HudsonAlpha Institute for Biotechnology

Despite the growing interest in implementing genomic medicine into primary care, numerous barriers persist. Herein we present formative work conducted at three primary care clinics to identify barriers to implementation as part of a state-funded genomic medicine initiative. A convergent mixed-methods case study was conducted across three family practice clinics participating in the Alabama Genomic Health Initiative. Quantitative, cross-sectional surveys evaluated clinic personnel attitudes, implementation climate, and feasibility. Additionally, qualitative, semi-structured interviews examined clinic personnel's perceptions of genomic medicine implementation into routine care, identified barriers to this integration, and investigated strategies for overcoming these barriers. Respondents exhibited positive attitudes towards genomic medicine while voicing concerns regarding time constraints, technological integration, lack of actionable results for certain patients, and inadequate implementation knowledge. Although the primary care climate was generally supportive of change, it was less conducive to the specific implementation of genomic medicine. External facilitators and educational resources were identified as critical for successful implementation. Despite primary care personnel acknowledging the preventive and diagnostic value of genomic medicine, the effective integration of this practice requires practical solutions to barriers such as resource limitations and specialized training needs, indicating that enhanced infrastructure and focused educational initiatives could facilitate its implementation.

Abstract Peanut smut, caused by the fungus Thecaphora frezzii , is a significant disease of peanuts in Argentina. Infected plants have seeds replaced by a mass of dark teliospores, reducing yield and seed quality. To prevent the spread of the pathogen, several countries have limited import of raw peanuts from Argentina, a major grower and exporter. Following successful in vitro culture of the fungus in its haploid stage, we produced a chromosome-level genome assembly of the species for the first time. We compare this genome with those of 49 other species of true smut fungi, or Ustilaginomycetes, including species of medical, agricultural, and industrial importance, some of which are known as pathogens and others only as saprotrophic yeasts. At almost 39 Mb, T. frezzii has the largest genome of the smut fungi sequenced to date and the highest repetitive content. While it shares some core effectors with species of the distantly related and better studied Ustilago and related fungi, predicted effectors only found in T. frezzii or in Thecaphora suggest unique infection strategies. Comparisons among the 50 smut genomes also show that the 14 smut fungi observed only as yeasts share genomic traits such as low repeat content and generally smaller genomes. This supports the hypothesis that some smut fungi are adapted to saprotrophic growth as yeasts. The high-quality, annotated genome for T. frezzii will be a valuable resource for investigating the population dynamics and evolution of an economically important pathogen, as well as illuminating an understudied clade of smut fungi. Article summary Peanut smut is a destructive and costly disease of peanuts in Argentina. For the first time, a high quality, annotated genome is presented for the causal agent, Thecaphora frezzii . This fungus has the largest and most repetitive genome of the true smut fungi, thus prompting comparison with 49 other species of smut fungi with available genomes, including non-pathogenic ones. While it shares some likely pathogenicity factors with well-studied smuts, it has many unique genes, a trait reflective of its evolutionary distance and likely novel mechanism for infection. This important genomic resource will benefit research regarding the evolution and adaption of T. frezzii , the development of diagnostic tools that enable rapid detection of it, and the study of smut fungi broadly.

Protein truncating variants (PTVs) in SORL1 are observed almost exclusively in Alzheimer’s Disease (AD) cases, but the effect of rare SORL1 missense variants is unclear. To identify high-priority missense variants (HPVs), we applied ‘domain mapping of disease mutations’ for the 637 unique coding SORL1 variants detected in 18,959 AD-cases and 21,893 non-demented controls. In this sample, PTVs and HPVs associated with respectively a 35- and 10-fold increased risk of early onset AD and 17- and 6-fold increased risk of overall AD. The median age at onset (AAO) of PTV- and HPV-carriers was 62 and 64 years, and APOE-genotype contributed to AAO-variability. The median AAO of PTV- and HPV-carriers is ~8–10 years earlier than wild-type SORL1 carriers, matched for APOE-genotype. Specific HPVs are highly penetrant and lead to earlier AAOs than PTVs, suggesting possible dominant negative effects. Our results justify a debate on whether HPV carriers should be considered for clinical counseling.

Spike-in normalization enables quantitative analysis of ChIP-sequencing (ChIP-seq) signal. Here we introduce a novel robust dual-spike-in normalization approach for ChIP-seq (ChIP-wrangler). We identify optimal conditions, such as the ratio between the spike-in species and the target, demonstrate the ability of this approach to detect technical artefacts, and use ChIP-wrangler to revisit recent claims that active histone marks are dependent on transcription. Concerned that previous studies improperly used spike-in normalization to arrive at their conclusions, we used ChIP-wrangler to show that acute depletion of RNA polymerase II (RNAPII) has only a modest impact on the levels of H3K4me3 and H3K27ac. In line with other studies, our results provide proof that the maintenance of histone acetylation is not merely a consequence of ongoing transcription. Further, we show that promoters and enhancers are differentially impacted by inhibiting transcription. Specifically, of the 5.9% peaks that showed a decrease in H3K27ac following depletion of RNAPII, 82% are promoter-distal and contain enhancer-related DNA binding motifs. Further, the small subset of regions that gain acetylation (0.35%) were enriched for stress response motifs. Our innovative ChIP-seq normalization approach provides increased rigor and “guardrails” for successful spike-in normalization, and as applied here refines the understanding of the intricate crosstalk between RNAPII activity and histone marks associated with transcription.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to study the genomic localization of DNA-associated proteins. However, conventional protocols include multiple manual steps that can introduce inconsistency and limit scalability, thereby restricting the inclusion of appropriate replicates and controls. Although the introduction of liquid handling platforms has improved reproducibility, most existing efforts have automated only a subset of the workflow, and extending automation to efficiently map non-histone proteins, such as chromatin regulators, remains challenging. Here, we present a fully automated implementation of our previously developed single-pot ChIP-seq protocol (Texari et al. 2021), named spa-ChIP-seq, which enables scalable processing of 8 to 96 ChIP-seq samples from crosslinked cells to sequencing-ready library in approximately three days with an estimated cost of $70 per sample. Benchmarking spa-ChIP-seq against manual ChIP-seq performed in parallel demonstrates comparable signal-to-noise ratio between the two workflows. Using spa-ChIP-seq, we systematically evaluate multiple parameters including shearing and crosslinking conditions, buffer compositions, and the ratio of antibody to cell-number. We find, for the first time to our knowledge, that weaker genomic localization signals are sensitive to changing the antibody to cell-number ratio, whereas the stronger signals remain unaffected. This finding underscores the importance of maintaining consistent antibody-to-cell-number ratio for comparative studies, such as treatment responses or chromatin-QTL mapping. The spa-ChIP-seq protocol is publicly available, including deck setups, operational parameters, and scripts. We envision that this robust, cost-efficient protocol will facilitate high-throughput, reproducible ChIP-seq analyses, supporting large-scale studies of antibody validation, compound screening, population genomics, and diagnostic frameworks.

Tall fescue toxicosis, caused by ingestion of endophyte-infected tall fescue (Lolium arundinaceum), impairs growth and reproduction in beef cattle and results in over USD 3 billion annual loss to the U.S. livestock industry. While the effects on host metabolism and rumen function have been described, the impact on the rectal microbiome remains poorly understood. In this study, we performed whole-genome shotgun metagenomic sequencing on fecal samples collected before and after a 30-day toxic fescue seed supplementation from eight pregnant Angus × Simmental cows and heifers. We generated 157 Gbp of sequencing data in 16 metagenomes, and assembled 13.1 Gbp de novo microbial contigs, identifying 22 million non-redundant microbial genes from the cattle rectum microbiome. Fescue toxicosis significantly reduced alpha diversity (p < 0.01) and altered beta diversity (PERMANOVA p < 0.01), indicating microbial dysbiosis. We discovered significant enrichment of 31 bacterial species post-treatment, including multiple core rumen taxa. Ruminococcaceae bacterium P7 showed an average of 16-fold increase in fecal abundance (p < 0.01), making it the top-featured species in linear discriminant analysis. Functional pathway analysis revealed a shift from energy metabolism to antimicrobial resistance and DNA replication following toxic seed consumption. Comparative analysis showed increased representation of core rumen taxa in rectal microbiota post-treatment, suggesting disrupted rumen function. These findings demonstrate that fescue toxicosis alters both the composition and functional landscape of the hindgut microbiota. Ruminococcaceae bacterium P7 emerges as a promising biomarker for monitoring fescue toxicosis through non-invasive fecal sampling, with potential applications in herd-level diagnostics and mitigation strategies.

Abstract While graph-based pangenomes have become a standard and interoperable foundation for comparisons across multiple reference genomes, integrating protein-coding gene annotations across pangenomes in a single ‘pangene set’ remains challenging, both because of methodological inconsistency and biological presence-absence variation (PAV). Here, we review and experimentally evaluate the root of genome annotation and pangene set inconsistency using two polyploid plant pangenomes: cotton and soybean, which were chosen because of their existing diverse high-quality genomic resources and the known importance of gene presence-absence variation in their respective breeding programs. We first demonstrate that building pangene sets across different genome resources is highly error prone: PAV calculated directly from the genome annotations hosted on public repositories recapitulates structure in annotation methods and not biological sequence differences. Re-annotation of all genomes with a single identical pipeline largely resolves the broadest stroke issues; however, substantial challenges remain, including a surprisingly common case where exactly identical sequences have different gene model structural annotations. Combined, these results clearly show that pangenome gene model annotations must be carefully integrated before any biological inference can be made regarding sequence evolution, gene copy-number, or presence-absence variation.

There is increasing evidence that the methyl-binding domain (MBD) is a protein-protein interaction motif that can function independently of methylated DNA binding. The MBD proteins found throughout plants and invertebrates duplicated into multiple vertebrate DNA and non-DNA-binding members (MBD1, MBD2, MBD3, MBD4, MBD5, MBD6, MECP2, BAZ2A, BAZ2B, SETDB1, and SETDB2). Although many invertebrate species possess MBD proteins that can bind and recognize DNA methylation, the DNA-binding function has been independently lost multiple times, with only minor alterations to the protein interaction residues. The nucleosome remodeling and deacetylase (NuRD) complex, which interacts with MBD2/3 and is colocalized with MBD1/4 ChIP-Seq, is maintained in species where MBD2/3 cannot bind to DNA. NuRD ChIP-seq data from HepG2 cell line, human induced pluripotent stem cells (iPSCs), and human iPSC-derived liver cells suggest that the NuRD complex is highly localized to nonmethylated CpG-rich housekeeping gene promoter elements, which are essential in organogenesis and maintained within the Drosophila melanogaster MBD2/3 non-DNA-binding system. Integration of MBD interaction proteins and NuRD gene expression from >115 million cells of single-cell RNA-seq, along with thousands of bulk tissue profiles, highlights a critical role of MBD3, MECP2, and GATAD2B in brain development and intellectual disability syndromes that is maintained throughout invertebrate neural development and likely involves evolutionary expanded entanglement as the vertebrate MBD proteins expanded. This work suggests that MBD has a largely unexplored role as a critical protein interaction motif that is evolutionarily conserved for regulating enhancers and promoters.NEW & NOTEWORTHY The evolution of the methyl-binding domain (MBD) suggests a shared function in gene regulation, from plants to humans, with the conservation of non-DNA-interacting amino acids critical for protein-protein interactions. The MBD-regulated NuRD complex localizes to promoters of housekeeping genes with hypomethylated CpG islands. Expression profiles suggest a shared role for NuRD complex components in neurodevelopment, where the MBD3 and GATAD2B subcomplex of NuRD may be underexplored due to its non-DNA-binding biology.