Allele-specific Expression Research Articles

A spectrum of nonsense-mediated mRNA decay efficiency along the degree of mutational constraint

Despite its importance for regulating gene expression, nonsense-mediated mRNA decay (NMD) remains poorly understood. Here, we extend the findings of a previous landmark study that proposed several factors associated with NMD efficiency using matched genome and transcriptome data from The Cancer Genome Atlas Program (TCGA) by incorporating additional data including Genotype-Tissue Expression (GTEx), gnomAD, and metrics for mutational constraints. Factors affecting NMD efficiency are analyzed using an allele-specific expression (ASE)-based measure to reduce noise caused by random variations. Additionally, by combining our data with the updated allele frequency database of gnomAD, we demonstrate the spectrum of NMD efficiency according to the degree of gene-level mutational constraints (degree of a gene-tolerating loss-of-function variants). The NMD prediction model, trained on TCGA data, shows that gene-level mutational constraint is an important predictor of NMD efficiency. Findings of this study suggest the potential role of NMD on shaping the landscape of mutational constraints.

Phased T2T genome assemblies facilitate the mining of disease-resistance genes in Vitis davidii

Abstract Grape is an important fruit crop, and its production faces significant threat from diseases, resulting in substantial economic loss. Wild grape relatives are valuable resources for the restoration of disease-resistance loci. However, available resistance loci in wild grape genomes remain largely unexplored. In this study, we assembled two phased genomes, including a high resistant Chinese wild grape, Vitis davidii Föex, and a susceptible cultivar, V. vinifera L. cv. “Manicure Finger”. We detected a total of 36,688 structural variations (SVs), with the genes associated with heterozygous SVs showing an enrichment in allele-specific expression (ASE). Furthermore, we identified eight subgroups of R genes and found that 74.2% of R genes overlap with transposable elements (TEs). Among R genes, NBS-type genes exhibit higher expression profiles in the wild grape genome compared with those in the grape cultivar. Additionally, five specific NBS-type R gene clusters were identified in the wild grape genome that are absent in the cultivar. Through genetic mapping, we identified four quantitative trait loci (QTLs) associated with grape white rot resistance based on the Vitis davidii genome, within which six NBS-type R genes exhibit differential expression between wild and cultivated grapes. Overall, our study revealed the landscape of resistance genes in grape genomes, providing valuable genetic resources for further breeding programs.

A compendium of methods: Searching allele specific expression via RNA sequencing

A compendium of methods: Searching allele specific expression via RNA sequencing

Long-read RNA sequencing reveals allele-specific N6-methyladenosine modifications.

Long-read sequencing technology enables highly accurate detection of allele-specific RNA expression, providing insights into the effects of genetic variation on splicing and RNA abundance. Furthermore, the ability to directly sequence RNA using the Oxford Nanopore technology promises the detection of RNA modifications in tandem with ascertaining the allelic origin of each molecule. Here, we leverage these advantages to determine allele-biased patterns of N6-methyladenosine (m6A) modifications in native mRNA. We utilized human and mouse cells with known genetic variants to assign allelic origin of each mRNA molecule combined with a supervised machine learning model to detect read-level m6A modification ratios. Our analyses revealed the importance of sequences adjacent to the DRACH-motif in determining m6A deposition, in addition to allelic differences that directly alter the motif. Moreover, we discovered allele-specific m6A modification (ASM) events with no genetic variants in close proximity to the differentially modified nucleotide, demonstrating the unique advantage of using long reads and surpassing the capabilities of antibody-based short-read approaches. This technological advancement promises to advance our understanding of the role of genetics in determining mRNA modifications.

Candida albicans isolates contain frequent heterozygous structural variants and transposable elements within genes and centromeres.

The human fungal pathogen Candida albicans poses a significant burden on global health, causing high rates of mortality and antifungal drug resistance. C. albicans is a heterozygous diploid organism that reproduces asexually. Structural variants (SVs) are an important source of genomic rearrangement, particularly in species that lack sexual recombination. To comprehensively investigate SVs across clinical isolates of C. albicans, we conducted long read sequencing and genome-wide SV analysis in three distantly related clinical isolates. Our work included a new, comprehensive analysis of transposable element (TE) composition, location and diversity. SVs and TEs are frequently close to coding sequences and many SVs are heterozygous, suggesting that SVs might impact gene and allele-specific expression. Most SVs are uniquely present in only one clinical isolate, indicating that SVs represent a significant source of intra-species genetic variation. We identified multiple, distinct SVs at the centromeres of Chromosome 4 and Chromosome 5, including inversions and transposon polymorphisms. These two chromosomes are often aneuploid in drug resistant clinical isolates, and can form isochromosome structures with breakpoints near the centromere. Further screening of 100 clinical isolates confirmed the widespread presence of centromeric SVs in C. albicans, often appearing in a heterozygous state, indicating that SVs are contributing to centromere evolution in C. albicans Together, these findings highlight that SVs and TEs are common across diverse clinical isolates of C. albicans and that the centromeres of this organism are important sites of genome rearrangement.

Stabilizing selection and adaptation shape cis and trans gene expression variation in C. elegans.

An outstanding question in the evolution of gene expression is the relative influence of neutral processes versus natural selection, including adaptive change driven by directional selection as well as stabilizing selection, which may include compensatory dynamics. These forces shape patterns of gene expression variation within and between species, including the regulatory mechanisms governing expression in cis and trans . In this study, we interrogate intraspecific gene expression variation among seven wild C. elegans strains, with varying degrees of genomic divergence from the reference strain N2, leveraging this system's unique advantages to comprehensively evaluate gene expression evolution. By capturing allele-specific and between-strain changes in expression, we characterize the regulatory architecture and inheritance mode of gene expression variation within C. elegans and assess their relationship to nucleotide diversity, genome evolutionary history, gene essentiality, and other biological factors. We conclude that stabilizing selection is a dominant influence in maintaining expression phenotypes within the species, and the discovery that genes with higher overall expression tend to exhibit fewer expression differences supports this conclusion, as do widespread instances of cis differences compensated in trans . Moreover, analyses of human expression data replicate our finding that higher expression genes have less variable expression. We also observe evidence for directional selection driving expression divergence, and that expression divergence accelerates with increasing genomic divergence. To provide community access to the data from this first analysis of allele-specific expression in C. elegans , we introduce an interactive web application, where users can submit gene-specific queries to view expression, regulatory pattern, inheritance mode, and other information: https://wildworm.biosci.gatech.edu/ase/ .

Trans regulation of an odorant binding protein by a proto-Y chromosome affects male courtship in house fly.

The male-limited inheritance of Y chromosomes favors alleles that increase male fitness, often at the expense of female fitness. Determining the mechanisms underlying these sexually antagonistic effects is challenging because it can require studying Y-linked alleles while they still segregate as polymorphisms. We used a Y chromosome polymorphism in the house fly, Musca domestica, to address this challenge. Two male determining Y chromosomes (YM and IIIM) segregate as stable polymorphisms in natural populations, and they differentially affect multiple traits, including male courtship performance. We identified differentially expressed genes encoding odorant binding proteins (in the Obp56h family) as candidate agents for the courtship differences. Through network analysis and allele-specific expression measurements, we identified multiple genes on the house fly IIIM chromosome that could serve as trans regulators of Obp56h gene expression. One of those genes is homologous to Drosophila melanogaster CG2120, which encodes a transcription factor that binds near Obp56h. Upregulation of CG2120 in D. melanogaster nervous tissues reduces copulation latency, consistent with this transcription factor acting as a negative regulator of Obp56h expression. The transcription factor gene, which we name speed date, demonstrates a molecular mechanism by which a Y-linked gene can evolve male-beneficial effects.

Genotyping sequence-resolved copy-number variations using pangenomes reveals paralog-specific global diversity and expression divergence of duplicated genes.

Copy-number variable (CNV) genes are important in evolution and disease, yet sequence variation in CNV genes is a blindspot for large-scale studies. We present a method, ctyper, that leverages pangenomes to produce copy-number maps with allele-specific sequences containing locally phased variants of CNV genes from NGS reads. We extensively characterized accuracy and efficiency on a database of 3,351 CNV genes including HLA , SMN , and CYP2D6 as well as 212 non-CNV medically-relevant challenging genes. The genotypes capture 96.5% of underlying variants in new genomes, requiring 0.9 seconds per gene. Expression analysis of ctyper genotypes explains more variance than known eQTL variants. Comparing allele-specific expression quantified divergent expression on 7.94% of paralogs and tissue-specific biases on 4.7% of paralogs. We found reduced expression of SMN-1 converted from SMN-2, which potentially affects diagnosis of spinal muscular atrophy, and increased expression of a duplicative translocation of AMY2B . Overall, ctyper enables biobank-scale genotyping of CNV and challenging genes.

Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the POG program and the Marathon of Hope Cancer Centres Network includes DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind homologous recombination deficiency where no coding driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology.

Linking candidate causal autoimmune variants to T cell networks using genetic and epigenetic screens in primary human T cells.

Genetic variants associated with autoimmune diseases are highly enriched within putative cis -regulatory regions of CD4 + T cells, suggesting that they alter disease risk via changes in gene regulation. However, very few genetic variants have been shown to affect T cell gene expression or function. We tested >18,000 autoimmune disease-associated variants for allele-specific expression using massively parallel reporter assays in primary human CD4 + T cells. The 545 expression-modulating variants (emVars) identified greatly enrich for likely causal variants. We provide evidence that many emVars are mediated by common upstream regulatory conduits, and that putative target genes of primary T cell emVars are highly enriched within a lymphocyte activation network. Using bulk and single-cell CRISPR-interference screens, we confirm that emVar-containing T cell cis -regulatory elements modulate both known and novel target genes that regulate T cell proliferation, providing plausible mechanisms by which these variants alter autoimmune disease risk.

Characterizing the allele-specific gene expression landscape in high hyperdiploid acute lymphoblastic leukemia with BASE.

Somatic copy number variations (CNVs), including abnormal chromosome numbers and structural changes leading to gain or loss of genetic material, play a crucial role in initiation and progression of cancer. CNVs are believed to cause gene dosage imbalances and modify cis-regulatory elements, leading to allelic expression imbalances in genes that influence cell division and thereby contribute to cancer development. However, the impact of CNVs on allelic gene expression in cancer remains unclear. Allele-specific expression (ASE) analysis, a potent method for investigating genome-wide allelic imbalance profiles in tumors, assesses the relative expression of two alleles using high-throughput sequencing data. However, many existing methods for gene-level ASE detection rely on only RNA sequencing data, which present challenges in interpreting the genetic mechanisms underlying ASE in cancer. To address this issue, we developed a robust framework that integrates allele-specific copy number calls into ASE calling algorithms by leveraging paired genome and transcriptome data from the same sample. This integration enhances the interpretability of the genetic mechanisms driving ASE, thereby facilitating the identification of driver events triggered by CNVs in cancer. In this study, we utilized BASE to conduct a comprehensive analysis of ASE in high hyperdiploid acute lymphoblastic leukemia (HeH ALL), a prevalent childhood malignancy characterized by gains of chromosomes X, 4, 6, 10, 14, 17, 18, and 21. Our analysis unveiled the comprehensive ASE landscape in HeH ALL. Through a multi-perspective examination of HeH ASEs, we offer a systematic understanding of how CNVs impact ASE in HeH, providing valuable insights to guide ASE studies in cancer.

Promoter Deletion Leading to Allele Specific Expression in a Genetically Unsolved Case of Primary Ciliary Dyskinesia.

Variation in the non-coding genome represents an understudied mechanism of disease and it remains challenging to predict if single nucleotide variants, small insertions and deletions, or structural variants in non-coding genomic regions will be detrimental. Our approach using complementary RNA-seq and targeted long-read DNA sequencing can prioritize identification of non-coding variants that lead to disease via alteration of gene splicing or expression. We have identified a patient with primary ciliary dyskinesia with a pathogenic coding variant on one allele of the SPAG1 gene, while the second allele appears normal by whole exome sequencing despite an autosomal recessive inheritance pattern. RNA sequencing revealed reduced SPAG1 transcript levels and exclusive allele specific expression of the known pathogenic allele, suggesting the presence of a non-coding variant on the second allele that impacts transcription. Targeted long-read DNA sequencing identified a heterozygous 3 kilobase deletion of the 5' untranslated region of SPAG1, overlapping the promoter and first non-coding exon. This non-coding deletion was missed by whole exome sequencing and gene-specific deletion/duplication analysis, highlighting the importance of investigating the non-coding genome in patients with "missing" disease-causing variation. This paradigm demonstrates the utility of both RNA and long-read DNA sequencing in identifying pathogenic non-coding variants in patients with unexplained genetic disease.

Maintenance of X chromosome inactivation after T cell activation requires NF-κB signaling.

X chromosome inactivation (XCI) balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of X-inactive specific transcript (Xist) RNA and heterochromatic modifications on the inactive X chromosome (Xi), which are involved in maintenance of XCI, and these modifications return to the Xi after stimulation. Here, we examined allele-specific gene expression and epigenomic profiles of the Xi in T cells. We found that the Xi in unstimulated T cells is largely dosage compensated and enriched with the repressive H3K27me3 modification but not the H2AK119-ubiquitin (Ub) mark. Upon T cell stimulation mediated by both CD3 and CD28, the Xi accumulated H2AK119-Ub at gene regions of previous H3K27me3 enrichment. T cell receptor (TCR) engagement, specifically NF-κB signaling downstream of the TCR, was required for Xist RNA localization to the Xi. Disruption of NF-κB signaling in mouse and human T cells using genetic deletion, chemical inhibitors, and patients with immunodeficiencies prevented Xist/XIST RNA accumulation at the Xi and altered X-linked gene expression. Our findings reveal a previously undescribed connection between NF-κB signaling pathways, which affects XCI maintenance in T cells in females.

PRKACB is a novel imprinted gene in marsupials

BackgroundGenomic imprinting results in parent-of-origin-specific gene expression and, among vertebrates, is found only in therian mammals: marsupials and eutherians. A differentially methylated region (DMR), in which the methylation status of CpG dinucleotides differs between the two alleles, can mark the parental identity of imprinted genes. We developed a computational pipeline that detected CpG islands (CGIs) marked by both methylated and unmethylated signals in whole genome bisulfite sequencing data. This approach identified candidate marsupial DMRs in a publicly available koala methylome. One of these candidate DMRs was associated with PRKACB, a gene encoding the protein kinase A catalytic subunit beta. Nothing is known about the imprinting status of PRKACB in eutherian mammals although mutations of this gene are associated with endocrine neoplasia and other developmental disorders.ResultsIn the tammar wallaby and brushtail possum there was parent-of-origin-specific DNA methylation in the PRKACB DMR in which the maternal allele was methylated and the paternal allele was unmethylated. There were multiple RNAs transcribed from this locus. Allele-specific expression analysis identified paternal expression of a PRKACB lncRNA and an mRNA isoform. Comparison of the PRKACB gene start site between marsupials and eutherians demonstrated that the CGI is longer in marsupials. The PRKACB gene product functions in the same signalling pathway as the guanine nucleotide-binding protein alpha subunit encoded at the GNAS locus, a known eutherian imprinted gene. In a mouse methylome Gnas had three differentially methylated CGIs, while in the koala methylome the GNAS locus had two unmethylated CGIs.ConclusionsWe conclude that PRKACB is a novel, DMR-associated marsupial imprinted gene. Imprinting of PRKACB in marsupials and GNAS in eutherians may indicate a conserved selection pressure for imprinting of the protein kinase A signalling pathway in therians with the two lineages adapting by imprinting different genes.

Structural genomic variation and behavioral interactions underpin a balanced sexual mimicry polymorphism

How phenotypic diversity originates and persists within populations are classic puzzles in evolutionary biology. While balanced polymorphisms segregate within many species, it remains rare for both the genetic basis and the selective forces to be known, leading to an incomplete understanding of many classes of traits under balancing selection. Here, we uncover the genetic architecture of a balanced sexual mimicry polymorphism and identify behavioral mechanisms that may be involved in its maintenance in the swordtail fish Xiphophorus birchmanni. We find that ∼40% of X.birchmanni males develop a "false gravid spot," a melanic pigmentation pattern that mimics the "pregnancy spot" associated with sexual maturity in female live-bearing fish. Using genome-wide association mapping, we detect a single intergenic region associated with variation in the false gravid spot phenotype, which is upstream of kitlga, a melanophore patterning gene. By performing long-read sequencing within and across populations, we identify complex structural rearrangements between alternate alleles at this locus. The false gravid spot haplotype drives increased allele-specific expression of kitlga, which provides a mechanistic explanation for the increased melanophore abundance that causes the spot. By studying social interactions in the laboratory and in nature, we find that males with the false gravid spot experience less aggression; however, they also receive increased attention from other males and are disdained by females. These behavioral interactions may contribute to the maintenance of this phenotypic polymorphism in natural populations. We speculate that structural variants affecting gene regulation may be an underappreciated driver of balanced polymorphisms across diverse species.

Allele-specific transcription factor binding across human brain regions offers mechanistic insight into eQTLs.

Transcription factors (TFs) regulate gene expression by facilitating or disrupting the formation of transcription initiation machinery at particular genomic loci. Because TF occupancy is driven in part by recognition of DNA sequence, genetic variation can influence TF-DNA associations and gene regulation. To identify variants that impact TF binding in human brain tissues, we assessed allele-specific binding (ASB) at heterozygous variants for 94 TFs in nine brain regions from two donors. Leveraging graph genomes constructed from phased genomic sequence data, we compared ChIP-seq signals between alleles at heterozygous variants within each brain region and identified thousands of variants exhibiting ASB for at least one TF. ASB reproducibility was measured by comparisons between independent experiments both within and between donors. We found that rare alleles in the general population more frequently led to reduced TF binding, whereas common alleles had an equal likelihood of increasing or decreasing binding. Further, for ASB variants in predicted binding motifs, the favored allele tended to be the one with the stronger expected motif match, but this concordance was not observed within highly occupied sites. We also found that neuron-specific cis-regulatory elements (cCREs), in contrast with oligodendrocyte-specific cCREs, showed depletion of ASB variants. We identified 2670 ASB variants associated with evidence for allele-specific gene expression in the brain from GTEx data and observed increasing eQTL effect direction concordance as ASB significance increases. These results provide a valuable and unique resource for mechanistic analysis of cis-regulatory variation in human brain tissue.

Haplotype-based pangenomes reveal genetic variations and climate adaptations in moso bamboo populations

Moso bamboo (Phyllostachys edulis), an ecologically and economically important forest species in East Asia, plays vital roles in carbon sequestration and climate change mitigation. However, intensifying climate change threatens moso bamboo survival. Here we generate high-quality haplotype-based pangenome assemblies for 16 representative moso bamboo accessions and integrated these assemblies with 427 previously resequenced accessions. Characterization of the haplotype-based pangenome reveals extensive genetic variation, predominantly between haplotypes rather than within accessions. Many genes with allele-specific expression patterns are implicated in climate responses. Integrating spatiotemporal climate data reveals more than 1050 variations associated with pivotal climate factors, including temperature and precipitation. Climate-associated variations enable the prediction of increased genetic risk across the northern and western regions of China under future emissions scenarios, underscoring the threats posed by rising temperatures. Our integrated haplotype-based pangenome elucidates moso bamboo’s local climate adaptation mechanisms and provides critical genomic resources for addressing intensifying climate pressures on this essential bamboo. More broadly, this study demonstrates the power of long-read sequencing in dissecting adaptive traits in climate-sensitive species, advancing evolutionary knowledge to support conservation.

PSVII-1 Widespread allele-specific expression across tissues and cells of the chicken genome

Abstract Chickens are a valuable livestock species, providing an affordable and nutritious food source worldwide through eggs and meat. Additionally, chickens are extensively used in scientific research as model organisms in virology, immunology, epigenetics, development, and conservation biology. Considering this, there are increasing efforts to deepen our understanding of their genome complexity, such as efforts by the Functional Annotation of Animal Genomes (FAANG) Consortium. Toward this goal, we are working to contribute to the annotation of the chicken transcriptome. Allele-specific expression (ASE) is an imbalance of allelic gene expression often driven by genetic and epigenetic changes in cis-regulatory regions. We, therefore, aimed to determine allelic imbalances in gene expression across 20 tissues and cells to shed light on mechanisms regulating gene expression. Using replicate paired-end (PE) RNA-seq samples, we identified around 7 million variants across 20 tissues and cells (e.g., reproductive tissues, intestine tissues, muscle, and immune tissues and cells). We applied quality control measures and filtered out monoallelic and homozygous variants. After that, we calculated read counts per allele to determine allelic expression. We found 365,894 significant ASE SNPs and 11,530 ASE genes in at least one tissue or cell type (FDR ≤ 0.05). We discovered that ASE SNPs are widely distributed throughout the chicken genome, and ASE SNPs and ASE genes showed a varied distribution across tissues and cells. Primary macrophage exhibited the most abundant, while the pectoralis major (breast muscle) had the lowest ASE genes and ASE SNPs, ranging from 7,592 to 32,505 ASE SNPs and 773 to 2,387 ASE genes. Our findings reveal several important pathways affected by allelic imbalance of expression. These pathways included fatty acid biosynthesis and regulation, cell proliferation and differentiation, cell metabolism, adipocytokine signaling, proteolysis and autophagy, and focal adhesion. In summary, our study provides insight into relevant biological processes critical to chickens and can contribute to improving genome annotation, helping to expand the transcriptomic reference source.

DNA Methylation and Subgenome Dominance Reveal the Role of Lipid Metabolism in Jinhu Grouper Heterosis.

Heterosis of growth traits in economic fish has benefited the production of aquaculture for many years, yet its genetic and molecular basis has remained obscure. Nowadays, a new germplasm of hybrid Jinhu grouper (Epinephelus fuscoguttatus ♀ × E. tukula ♂), abbreviated as EFT, exhibiting paternal-biased growth heterosis, has provided an excellent model for investigating the potential regulatory mechanisms of heterosis. We integrated transcriptome and methylome to unravel the changes of gene expression, epigenetic modification, and subgenome dominance in EFT compared with maternal E. fuscoguttatus. Integration analyses showed that the heterotic hybrids showed lower genomic DNA methylation levels than the purebred parent, and the up-regulated genes were mostly DNA hypomethylation. Furthermore, allele-specific expression (ASE) detected paternal subgenome dominance-regulated paternal-biased heterosis, and paternal bias differentially expressed genes (DEGs) were wholly up-regulated in the muscle. Multi-omics results highlighted the role of lipid metabolism, particularly "Fatty acid synthesis", "EPA biosynthesis", and "Signaling lipids", in EFT heterosis formation. Coherently, our studies have proved that the eicosapentaenoic acid (EPA) of EFT was greater than that of maternal E. fuscoguttatus (8.46% vs. 7.46%). Finally, we constructed a potential regulatory network for control of the heterosis formation in EFT. Among them, fasn, pparg, dgat1, igf1, pomca, fgf8a, and fgfr4 were identified as key genes. Our results provide new and valuable clues for understanding paternal-biased growth heterosis in EFT, taking a significant step towards the molecular basis of heterosis.

Long-read RNA sequencing reveals allele-specific N6-methyladenosine modifications.

Long-read sequencing technology enables highly accurate detection of allele-specific RNA expression, providing insights into the effects of genetic variation on splicing and RNA abundance. Furthermore, the ability to directly sequence RNA promises the detection of RNA modifications in tandem with ascertaining the allelic origin of each molecule. Here, we leverage these advantages to determine allele-biased patterns of N6-methyladenosine (m6A) modifications in native mRNA. We utilized human and mouse cells with known genetic variants to assign allelic origin of each mRNA molecule combined with a supervised machine learning model to detect read-level m6A modification ratios. Our analyses revealed the importance of sequences adjacent to the DRACH-motif in determining m6A deposition, in addition to allelic differences that directly alter the motif. Moreover, we discovered allele-specific m6A modification (ASM) events with no genetic variants in close proximity to the differentially modified nucleotide, demonstrating the unique advantage of using long reads and surpassing the capabilities of antibody-based short-read approaches. This technological advancement promises to advance our understanding of the role of genetics in determining mRNA modifications.