Accurate quantification and detection of intron retention levels require specialized software. Building on our previous software, we create a suite of tools called IRFinder-S, to analyze and explore intron retention events in multiple samples. Specifically, IRFinder-S allows a better identification of true intron retention events using a convolutional neural network, allows the sharing of intron retention results between labs, integrates a dynamic database to explore and contrast available samples, and provides a tested method to detect differential levels of intron retention.

BACKGROUND: Topologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs has been shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing lead to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains, which are highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies. Recent computational modeling studies suggest that epigenetic interactions may underlie the formation of the single-cell domains. RESULTS: Here we use chromatin tracing to visualize in female human cells the fine-scale chromatin folding of inactive and active X chromosomes, which are known to have distinct global epigenetic landscapes and distinct population-averaged TAD profiles, with inactive X chromosomes largely devoid of TADs and cohesin. We show that both inactive and active X chromosomes possess highly variant single-cell domains across the same genomic region despite the fact that only active X chromosomes show clear TAD structures at the population level. These X chromosome single-cell domains exist in distinct cell lines. Perturbations of major epigenetic components and transcription mostly do not affect the frequency or strength of the single-cell domains. Increased chromatin compaction of inactive X chromosomes occurs at a length scale above that of the single-cell domains. CONCLUSIONS: In sum, this study suggests that single-cell domains are genome architecture building blocks independent of the tested major epigenetic components.

BACKGROUND: Enhancers are non-coding regions of the genome that control the activity of target genes. Recent efforts to identify active enhancers experimentally and in silico have proven effective. While these tools can predict the locations of enhancers with a high degree of accuracy, the mechanisms underpinning the activity of enhancers are often unclear. RESULTS: Using machine learning (ML) and a rule-based explainable artificial intelligence (XAI) model, we demonstrate that we can predict the location of known enhancers in Drosophila with a high degree of accuracy. Most importantly, we use the rules of the XAI model to provide insight into the underlying combinatorial histone modifications code of enhancers. In addition, we identified a large set of putative enhancers that display the same epigenetic signature as enhancers identified experimentally. These putative enhancers are enriched in nascent transcription, divergent transcription and have 3D contacts with promoters of transcribed genes. However, they display only intermediary enrichment of mediator and cohesin complexes compared to previously characterised active enhancers. We also found that 10-15% of the predicted enhancers display similar characteristics to super enhancers observed in other species. CONCLUSIONS: Here, we applied an explainable AI model to predict enhancers with high accuracy. Most importantly, we identified that different combinations of epigenetic marks characterise different groups of enhancers. Finally, we discovered a large set of putative enhancers which display similar characteristics with previously characterised active enhancers.

BACKGROUND: Coconut is an important tropical oil and fruit crop whose evolutionary position renders it a fantastic species for the investigation of the evolution of monocot chromosomes and the subsequent differentiation of ancient plants. RESULTS: Here, we report the assembly and annotation of reference-grade genomes of Cn. tall and Cn. dwarf, whose genome sizes are 2.40 Gb and 2.39 Gb, respectively. The comparative analysis reveals that the two coconut subspecies diverge about 2-8 Mya while the conserved Arecaceae-specific whole-genome duplication (ω WGD) occurs approximately 47-53 Mya. It additionally allows us to reconstruct the ancestral karyotypes of the ten ancient monocot chromosomes and the evolutionary trajectories of the 16 modern coconut chromosomes. Fiber synthesis genes in Cn. tall, related to lignin and cellulose synthesis, are found at a higher copy number and expression level than dwarf coconuts. Integrated multi-omics analysis reveals that the difference in coconut plant height is the result of altered gibberellin metabolism, with both the GA20ox copy number and a single-nucleotide change in the promoter together leading to the difference in plant height between Cn. tall and Cn. dwarf. CONCLUSION: We provide high-quality coconut genomes and reveal the genetic basis of trait differences between two coconuts through multi-omics analysis. We also reveal that the selection of plant height has been targeted for the same gene for millions of years, not only in natural selection of ancient plant as illustrated in coconut, but also for artificial selection in cultivated crops such as rice and maize.

BACKGROUND: Polyploidy, especially allopolyploidy, which entails merging divergent genomes via hybridization and whole-genome duplication (WGD), is a major route to speciation in plants. The duplication among the parental genomes (subgenomes) often leads to one subgenome becoming dominant over the other(s), resulting in subgenome asymmetry in gene content and expression. Polyploid wheats are allopolyploids with most genes present in two (tetraploid) or three (hexaploid) functional copies, which commonly show subgenome expression asymmetry. It is unknown whether a similar subgenome asymmetry exists during translation. We aim to address this key biological question and explore the major contributing factors to subgenome translation asymmetry. RESULTS: Here, we obtain the first tetraploid wheat translatome and reveal that subgenome expression asymmetry exists at the translational level. We further perform in vivo RNA structure profiling to obtain the wheat RNA structure landscape and find that mRNA structure has a strong impact on translation, independent of GC content. We discover a previously uncharacterized contribution of RNA structure in subgenome translation asymmetry. We identify 3564 single-nucleotide variations (SNVs) across the transcriptomes between the two tetraploid wheat subgenomes, which induce large RNA structure disparities. These SNVs are highly conserved within durum wheat cultivars but are divergent in both domesticated and wild emmer wheat. CONCLUSIONS: We successfully determine both the translatome and in vivo RNA structurome in tetraploid wheat. We reveal that RNA structure serves as an important modulator of translational subgenome expression asymmetry in polyploids. Our work provides a new perspective for molecular breeding of major polyploid crops.

BACKGROUND: Intermixing of genomes through meiotic reassortment and recombination of homologous chromosomes is a unifying theme of sexual reproduction in eukaryotic organisms and is considered crucial for their adaptive evolution. Previous studies of the budding yeast species Saccharomycodes ludwigii suggested that meiotic crossing over might be absent from its sexual life cycle, which is predominated by fertilization within the meiotic tetrad. RESULTS: We demonstrate that recombination is extremely suppressed during meiosis in Sd. ludwigii. DNA double-strand break formation by the conserved transesterase Spo11, processing and repair involving interhomolog interactions are required for normal meiosis but do not lead to crossing over. Although the species has retained an intact meiotic gene repertoire, genetic and population analyses suggest the exceptionally rare occurrence of meiotic crossovers in its genome. A strong AT bias of spontaneous mutations and the absence of recombination are likely responsible for its unusually low genomic GC level. CONCLUSIONS: Sd. ludwigii has followed a unique evolutionary trajectory that possibly derives fitness benefits from the combination of frequent mating between products of the same meiotic event with the extreme suppression of meiotic recombination. This life style ensures preservation of heterozygosity throughout its genome and may enable the species to adapt to its environment and survive with only minimal levels of rare meiotic recombination. We propose Sd. ludwigii as an excellent natural forum for the study of genome evolution and recombination rates.

We present recount3, a resource consisting of over 750,000 publicly available human and mouse RNA sequencing (RNA-seq) samples uniformly processed by our new Monorail analysis pipeline. To facilitate access to the data, we provide the recount3 and snapcount R/Bioconductor packages as well as complementary web resources. Using these tools, data can be downloaded as study-level summaries or queried for specific exon-exon junctions, genes, samples, or other features. Monorail can be used to process local and/or private data, allowing results to be directly compared to any study in recount3. Taken together, our tools help biologists maximize the utility of publicly available RNA-seq data, especially to improve their understanding of newly collected data. recount3 is available from http://rna.recount.bio .

High-throughput single-cell technologies hold the promise of discovering novel cellular relationships with disease. However, analytical workflows constructed for these technologies to associate cell proportions with disease often employ unsupervised clustering techniques that overlook the valuable hierarchical structures that have been used to define cell types. We present treekoR, a framework that empirically recapitulates these structures, facilitating multiple quantifications and comparisons of cell type proportions. Our results from twelve case studies reinforce the importance of quantifying proportions relative to parent populations in the analyses of cytometry data - as failing to do so can lead to missing important biological insights.

BACKGROUND: The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods. RESULTS: Here, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5' Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant. CONCLUSIONS: Our method is applicable to establish a comprehensive termination landscape in a broad range of species.

BACKGROUND: Plant secondary growth depends on the activity of the vascular cambium, which produces xylem and phloem. Wood derived from xylem is the most abundant form of biomass globally and has played key socio-economic and subsistence roles throughout human history. However, despite intensive study of vascular development, the full diversity of cell types and the gene networks engaged are still poorly understood. RESULTS: Here, we have applied an optimized protoplast isolation protocol and RNA sequencing to characterize the high-resolution single-cell transcriptional landscape of highly lignified poplar stems. We identify 20 putative cell clusters with a series of novel cluster-specific marker genes and find that these cells are highly heterogeneous based on the transcriptome. Analysis of these marker genes' expression dynamics enables reconstruction of the cell differentiation trajectories involved in phloem and xylem development. We find that different cell clusters exhibit distinct patterns of phytohormone responses and emphasize the use of our data to predict potential gene redundancy and identify candidate genes related to vascular development in trees. CONCLUSIONS: These findings establish the transcriptional landscape of major cell types of poplar stems at single-cell resolution and provide a valuable resource for investigating basic principles of vascular cell specification and differentiation in trees.

Promoters are genomic regions where the transcription machinery binds to initiate the transcription of specific genes. Computational tools for identifying bacterial promoters have been around for decades. However, most of these tools were designed to recognize promoters in one or few bacterial species. Here, we present Promotech, a machine-learning-based method for promoter recognition in a wide range of bacterial species. We compare Promotech's performance with the performance of five other promoter prediction methods. Promotech outperforms these other programs in terms of area under the precision-recall curve (AUPRC) or precision at the same level of recall. Promotech is available at https://github.com/BioinformaticsLabAtMUN/PromoTech .

BACKGROUND: Fragile X syndrome (FXS) is an intellectual disability attributable to loss of fragile X protein (FMRP). We previously demonstrated that FMRP binds mRNAs targeted for nonsense-mediated mRNA decay (NMD) and that FMRP loss results in hyperactivated NMD and inhibition of neuronal differentiation in human stem cells. RESULTS: We show here that NMD is hyperactivated during the development of the cerebral cortex, hippocampus, and cerebellum in the Fmr1-knockout (KO) mouse during embryonic and early postnatal periods. Our findings demonstrate that NMD regulates many neuronal mRNAs that are important for mouse brain development. CONCLUSIONS: We reveal the abnormal regulation of these mRNAs in the Fmr1-KO mouse, a model of FXS, and highlight the importance of early intervention.

BACKGROUND: Heterozygous genomes are widespread in outcrossing and clonally propagated crops. However, the variation in heterozygosity underlying key agronomic traits and crop domestication remains largely unknown. Cassava is a staple crop in Africa and other tropical regions and has a highly heterozygous genome. RESULTS: We describe a genomic variation map from 388 resequenced genomes of cassava cultivars and wild accessions. We identify 52 loci for 23 agronomic traits through a genome-wide association study. Eighteen allelic variations in heterozygosity for nine candidate genes are significantly associated with seven key agronomic traits. We detect 81 selective sweeps with decreasing heterozygosity and nucleotide diversity, harboring 548 genes, which are enriched in multiple biological processes including growth, development, hormone metabolisms and responses, and immune-related processes. Artificial selection for decreased heterozygosity has contributed to the domestication of the large starchy storage root of cassava. Selection for homozygous GG allele in MeTIR1 during domestication contributes to increased starch content. Selection of homozygous AA allele in MeAHL17 is associated with increased storage root weight and cassava bacterial blight (CBB) susceptibility. We have verified the positive roles of MeTIR1 in increasing starch content and MeAHL17 in resistance to CBB by transient overexpression and silencing analysis. The allelic combinations in MeTIR1 and MeAHL17 may result in high starch content and resistance to CBB. CONCLUSIONS: This study provides insights into allelic variation in heterozygosity associated with key agronomic traits and cassava domestication. It also offers valuable resources for the improvement of cassava and other highly heterozygous crops.

BACKGROUND: Three-dimensional (3D) chromatin organization provides a critical foundation to investigate gene expression regulation and cellular homeostasis. RESULTS: Here, we present the first 3D genome architecture maps in wild type and mutant allotetraploid peanut lines, which illustrate A/B compartments, topologically associated domains (TADs), and widespread chromatin interactions. Most peanut chromosomal arms (52.3%) have active regions (A compartments) with relatively high gene density and high transcriptional levels. About 2.0% of chromosomal regions switch from inactive to active (B-to-A) in the mutant line, harboring 58 differentially expressed genes enriched in flavonoid biosynthesis and circadian rhythm functions. The mutant peanut line shows a higher number of genome-wide cis-interactions than its wild-type. The present study reveals a new TAD in the mutant line that generates different chromatin loops and harbors a specific upstream AP2EREBP-binding motif which might upregulate the expression of the GA2ox gene and decrease active gibberellin (GA) content, presumably making the mutant plant dwarf. CONCLUSIONS: Our findings will shed new light on the relationship between 3D chromatin architecture and transcriptional regulation in plants.

BACKGROUND: The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. RESULTS: Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. CONCLUSIONS: Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

Long-read de novo genome assembly continues to advance rapidly. However, there is a lack of effective tools to accurately evaluate the assembly results, especially for structural errors. We present Inspector, a reference-free long-read de novo assembly evaluator which faithfully reports types of errors and their precise locations. Notably, Inspector can correct the assembly errors based on consensus sequences derived from raw reads covering erroneous regions. Based on in silico and long-read assembly results from multiple long-read data and assemblers, we demonstrate that in addition to providing generic metrics, Inspector can accurately identify both large-scale and small-scale assembly errors.

BACKGROUND: Stone cells in fruits of pear (Pyrus pyrifolia) negatively influence fruit quality because their lignified cell walls impart a coarse and granular texture to the fruit flesh. RESULTS: We generate RNA-seq data from the developing fruits of 206 pear cultivars with a wide range of stone cell contents and use a systems genetics approach to integrate co-expression networks and expression quantitative trait loci (eQTLs) to characterize the regulatory mechanisms controlling lignocellulose formation in the stone cells of pear fruits. Our data with a total of 35,897 expressed genes and 974,404 SNPs support the identification of seven stone cell formation modules and the detection of 139,515 eQTLs for 3229 genes in these modules. Focusing on regulatory factors and using a co-expression network comprising 39 structural genes, we identify PbrNSC as a candidate regulator of stone cell formation. We then verify the function of PbrNSC in regulating lignocellulose formation using both pear fruit and Arabidopsis plants and further show that PbrNSC can transcriptionally activate multiple target genes involved in secondary cell wall formation. CONCLUSIONS: This study generates a large resource for studying stone cell formation and provides insights into gene regulatory networks controlling the formation of stone cell and lignocellulose.

BACKGROUND: Recent single-cell transcriptomic studies report that IDH-mutant gliomas share a common hierarchy of cellular phenotypes, independent of genetic subtype. However, the genetic differences between IDH-mutant glioma subtypes are prognostic, predictive of response to chemotherapy, and correlate with distinct tumor microenvironments. RESULTS: To reconcile these findings, we profile 22 human IDH-mutant gliomas using scATAC-seq and scRNA-seq. We determine the cell-type-specific differences in transcription factor expression and associated regulatory grammars between IDH-mutant glioma subtypes. We find that while IDH-mutant gliomas do share a common distribution of cell types, there are significant differences in the expression and targeting of transcription factors that regulate glial identity and cytokine elaboration. We knock out the chromatin remodeler ATRX, which suffers loss-of-function alterations in most IDH-mutant astrocytomas, in an IDH-mutant immunocompetent intracranial murine model. We find that both human ATRX-mutant gliomas and murine ATRX-knockout gliomas are more heavily infiltrated by immunosuppressive monocytic-lineage cells derived from circulation than ATRX-intact gliomas, in an IDH-mutant background. ATRX knockout in murine glioma recapitulates gene expression and open chromatin signatures that are specific to human ATRX-mutant astrocytomas, including drivers of astrocytic lineage and immune-cell chemotaxis. Through single-cell cleavage under targets and tagmentation assays and meta-analysis of public data, we show that ATRX loss leads to a global depletion in CCCTC-binding factor association with DNA, gene dysregulation along associated chromatin loops, and protection from therapy-induced senescence. CONCLUSIONS: These studies explain how IDH-mutant gliomas from different subtypes maintain distinct phenotypes and tumor microenvironments despite a common lineage hierarchy.

A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.

BACKGROUND: Precise gene dosage of the X chromosomes is critical for normal development and cellular function. In mice, XX female somatic cells show transcriptional X chromosome upregulation of their single active X chromosome, while the other X chromosome is inactive. Moreover, the inactive X chromosome is reactivated during development in the inner cell mass and in germ cells through X chromosome reactivation, which can be studied in vitro by reprogramming of somatic cells to pluripotency. How chromatin processes and gene regulatory networks evolved to regulate X chromosome dosage in the somatic state and during X chromosome reactivation remains unclear. RESULTS: Using genome-wide approaches, allele-specific ATAC-seq and single-cell RNA-seq, in female embryonic fibroblasts and during reprogramming to pluripotency, we show that chromatin accessibility on the upregulated mammalian active X chromosome is increased compared to autosomes. We further show that increased accessibility on the active X chromosome is erased by reprogramming, accompanied by erasure of transcriptional X chromosome upregulation and the loss of increased transcriptional burst frequency. In addition, we characterize gene regulatory networks during reprogramming and X chromosome reactivation, revealing changes in regulatory states. Our data show that ZFP42/REX1, a pluripotency-associated gene that evolved specifically in placental mammals, targets multiple X-linked genes, suggesting an evolutionary link between ZFP42/REX1, X chromosome reactivation, and pluripotency. CONCLUSIONS: Our data reveal the existence of intrinsic compensatory mechanisms that involve modulation of chromatin accessibility to counteract X-to-Autosome gene dosage imbalances caused by evolutionary or in vitro X chromosome loss and X chromosome inactivation in mammalian cells.