Expression Atlas update: insights from sequencing data at both bulk and single cell level.

Expression Atlas (www.ebi.ac.uk/gxa) and its newest counterpart the Single Cell Expression Atlas (www.ebi.ac.uk/gxa/sc) are EMBL-EBI's knowledgebases for gene and protein expression and localisation in bulk and at single cell level. These resources aim to allow users to investigate their expression in normal tissue (baseline) or in response to perturbations such as disease or changes to genotype (differential) across multiple species. Users are invited to search for genes or metadata terms across species or biological conditions in a standardised consistent interface. Alongside these data, new features in Single Cell Expression Atlas allow users to query metadata through our new cell type wheel search. At the experiment level data can be explored through two types of dimensionality reduction plots, t-distributed Stochastic Neighbor Embedding (tSNE) and Uniform Manifold Approximation and Projection (UMAP), overlaid with either clustering or metadata information to assist users' understanding. Data are also visualised as marker gene heatmaps identifying genes that help confer cluster identity. For some data, additional visualisations are available as interactive cell level anatomograms and cell type gene expression heatmaps.

A large sequenced mutant library - valuable reverse genetic resource that covers 98% of sorghum genes.

Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat-tolerant C4 species, requires a vast, large-scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large-scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)-induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis-regulatory elements. A comparison of the distribution of mutations in the large-scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS-induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5' UTR, gene body, and 3'-UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the Ka /Ks value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB (https://www.depts.ttu.edu/igcast/sorbmutdb.php) and SorghumBase (https://sorghumbase.org/). This current large-scale sequence-indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

An improved reference of the grapevine genome reasserts the origin of the PN40024 highly homozygous genotype.

The genome sequence of the diploid and highly homozygous Vitis vinifera genotype PN40024 serves as the reference for many grapevine studies. Despite several improvements to the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, being nearly homozygous, this genome contains several heterozygous regions that are yet to be resolved. Taking the opportunity of improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences, an improved version of the reference, called PN40024.v4, was generated. Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased with a total number decreasing from 2,059 to 640 and a reduction in N bases of 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the number of unplaced scaffolds was reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow for Vitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation of 35,230 genes to date. Finally, we demonstrated that PN40024 resulted from 9 selfings of cv. "Helfensteiner" (cross of cv. "Pinot noir" and "Schiava grossa") instead of a single "Pinot noir". These advances will help maintain the PN40024 genome as a gold-standard reference, also contributing toward the eventual elaboration of the grapevine pangenome.

De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes.

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The number of pan-genes in these diverse genomes exceeds 103,000, with approximately a third found across all genotypes. The results demonstrate that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres revealed additional variation in major cytological landmarks. We show that combining structural variation with single-nucleotide polymorphisms can improve the power of quantitative mapping studies. We also document variation at the level of DNA methylation and demonstrate that unmethylated regions are enriched for cis-regulatory elements that contribute to phenotypic variation.

Gramene 2021: harnessing the power of comparative genomics and pathways for plant research.

Gramene (http://www.gramene.org), a knowledgebase founded on comparative functional analyses of genomic and pathway data for model plants and major crops, supports agricultural researchers worldwide. The resource is committed to open access and reproducible science based on the FAIR data principles. Since the last NAR update, we made nine releases; doubled the genome portal's content; expanded curated genes, pathways and expression sets; and implemented the Domain Informational Vocabulary Extraction (DIVE) algorithm for extracting gene function information from publications. The current release, #63 (October 2020), hosts 93 reference genomes-over 3.9 million genes in 122 947 families with orthologous and paralogous classifications. Plant Reactome portrays pathway networks using a combination of manual biocuration in rice (320 reference pathways) and orthology-based projections to 106 species. The Reactome platform facilitates comparison between reference and projected pathways, gene expression analyses and overlays of gene-gene interactions. Gramene integrates ontology-based protein structure-function annotation; information on genetic, epigenetic, expression, and phenotypic diversity; and gene functional annotations extracted from plant-focused journals using DIVE. We train plant researchers in biocuration of genes and pathways; host curated maize gene structures as tracks in the maize genome browser; and integrate curated rice genes and pathways in the Plant Reactome.

Ensembl Genomes 2020-enabling non-vertebrate genomic research.

Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species, complementing the resources for vertebrate genomics developed in the context of the Ensembl project (http://www.ensembl.org). Together, the two resources provide a consistent set of interfaces to genomic data across the tree of life, including reference genome sequence, gene models, transcriptional data, genetic variation and comparative analysis. Data may be accessed via our website, online tools platform and programmatic interfaces, with updates made four times per year (in synchrony with Ensembl). Here, we provide an overview of Ensembl Genomes, with a focus on recent developments. These include the continued growth, more robust and reproducible sets of orthologues and paralogues, and enriched views of gene expression and gene function in plants. Finally, we report on our continued deeper integration with the Ensembl project, which forms a key part of our future strategy for dealing with the increasing quantity of available genome-scale data across the tree of life.

Plant Reactome: a knowledgebase and resource for comparative pathway analysis.

Plant Reactome (https://plantreactome.gramene.org) is an open-source, comparative plant pathway knowledgebase of the Gramene project. It uses Oryza sativa (rice) as a reference species for manual curation of pathways and extends pathway knowledge to another 82 plant species via gene-orthology projection using the Reactome data model and framework. It currently hosts 298 reference pathways, including metabolic and transport pathways, transcriptional networks, hormone signaling pathways, and plant developmental processes. In addition to browsing plant pathways, users can upload and analyze their omics data, such as the gene-expression data, and overlay curated or experimental gene-gene interaction data to extend pathway knowledge. The curation team actively engages researchers and students on gene and pathway curation by offering workshops and online tutorials. The Plant Reactome supports, implements and collaborates with the wider community to make data and tools related to genes, genomes, and pathways Findable, Accessible, Interoperable and Re-usable (FAIR).

Double triage to identify poorly annotated genes in maize: The missing link in community curation.

The sophistication of gene prediction algorithms and the abundance of RNA-based evidence for the maize genome may suggest that manual curation of gene models is no longer necessary. However, quality metrics generated by the MAKER-P gene annotation pipeline identified 17,225 of 130,330 (13%) protein-coding transcripts in the B73 Reference Genome V4 gene set with models of low concordance to available biological evidence. Working with eight graduate students, we used the Apollo annotation editor to curate 86 transcript models flagged by quality metrics and a complimentary method using the Gramene gene tree visualizer. All of the triaged models had significant errors-including missing or extra exons, non-canonical splice sites, and incorrect UTRs. A correct transcript model existed for about 60% of genes (or transcripts) flagged by quality metrics; we attribute this to the convention of elevating the transcript with the longest coding sequence (CDS) to the canonical, or first, position. The remaining 40% of flagged genes resulted in novel annotations and represent a manual curation space of about 10% of the maize genome (~4,000 protein-coding genes). MAKER-P metrics have a specificity of 100%, and a sensitivity of 85%; the gene tree visualizer has a specificity of 100%. Together with the Apollo graphical editor, our double triage provides an infrastructure to support the community curation of eukaryotic genomes by scientists, students, and potentially even citizen scientists.

Gramene 2018: unifying comparative genomics and pathway resources for plant research.

Gramene (http://www.gramene.org) is a knowledgebase for comparative functional analysis in major crops and model plant species. The current release, #54, includes over 1.7 million genes from 44 reference genomes, most of which were organized into 62,367 gene families through orthologous and paralogous gene classification, whole-genome alignments, and synteny. Additional gene annotations include ontology-based protein structure and function; genetic, epigenetic, and phenotypic diversity; and pathway associations. Gramene's Plant Reactome provides a knowledgebase of cellular-level plant pathway networks. Specifically, it uses curated rice reference pathways to derive pathway projections for an additional 66 species based on gene orthology, and facilitates display of gene expression, gene-gene interactions, and user-defined omics data in the context of these pathways. As a community portal, Gramene integrates best-of-class software and infrastructure components including the Ensembl genome browser, Reactome pathway browser, and Expression Atlas widgets, and undergoes periodic data and software upgrades. Via powerful, intuitive search interfaces, users can easily query across various portals and interactively analyze search results by clicking on diverse features such as genomic context, highly augmented gene trees, gene expression anatomograms, associated pathways, and external informatics resources. All data in Gramene are accessible through both visual and programmatic interfaces.

Gramene 2016: comparative plant genomics and pathway resources.

Gramene (http://www.gramene.org) is an online resource for comparative functional genomics in crops and model plant species. Its two main frameworks are genomes (collaboration with Ensembl Plants) and pathways (The Plant Reactome and archival BioCyc databases). Since our last NAR update, the database website adopted a new Drupal management platform. The genomes section features 39 fully assembled reference genomes that are integrated using ontology-based annotation and comparative analyses, and accessed through both visual and programmatic interfaces. Additional community data, such as genetic variation, expression and methylation, are also mapped for a subset of genomes. The Plant Reactome pathway portal (http://plantreactome.gramene.org) provides a reference resource for analyzing plant metabolic and regulatory pathways. In addition to ∼ 200 curated rice reference pathways, the portal hosts gene homology-based pathway projections for 33 plant species. Both the genome and pathway browsers interface with the EMBL-EBI's Expression Atlas to enable the projection of baseline and differential expression data from curated expression studies in plants. Gramene's archive website (http://archive.gramene.org) continues to provide previously reported resources on comparative maps, markers and QTL. To further aid our users, we have also introduced a live monthly educational webinar series and a Gramene YouTube channel carrying video tutorials.