Elena Buena-Atienza

Spinocerebellar ataxia type 3 (SCA3), also called Machado-Joseph disease, the most common form of autosomal dominant cerebellar ataxia, is caused by a CAG repeat expansion in ATXN3 . The mutant protein is prone to aggregation and exerts toxic gain-of-function effects, whereas the wild-type protein has important neuroprotective roles as a deubiquitinase. Current treatment strategies for SCA3 lack specificity for the mutant allele. Identifying genetic variants such as single-nucleotide polymorphisms (SNPs) in linkage with the pathogenic CAG expansion offers a promising avenue to develop allele-specific therapeutic interventions, like antisense oligonucleotides (ASOs). To address this, we conducted allele-specific, targeted, multiplexed nanopore sequencing on 79 patients with SCA3 from Germany, UK, and Portugal. We identified almost 200 polymorphisms and grouped them into three haplotype blocks on the basis of linkage disequilibrium. These SNPs were strongly associated with the CAG expansion. Genotypic modeling of our subcohorts indicated that targeting only two SNPs from different blocks could enable allele-specific treatment of the majority of European patients with SCA3. We evaluated 17 candidate ASOs targeting SNPs from different haplotype blocks. Seven of these ASOs caused allele-preferential reduction of more than 65%. A single ASO application in two SCA3 iPSC-derived cortical neurons (AX1, AX2) achieved up to 90% reduction of mutant ATXN3. Toxicity assessments together with RNA sequencing analyses in AX1 did not indicate evidence of toxicity or widespread transcriptional perturbations. These findings highlight that characterizing heterozygosity rates offers valuable opportunities for selective ASO therapies. Our proof-of-principle targeting with ASOs demonstrates the feasibility of haplotype-informed allele-specific silencing in ATXN3 .

Background: DNA methylation can be profiled using multiple technologies that vary in resolution, coverage and cost. Yet systematic benchmarks across these methods remain scarce. Methods: We compared six widely used technologies — Illumina EPIC array, TWIST, Whole-Genome Enzymatic Conversion (WGEC), Reduced Representation Bisulfite Sequencing (RRBS), long-read genome sequencing (LR-GS) with Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) — using Genome in a Bottle (GIAB) reference samples and ten samples derived of blood and fibroblast cultures of 5 individuals. We assessed CpG coverage, consistency of differentially methylated cytosine (DMC) detection and genomic annotation, with particular attention to overlapping signals across assays. Results: Despite major differences in assay design, all technologies consistently identified DMCs enriched in promoter and intronic regions, highlighting these loci as robust hotspots of epigenetic variability. Annotation redundancy strongly influenced initial interpretations, with CpG island–related categories largely disappearing once annotations were collapsed to unique features. Sequencing-based methods (WGEC, TWIST, ONT) achieved the most comprehensive coverage, whereas EPIC arrays reproducibly captured promoter-associated differences despite limited scope. ONT sequencing enabled direct, long-read–based methylation profiling with phasing capability and showed strong concordance with short-read sequencing methods after coverage filtering, but required higher and more uniform coverage to achieve reproducible CpG-level agreement. PacBio methylation profiles showed a coverage-dependent discrepancy, with cross-platform concordance plateauing in GIAB samples despite high mean coverage, indicating residual technology-specific biases beyond simple coverage effects. Conclusions: Cross-platform benchmarking yields coherent biological insights when coverage and annotation redundancies are carefully addressed. Practically, EPIC arrays remain valuable for promoter-focused cohort studies, WGEC and TWIST enable genome-wide discovery and ONT provides unique phasing and multimodal potential. This comparative framework can guide method selection and support more robust interpretation of DNA methylation data across diverse platforms.

Long-read RNA sequencing has transformed transcriptome analysis by enabling comprehensive mapping of full-length transcripts, providing an unprecedented resolution of transcript diversity, alternative splicing and transcript-specific regulation. In this study, we employed nanopore long-read RNA sequencing to profile the transcriptomes of three cell types commonly used to model brain disorders, human fibroblasts, induced pluripotent stem cells and stem cell-derived cortical neurons, identifying extensive transcript diversity with 15 072 transcripts in stem cell-derived cortical neurons, 13 048 in fibroblasts and 12 759 in induced pluripotent stem cells. Our analyses uncovered 35 519 differential transcript expression events and 5135 differential transcript usage events, underscoring the complexity of transcriptomic regulation across these cell types. Importantly, by integrating differential transcript expression and usage analyses, we gained deeper insights into transcript dynamics that are not captured by gene-level expression analysis alone. Differential transcript usage analysis highlighted transcript-specific changes in disease-relevant genes such as APP , KIF2A and BSCL2 , associated with Alzheimer’s disease, neuronal migration disorders and degenerative axonopathies, respectively. This added resolution emphasizes the significance of transcript-level variations that often remain hidden in traditional differential gene expression analyses. Overall, our work provides a framework for understanding transcript diversity in both pluripotent and specialized cell types, which can be used to investigate transcriptomic changes in disease states in future work. Additionally, this study underscores the utility of differential transcript usage analysis in advancing our understanding of neurodevelopmental and neurodegenerative diseases, paving the way for identifying transcript-specific therapeutic targets.

Background Soil ecosystems have long been recognized as hotspots of microbial diversity, but most estimates of their complexity remain speculative, relying on limited data and extrapolation from shallow sequencing. Here, we revisit this question using one of the deepest metagenomic sequencing efforts to date, applying 148 Gbp of Nanopore long-read and 122 Gbp of Illumina short-read data to a single forest soil sample. Results Our hybrid assembly reconstructed 837 metagenome-assembled genomes (MAGs), including 466 high- and medium-quality genomes, nearly all lacking close relatives among cultivated taxa. Rarefaction and k-mer analyses reveal that, even at this depth, we capture only a fraction of the extant diversity: nonparametric models project that over 10 Tbp would be required to approach saturation. These findings offer a quantitative, technology-enabled update to long-standing diversity estimates and demonstrate that conventional metagenomic sequencing efforts likely miss the majority of microbial and biosynthetic potential in soil. We further identify over 11,000 biosynthetic gene clusters (BGCs), >99% of which have no match in current databases, underscoring the breadth of unexplored metabolic capacity. Conclusions Taken together, our results emphasize both the power and the present limitations of metagenomics in resolving natural microbial complexity, and they provide a new baseline for evaluating future advances in microbial genome recovery, taxonomic classification, and natural product discovery.

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder characterized by ataxia, sensory loss and pyramidal signs. While the majority of FRDA cases are caused by biallelic GAA trinucleotide repeat expansions in intron 1 of FXN, there is a subset of patients harboring a heterozygous pathogenic small variant compound-heterozygous with a GAA repeat expansion. We report on the diagnostic journey of a 21-year-old patient who was clinically suspected of having FRDA at the age of 12 years. Genetic testing included fragment analysis, gene panel analysis and exome sequencing, which only detected one pathogenic heterozygous missense variant (c.389 G>T,p.Gly130Val) in FXN. Although conventional repeat analyses failed to detect GAA expansions in our patient, subsequent short-read genome sequencing (GS) indicated a potential GAA repeat expansion. This finding was confirmed by long-read GS, which in addition revealed a complex pattern of interruptions. Both large and small GAA expansions with divergent interruptions containing G, A, GA, GAG and/or GAAG sequences were present within one allele, indicating mosaic sequence variations. Our findings underscore the complexity of repeat expansions which can exhibit both interruptions and somatic instability. We also highlight the utility of long-read GS in unraveling intricate genetic profiles, ultimately contributing to more accurate diagnoses in clinical practice.

Despite linkage to chromosome 16q in 1996, the mutation causing spinocerebellar ataxia type 4 (SCA4), a late-onset sensory and cerebellar ataxia, remained unknown. Here, using long-read single-strand whole-genome sequencing (LR-GS), we identified a heterozygous GGC-repeat expansion in a large Utah pedigree encoding polyglycine (polyG) in zinc finger homeobox protein 3 (ZFHX3), also known as AT-binding transcription factor 1 (ATBF1). We queried 6,495 genome sequencing datasets and identified the repeat expansion in seven additional pedigrees. Ultrarare DNA variants near the repeat expansion indicate a common distant founder event in Sweden. Intranuclear ZFHX3–p62–ubiquitin aggregates were abundant in SCA4 basis pontis neurons. In fibroblasts and induced pluripotent stem cells, the GGC expansion led to increased ZFHX3 protein levels and abnormal autophagy, which were normalized with small interfering RNA-mediated ZFHX3 knockdown in both cell types. Improving autophagy points to a therapeutic avenue for this novel polyG disease. The coding GGC-repeat expansion in an extremely G+C-rich region was not detectable by short-read whole-exome sequencing, which demonstrates the power of LR-GS for variant discovery.

A global genome database of all of Earth's species diversity could be a treasure trove of scientific discoveries. However, regardless of the major advances in genome sequencing technologies, only a tiny fraction of species have genomic information available. To contribute to a more complete planetary genomic database, scientists and institutions across the world have united under the Earth BioGenome Project (EBP), which plans to sequence and assemble high-quality reference genomes for all ~1.5 million recognized eukaryotic species through a stepwise phased approach. As the initiative transitions into Phase II, where 150,000 species are to be sequenced in just four years, worldwide participation in the project will be fundamental to success. As the European node of the EBP, the European Reference Genome Atlas (ERGA) seeks to implement a new decentralised, accessible, equitable and inclusive model for producing high-quality reference genomes, which will inform EBP as it scales. To embark on this mission, ERGA launched a Pilot Project to establish a network across Europe to develop and test the first infrastructure of its kind for the coordinated and distributed reference genome production on 98 European eukaryotic species from sample providers across 34 European countries. Here we outline the process and challenges faced during the development of a pilot infrastructure for the production of reference genome resources, and explore the effectiveness of this approach in terms of high-quality reference genome production, considering also equity and inclusion. The outcomes and lessons learned during this pilot provide a solid foundation for ERGA while offering key learnings to other transnational and national genomic resource projects.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease that mainly affects boys due to X-linked recessive inheritance. In most affected individuals, MLPA or sequencing-based techniques detect deletions, duplications, or point mutations in the dystrophin-encoding DMD gene. However, in a small subset of patients clinically diagnosed with DMD, the molecular cause is not identified with these routine methods. Evaluation of the 60 DMD patients in our center revealed three cases without a known genetic cause. DNA samples of these patients were analyzed using whole-exome sequencing (WES) and, if unconclusive, optical genome mapping (OGM). WES led to a diagnosis in two cases: one patient was found to carry a splice mutation in the DMD gene that had not been identified during previous Sanger sequencing. In the second patient, we detected two variants in the fukutin gene (FKTN) that were presumed to be disease-causing. In the third patient, WES was unremarkable, but OGM identified an inversion disrupting the DMD gene (~1.28 Mb) that was subsequently confirmed with long-read sequencing. These results highlight the importance of reanalyzing unsolved cases using WES and demonstrate that OGM is a useful method for identifying large structural variants in cases with unremarkable exome sequencing.

Ants are a highly diversified insect family of the order Hymenoptera, with many fascinating characteristics such as eusociality, chemical communication, farming, or social parasitism. Moreover, ants frequent a wide variety of habitats from dry deserts, grasslands, and savannas to cold temperate forests. The ability of ants to inhabit such diverse habitat ranges demonstrates their adaptability and ecological resilience. However, little is known about the genetic underpinnings of this vast array of traits and their adaptive potential. Here, we generated a high-quality genome assembly for the ant species Lasius platythorax using long-read PacBio HiFi in combination with chromatin conformation capture (Hi-C) sequencing. We successfully assembled the genome into 15 chromosome-level scaffolds ranging from 7.9 to 19.2 Mb and encompassing 204.6 Mb out of 235.3 Mb (total assembly), and a BUSCO score of 86% (Hymenoptera_odb10). Comparative genomics between the two sister species will provide insights into the genomic basis of trait differentiation.