by k-let preserving shuffling and deep generative language models. Our results reveal substantial differences between organismal genomes and their synthetic or shuffled counterparts, offering a novel approach to benchmark the biological plausibility of artificial genomes. Collectively, this work establishes new standards for modeling genomic k-mer distributions and provides insights relevant to synthetic biology, and evolutionary sequence analysis.

Prediction of patient survival using high-dimensional multi-omics data requires systematic feature selection methods that ensure predictive performance, sparsity, and reliability for prognostic biomarker discovery. We developed a hybrid ensemble feature selection (hEFS) approach that combines data subsampling with multiple prognostic models, integrating both embedded and wrapper-based strategies for survival prediction. Omics features are ranked using a voting-theory-inspired aggregation mechanism across models and subsamples, while the optimal number of features is selected via a Pareto front, balancing predictive accuracy and model sparsity without any user-defined thresholds. When applied to multi-omics datasets from three pancreatic cancer cohorts, hEFS identifies significantly fewer and more stable biomarkers compared to the conventional, late-fusion CoxLasso models, while maintaining comparable discrimination performance. Implemented within the open-source mlr3fselect R package, hEFS offers a robust, interpretable, and clinically valuable tool for prognostic modelling and biomarker discovery in high-dimensional survival settings.

Single-cell RNA sequencing (scRNA-seq) provides unprecedented insights into cellular heterogeneity, enabling detailed analysis of complex biological systems at single-cell resolution. However, the high dimensionality and technical noise inherent in scRNA-seq data pose significant analytical challenges. While current embedding methods focus primarily on gene expression levels, they often overlook crucial gene-gene interactions that govern cellular identity and function. To address this limitation, we present a novel embedding approach that integrates both gene expression profiles and data-driven gene-gene interactions. Our method first constructs a Cell-Leaf Graph (CLG) using random forest models to capture regulatory relationships between genes, while simultaneously building a K-Nearest Neighbor Graph (KNNG) to represent expression similarities between cells. These graphs are then combined into an Enriched Cell-Leaf Graph (ECLG), which serves as input for a graph neural network to compute cell embeddings. By incorporating both expression levels and gene-gene interactions, our approach provides a more comprehensive representation of cellular states. Extensive evaluation across multiple datasets demonstrates that our method enhances the detection of rare cell populations and improves downstream analyses such as visualization, clustering, and trajectory inference. This integrated approach represents a significant advance in single-cell data analysis, offering a more complete framework for understanding cellular diversity and dynamics.

Single-cell technologies provide an unprecedented opportunity for dissecting the interplay between the cancer cells and the associated tumor microenvironment, and the produced high-dimensional omics data should also augment existing survival modeling approaches for identifying tumor cell type-specific genes predictive of cancer patient survival. However, there is no statistical model to integrate multiscale data including individual-level survival data, multicellular-level cell composition data and cellular-level single-cell omics covariates. We propose a class of Bayesian generalized promotion time cure models (GPTCMs) for the multiscale data integration to identify cell-type-specific genes and improve cancer prognosis. We demonstrate with simulations in both low- and high-dimensional settings that the proposed Bayesian GPTCMs are able to identify cell-type-associated covariates and improve survival prediction.

Understanding the modular structure and central elements of complex biological networks is critical for uncovering system-level mechanisms in disease. Here, we constructed weighted gene co-expression networks from bulk RNA-seq data of rheumatoid arthritis (RA) synovial tissue, using pairwise correlation and a percolation-guided thresholding strategy. Community detection with Louvain and Leiden algorithms revealed robust modules, and node-strength ranking identified the top 50 hub genes globally and within communities. To assess novelty, we integrated genome-wide association studies (GWAS) with literature-based evidence from PubMed, highlighting five high-centrality genes with little to no prior RA-specific association. Functional enrichment confirmed their roles in immune-related processes, including adaptive immune response and lymphocyte regulation. Notably, these hubs showed strong positive correlations with T- and B-cell markers and negative correlations with NK-cell markers, consistent with RA immunopathology. Overall, our framework demonstrates how correlation-based network construction, modularity-driven clustering, and centrality-guided novelty scoring can jointly reveal informative structure in omics-scale data. This generalizable approach offers a scalable path to gene prioritization in RA and other autoimmune conditions.

Accurate prediction of virus-host interactions is critical for understanding viral ecology and developing applications like phage therapy. However, the growing number of computational tools has created a complex landscape, making direct performance comparison challenging due to inconsistent benchmarks and varying usability. Here, we provide a systematic review and a rigorous benchmark of 27 virus-host prediction tools. We formulate the host prediction task into two primary frameworks, link prediction and multi-class classification, and construct two benchmark datasets to evaluate tool performance in distinct scenarios: a database-centric dataset (RefSeq-VHDB) and a metagenomic discovery dataset (MetaHiC-VHDB). Our results reveal that no single tool is universally optimal. Performance is highly context-dependent, with tools like CHERRY and iPHoP demonstrating robust, broad applicability, while others, such as RaFAH and PHIST, excel in specific contexts. We further identify a critical trade-off between predictive accuracy, prediction rate, and computational cost. This work serves as a practical guide for researchers and establishes a standardized benchmark to drive future innovation in deciphering complex virus-host interactions.

Generative modeling of discrete variables is challenging yet crucial for applications in natural language processing and biological sequence design. We introduce the Shortlisting Model (SLM), a novel simplex-based diffusion model inspired by progressive candidate pruning. SLM operates on simplex centroids, reducing generation complexity and enhancing scalability. Additionally, SLM incorporates a flexible implementation of classifier-free guidance, enhancing unconditional generation performance. Extensive experiments on DNA promoter and enhancer design, protein design, character-level and large-vocabulary language modeling demonstrate the competitive performance and strong potential of SLM. Our code can be found at https://github.com/GenSI-THUAIR/SLM

Single-cell multi-omics data contain huge information of cellular states, and analyzing these data can reveal valuable insights into cellular heterogeneity, diseases, and biological processes. However, as cell differentiation \& development is a continuous and dynamic process, it remains challenging to computationally model and infer cell interaction patterns based on single-cell multi-omics data. This paper presents scI2CL, a new single-cell multi-omics fusion framework based on intra- and inter-omics contrastive learning, to learn comprehensive and discriminative cellular representations from complementary multi-omics data for various downstream tasks. Extensive experiments of four downstream tasks validate the effectiveness of scI2CL and its superiority over existing peers. Concretely, in cell clustering, scI2CL surpasses eight state-of-the-art methods on four widely-used real-world datasets. In cell subtyping, scI2CL effectively distinguishes three latent monocyte cell subpopulations, which are not discovered by existing methods. Simultaneously, scI2CL is the only method that correctly constructs the cell developmental trajectory from hematopoietic stem and progenitor cells to Memory B cells. In addition, scI2CL resolves the misclassification of cell types between two subpopulations of CD4+ T cells, while existing methods fail to precisely distinguish the mixed cells. In summary, scI2CL can accurately characterize cross-omics relationships among cells, thus effectively fuses multi-omics data and learns discriminative cellular representations to support various downstream analysis tasks.

Understanding which genes control which traits in an organism remains one of the central challenges in biology. Despite significant advances in data collection technology, our ability to map genes to traits is still limited. This genome-to-phenome (G2P) challenge spans several problem domains, including plant breeding, and requires models capable of reasoning over high-dimensional, heterogeneous, and biologically structured data. Currently, however, many datasets solely capture genetic information or solely capture phenotype information. Additionally, phenotype data is very heterogeneous, which many datasets do not fully capture. The critical drawback is that these datasets are not integrated, that is, they do not link with each other to describe the same biological specimens. This limits machine learning models' ability to be informed on the various aspects of these specimens, impacting the breadth of correlations learned, and therefore their ability to make more accurate predictions. To address this gap, we present the Arabidopsis Genomics-Phenomics (AGP) Dataset, a curated multi-modal dataset linking gene expression profiles with phenotypic trait measurements in Arabidopsis thaliana, a model organism in plant biology. AGP supports tasks such as phenotype prediction and interpretable graph learning. In addition, we benchmark conventional regression and explanatory baselines, including a biologically-informed hypergraph baseline, to validate gene-trait associations. To the best of our knowledge, this is the first dataset that provides multi-modal gene information and heterogeneous trait or phenotype data for the same Arabidopsis thaliana specimens. With AGP, we aim to foster the research community towards accurately understanding the connection between genotypes and phenotypes using gene information, higher-order gene pairings, and trait data from several sources.