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Abstract PremiseRobust standards to evaluate quality and completeness are lacking in eukaryotic structural genome annotation, as genome annotation software is developed using model organisms and typically lacks benchmarking to comprehensively evaluate the quality and accuracy of the final predictions. The annotation of plant genomes is particularly challenging due to their large sizes, abundant transposable elements, and variable ploidies. This study investigates the impact of genome quality, complexity, sequence read input, and method on protein‐coding gene predictions. MethodsThe impact of repeat masking, long‐read and short‐read inputs, and de novo and genome‐guided protein evidence was examined in the context of the popular BRAKER and MAKER workflows for five plant genomes. The annotations were benchmarked for structural traits and sequence similarity. ResultsBenchmarks that reflect gene structures, reciprocal similarity search alignments, and mono‐exonic/multi‐exonic gene counts provide a more complete view of annotation accuracy. Transcripts derived from RNA‐read alignments alone are not sufficient for genome annotation. Gene prediction workflows that combine evidence‐based and ab initio approaches are recommended, and a combination of short and long reads can improve genome annotation. Adding protein evidence from de novo assemblies, genome‐guided transcriptome assemblies, or full‐length proteins from OrthoDB generates more putative false positives as implemented in the current workflows. Post‐processing with functional and structural filters is highly recommended. DiscussionWhile the annotation of non‐model plant genomes remains complex, this study provides recommendations for inputs and methodological approaches. We discuss a set of best practices to generate an optimal plant genome annotation and present a more robust set of metrics to evaluate the resulting predictions. 

Abstract Background Protein inter-residue contact and distance prediction are two key intermediate steps essential to accurate protein structure prediction. Distance prediction comes in two forms: real-valued distances and ‘binned’ distograms, which are a more finely grained variant of the binary contact prediction problem. The latter has been introduced as a new challenge in the 14th Critical Assessment of Techniques for Protein Structure Prediction (CASP14) 2020 experiment. Despite the recent proliferation of methods for predicting distances, few methods exist for evaluating these predictions. Currently only numerical metrics, which evaluate the entire prediction at once, are used. These give no insight into the structural details of a prediction. For this reason, new methods and tools are needed. Results We have developed a web server for evaluating predicted inter-residue distances. Our server, DISTEVAL, accepts predicted contacts, distances, and a true structure as optional inputs to generate informative heatmaps, chord diagrams, and 3D models. All of these outputs facilitate visual and qualitative assessment. The server also evaluates predictions using other metrics such as mean absolute error, root mean squared error, and contact precision. Conclusions The visualizations generated by DISTEVAL complement each other and collectively serve as a powerful tool for both quantitative and qualitative assessments of predicted contacts and distances, even in the absence of a true 3D structure. 

Abstract Sequencing, assembly, and annotation of the 26.5 Gbp hexaploid genome of coast redwood (Sequoia sempervirens) was completed leading toward discovery of genes related to climate adaptation and investigation of the origin of the hexaploid genome. Deep-coverage short-read Illumina sequencing data from haploid tissue from a single seed were combined with long-read Oxford Nanopore Technologies sequencing data from diploid needle tissue to create an initial assembly, which was then scaffolded using proximity ligation data to produce a highly contiguous final assembly, SESE 2.1, with a scaffold N50 size of 44.9 Mbp. The assembly included several scaffolds that span entire chromosome arms, confirmed by the presence of telomere and centromere sequences on the ends of the scaffolds. The structural annotation produced 118,906 genes with 113 containing introns that exceed 500 Kbp in length and one reaching 2 Mb. Nearly 19 Gbp of the genome represented repetitive content with the vast majority characterized as long terminal repeats, with a 2.9:1 ratio of Copia to Gypsy elements that may aid in gene expression control. Comparison of coast redwood to other conifers revealed species-specific expansions for a plethora of abiotic and biotic stress response genes, including those involved in fungal disease resistance, detoxification, and physical injury/structural remodeling and others supporting flavonoid biosynthesis. Analysis of multiple genes that exist in triplicate in coast redwood but only once in its diploid relative, giant sequoia, supports a previous hypothesis that the hexaploidy is the result of autopolyploidy rather than any hybridizations with separate but closely related conifer species.