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Robust 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.

The 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.

Benchmarks 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.

While 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.

 

Douglas-fir (Pseudotsuga menziesii) is native to western North America. It grows in a wide range of environmental conditions and is an important timber tree. Although there are several studies on the gene expression responses of Douglas-fir to abiotic cues, the absence of high-quality transcriptome and genome data is a barrier to further investigation. Like for most conifers, the available transcriptome and genome reference dataset for Douglas-fir remains fragmented and requires refinement. We aimed to generate a highly accurate, and complete reference transcriptome and genome annotation. We deep-sequenced the transcriptome of Douglas-fir needles from seedlings that were grown under nonstress control conditions or a combination of heat and drought stress conditions using long-read (LR) and short-read (SR) sequencing platforms. We used 2 computational approaches, namely de novo and genome-guided LR transcriptome assembly. Using the LR de novo assembly, we identified 1.3X more high-quality transcripts, 1.85X more “complete” genes, and 2.7X more functionally annotated genes compared to the genome-guided assembly approach. We predicted 666 long noncoding RNAs and 12,778 unique protein-coding transcripts including 2,016 putative transcription factors. We leveraged the LR de novo assembled transcriptome with paired-end SR and a published single-end SR transcriptome to generate an improved genome annotation. This was conducted with BRAKER2 and refined based on functional annotation, repetitive content, and transcriptome alignment. This high-quality genome annotation has 51,419 unique gene models derived from 322,631 initial predictions. Overall, our informatics approach provides a new reference Douglas-fir transcriptome assembly and genome annotation with considerably improved completeness and functional annotation.

 

Abstract The giant sequoia (Sequoiadendron giganteum) of California are massive, long-lived trees that grow along the U.S. Sierra Nevada mountains. Genomic data are limited in giant sequoia and producing a reference genome sequence has been an important goal to allow marker development for restoration and management. Using deep-coverage Illumina and Oxford Nanopore sequencing, combined with Dovetail chromosome conformation capture libraries, the genome was assembled into eleven chromosome-scale scaffolds containing 8.125 Gbp of sequence. Iso-Seq transcripts, assembled from three distinct tissues, was used as evidence to annotate a total of 41,632 protein-coding genes. The genome was found to contain, distributed unevenly across all 11 chromosomes and in 63 orthogroups, over 900 complete or partial predicted NLR genes, of which 375 are supported by annotation derived from protein evidence and gene modeling. This giant sequoia reference genome sequence represents the first genome sequenced in the Cupressaceae family, and lays a foundation for using genomic tools to aid in giant sequoia conservation and management. 

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.