Genome Biology最新文献

Background: ALK gene fusions are key oncogenic drivers in cancers such as non-small cell lung cancer, where they define a molecular subtype responsive to ALK tyrosine kinase inhibitors (TKIs). However, resistance commonly arises due to single nucleotide variants (SNVs) within the ALK tyrosine kinase domain, many of which remain variants of uncertain significance (VUSs).

Results: To systematically profile resistance, we use prime editing to generate and assess 3,208 ALK variants covering 99% of all possible SNVs across exons 20-28, along with intronic variants. We evaluate drug resistance across three generations of ALK TKIs: alectinib, lorlatinib, and zotizalkib. These high-resolution resistance landscapes validate known resistance mutations (e.g., G1202R, L1196M), identify previously uncharacterized resistance-associated VUSs, and reveal distinct patterns of drug-specific and shared resistance across inhibitors. Structural mapping further contextualizes resistance-associated variants relative to the ATP-binding pocket and distal regions associated with resistance.

Conclusions: This study provides a comprehensive functional atlas of ALK tyrosine kinase domain variants under TKI selection, offering a valuable experimental framework for interpreting resistance-associated variants. Although derived from in vitro models and therefore context dependent, this resource complements existing clinical and genomic knowledge and may aid in the functional interpretation of ALK variants observed in ALK-driven cancers.

Spatial omics technologies localize individual molecules at subcellular resolution, yet growing numbers of molecules, features and replicates set analysis challenges. We present smoppix, a nonparametric analysis method based on the probabilistic index, to test for several uni- and bivariate localization patterns. It exploits the high-dimensionality of the data for variance weighting and for providing a background null distribution, unique for every molecule. Moreover, smoppix sidesteps segmentation, edge correction, warping and density estimation, and is scalable thanks to an exact permutation null distribution. We unearth spatial patterns in datasets from four kingdoms, and validate some findings experimentally on spikemoss roots.

Background: The field vole, an abundant and widespread microtine rodent, is a complex comprised of three cryptic species: the short-tailed field vole (Microtus agrestis) which is present over much of Eurasia, the Mediterranean field vole (Microtus lavernedii) in southern Europe, and the Portuguese field vole (Microtus rozianus) in western Spain and Portugal. Previous research has shown high genomic differentiation of these three lineages. However, the details of the process underlying their divergence remain unknown.

Results: We analyse 70 mitogenomes and 16 nuclear genomes of modern specimens, and 83 mitogenomes and 12 nuclear genomes of ancient specimens spanning the last 75 thousand years (ka). We estimate the divergence of Portuguese from short-tailed and Mediterranean field voles to be ca. 220 ka ago and of the latter two species to be ca. 110 ka ago, earlier than previous estimates involving only modern sequences. The divergence times we obtain match those between major mitochondrial lineages of cold-adapted and steppe rodents in Europe. We find signatures of gene flow within and between field vole lineages, with some analyses suggesting a hybrid origin of the Mediterranean lineage. Ancient specimens from the Italian Peninsula reveal a previously unrecognised lineage that show evidence of genetic exchange with other populations.

Conclusions: The pattern of genetic variation in the field vole species complex demonstrates the impact of stadial-interstadial cycles in generating recurrent episodes of allopatry and connectivity of populations, a situation which could only be revealed by our dense genomic sampling over time.

Background: Beta-thalassemia is among the most common monogenic disorders, posing a major global health challenge. Editing of genetic modifiers, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors (BE), are potentially safer and better suited for multiplexed application than DSB-dependent CRISPR/Cas technology. However, harmful on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements invisible to standard detection technologies.

Results: Using primary patient-derived CD34⁺ cells from three donors, we investigate simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115 bp) on the HBG promoter for DNA-level and functional studies at the RNA, protein, and morphological level. Analyses include direct comparison to DSB-based editing, the current clinically applied standard, and CAST-seq to assess recombination events, allowing wider inferences on relative safety. RNA-seq analyses for clones of primary CD34⁺ cells across all treatments confirm peak HBG induction for duplex BE and comparable effects on apoptotic and immune response signatures. Overall, duplex BE produces robust γ-globin and fetal hemoglobin induction, improves functional correction over simplex editing and results in low incidence of genomic alterations in both target loci.

Conclusions: Duplex BE targeting both BCL11A erythroid enhancer and HBG promoter enables functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach.

Alternative polyadenylation (APA) is a pervasive RNA-processing mechanism in eukaryotes that significantly promotes transcriptome and proteome diversity. Here we proposed PolyAseqTrap, an R package for probing polyA sites from diverse 3' sequencing data. PolyAseqTrap implements a polyA read prioritization strategy to determine precise positions of polyA sites. Particularly, it incorporates a transferrable cross-species deep learning model for mitigating the long-pending internal priming problem. Moreover, PolyAseqTrap employs a weighted density peak clustering method to reducing microheterogeneity impact in different species. We evaluated PolyAseqTrap using data from 16 different 3' sequencing techniques across multiple species, demonstrating the effectiveness and robustness of PolyAseqTrap.

Background: CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies, revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design.

Results: We systematically compare specific targeting and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNAs cause pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerges as the most efficient tool for stable promoter targeting, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations.

Conclusions: Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.

Background: Long dismissed as mere genomic parasites, transposable elements (TEs) are now recognized as major drivers of genome evolution. TEs serve as a source of cell-type specific cis-regulatory elements, influencing gene expression and observable phenotypes. However, the precise TE regulatory roles in different contexts remain largely unexplored and the impact of TEs on transcriptional regulatory networks and contribution to disease risk is likely deeply underestimated.

Results: Using a multimapper-aware strategy, we systematically characterize the epigenetic profile of TEs in human cell systems modeling neural development. This analysis reveals that MER57E3, a primate-specific TE subfamily, exhibits strong enrichment for active, and absence of repressive, histone modifications across six cultured human neural cell types. MER57E3 copies are predominantly located near zinc finger genes and enriched for homeodomain motifs recognized by brain-specific transcription factors, including GBX1 and BSX. Upon CRISPR interference (CRISPRi) targeting specific MER57E3 copies, RNA-seq analysis demonstrates downregulation of the key neurogenesis-related genes PAX6 and NEUROG2.

Conclusions: Our data indicate that members of the MER57E3 TE subfamily regulate the expression of critical neurogenesis genes during neural progenitor cell (NPC) development. Moreover, this study emphasizes the importance of investigating TEs, offering new insights into how their epigenetic dysregulation may contribute to pathogenesis of neurodevelopmental disorders.

Background: The ruminant gastrointestinal epithelium harbors a diverse and functionally critical remains poorly characterized microbial community due to persistent host-derived DNA contamination in metagenomic studies.

Results: We develop Dilute-MetaSeq (dilution-based metagenomic sequencing), a novel, metagenomic workflow integrating gradient dilution with multiple displacement amplification. Dilute-MetaSeq reduces host DNA interference by 52.4-fold and achieves > 90% microbial sequencing efficiency to assess gastrointestinal epithelium-associated microbiome. This enables the construction of the microbial genome atlas of gastrointestinal epithelium (MGA-GE). This comprehensive resource, comprising 1,907 nonredundant prokaryotic and 5,603 viral genomes, reveals extraordinary microbial diversity and novelty, with 41.4% of prokaryotic and 99.9% of viral genomes representing taxonomically unclassified lineages. Spatial profiling identifies the rumen and reticulum as a biodiversity hotspot dominated by epithelium-adapted Butyrivibrio and methylotrophic Methanomassiliicoccales, while functional annotation uncovers 1,200 biosynthetic gene clusters (primarily RiPPs and NRPSs) and 1,212 viral auxiliary metabolic genes linked to host metabolism modulation. Pangenome analysis of 987 strains, including a novel Butyrivibrio clade with reduced genome sizes, elevated GC content, and butyrate synthesis from amino acid-derived substrates (e.g., glutarate, lysine), highlights metabolic adaptations to the nutrient-scarce epithelial niche compared to digesta-associated microbes.

Conclusions: Collectively, the MGA-GE provides transformative insights into host-microbe-virus interactions and establishes a foundation for developing microbiome-based intervention strategies to enhance ruminant health, agricultural productivity, and bioactive discovery.

We introduce SCITO-seq2, an enhanced successor to SCITO-seq that integrates probe-based RNA detection with the established ultra-high-throughput protein profiling. SCITO-seq2 achieves robust quantification of transcripts and surface proteins across more than 100,000 cells, with a shared pool barcoding strategy ensuring precise matching of molecular profiles within multiplexed droplets. SCITO-seq2 is compatible with cell hashing technology, allowing efficient sample multiplexing. We demonstrate its utility in autoimmune diseases, including childhood systemic lupus erythematosus and CTLA4 haploinsufficiency with autoimmune infiltration, enabling the detection of minor immune clusters and disease-specific protein signatures. This platform establishes a scalable, streamlined, and cost-effective next-generation single-cell multi-omics workflow.