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For many years, machine learning-based ab initio gene finding approaches have been central components of eukaryotic genome annotation pipelines, and they remain so today. The reliance on these approaches was originally sustained by the high cost and low availability of gene expression data, a primary source of evidence for gene annotation along with protein homology. However, innovations in modern sequencing technologies have revolutionized the acquisition of gene expression data, allowing scientists to rely more heavily on this class of evidence. In addition, proteins found in a multitude of well-annotated genomes represent another invaluable resource for gene annotation. Existing annotation packages often underutilize these data sources, which prompted us to develop EviAnn (Evidence-based Annotator), a novel evidence-based eukaryotic gene annotation system. EviAnn takes a strongly data-driven approach, building the exon-intron structure of genes from transcript alignments or protein-sequence homology rather than from purely ab initio gene finding techniques. We show that when provided with the same input data, EviAnn consistently outperforms current state-of-the-art packages including BRAKER3, MAKER2, and FINDER, while utilizing considerably less computer time. Annotation of a mammalian genome can be completed in less than an hour on a single multi-core server. EviAnn is freely available under an open-source license from https://github.com/alekseyzimin/EviAnn_release and from Bioconda as “eviann”. 

Abstract Great Basin bristlecone pine (Pinus longaeva), one of two species of bristlecone pine, the other being Rocky Mountain bristlecone pine (P. aristata), is endemic to the high Great Basin mountains in eastern California, Nevada, and Utah. It is the upper treeline forest tree in this region, found mostly between 2900 and 3600 m. The primary goal of this project was to generate a reference genome sequence for P. longaeva that, among its many possible applications, will serve as an important genetic resource to better understand the genetic mechanisms underlying its extreme longevity and its adaptation to the extreme environmental conditions where it is found. A combination of short-read and long-read sequences were generated from haploid megagametophyte and diploid needle tissues, respectively. A customized genome assembly approach was used to construct a highly contiguous 23.8-gigabase genome with a scaffold N50 size of 1.2 gigabases. The chloroplast and mitochondrial genomes were assembled separately into circular chromosomes with lengths of 120 kilobases and 8.68 megabases, respectively. While the number of disease resistance genes known as nucleotide-binding leucine-rich repeat receptors (NLRs) and larger-than-average telomere lengths relative to other conifers have been suggested as genetic mechanisms for controlling longevity, we did not find strong evidence for their involvement. Clearly further study is needed. 

Background Long-read RNA sequencing technologies can produce complete or near-complete transcript sequences. Recently introduced methods for direct RNA and cDNA sequencing can provide a high-throughput strategy for the discovery of novel and rare gene isoforms. However, the high error rates in ONT sequences limit the ability to exactly pinpoint splice site boundaries when aligning reads to the genome. Methods In this paper, we present a novel tool called NIFFLR (Novel IsoForm Finder using Long Reads) that identifies and quantifies both known and novel isoforms using long-read RNA sequencing data. NIFFLR recovers known transcripts and assembles novel transcripts present in the data by aligning exons from a reference annotation to the long reads. Results NIFFLR effectively recovers correct transcripts from simulated reads based on known transcript annotations, achieving higher sensitivity and precision compared to several previously published tools. On real data, NIFFLR shows high accuracy as measured by concordance of isoform counts to the counts computed from Illumina data for the same sample. We applied NIFFLR to a set of 92 GTEx long-read samples and produced transcript counts for both novel and known isoforms. In total, we identified and quantified 119,928 isoforms present in the RefSeq annotation of GRCh38 and 42,868 novel isoforms across 34,383 genes, more than previous studies identified in this dataset. Conclusions NIFFLR is an effective tool aimed at assembly and quantification of transcripts present in the long, high error transcriptome reads. NIFFLR is released under an open-source license (GPL 3.0) and is available on GitHub at https://github.com/alguoo314/NIFFLR/releases, and from BioConda as “nifflr”. 

Abstract We used long-read DNA sequencing to assemble the genome of a Southern Han Chinese male. We organized the sequence into chromosomes and filled in gaps using the recently completed T2T-CHM13 genome as a guide, yielding a gap-free genome, Han1, containing 3,099,707,698 bases. Using the T2T-CHM13 annotation as a reference, we mapped all genes onto the Han1 genome and identified additional gene copies, generating a total of 60,708 putative genes, of which 20,003 are protein-coding. A comprehensive comparison between the genes revealed that 235 protein-coding genes were substantially different between the individuals, with frameshifts or truncations affecting the protein-coding sequence. Most of these were heterozygous variants in which one gene copy was unaffected. This represents the first gene-level comparison between two finished, annotated individual human genomes. 

Abstract Whitebark pine (WBP, Pinus albicaulis) is a white pine of subalpine regions in the Western contiguous United States and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola) and additional threats from mountain pine beetle (Dendroctonus ponderosae), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short reads of haploid megagametophyte tissue and Oxford Nanopore long reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gb of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gb). Approximately 87.2% (24.0 Gb) of total sequence was placed on the 12 WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the 3 subclasses of NLRs. Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo-assembled transcriptomes. 

Abstract Stalk-eyed flies in the genus Teleopsis carry selfish genetic elements that induce sex ratio (SR) meiotic drive and impact the fitness of male and female carriers. Here, we assemble and describe a chromosome-level genome assembly of the stalk-eyed fly, Teleopsis dalmanni, to elucidate patterns of divergence associated with SR. The genome contains tens of thousands of transposable element (TE) insertions and hundreds of transcriptionally and insertionally active TE families. By resequencing pools of SR and ST males using short and long reads, we find widespread differentiation and divergence between XSR and XST associated with multiple nested inversions involving most of the SR haplotype. Examination of genomic coverage and gene expression data revealed seven X-linked genes with elevated expression and coverage in SR males. The most extreme and likely drive candidate involves an XSR-specific expansion of an array of partial copies of JASPer, a gene necessary for maintenance of euchromatin and associated with regulation of TE expression. In addition, we find evidence for rapid protein evolution between XSR and XST for testis expressed and novel genes, that is, either recent duplicates or lacking a Dipteran ortholog, including an X-linked duplicate of maelstrom, which is also involved in TE silencing. Overall, the evidence suggests that this ancient XSR polymorphism has had a variety of impacts on repetitive DNA and its regulation in this species. 

Third-generation sequencing technologies can generate very long reads with relatively high error rates. The lengths of the reads, which sometimes exceed one million bases, make them invaluable for resolving complex repeats that cannot be assembled using shorter reads. Many high-quality genome assemblies have already been produced, curated, and annotated using the previous generation of sequencing data, and full re-assembly of these genomes with long reads is not always practical or cost-effective. One strategy to upgrade existing assemblies is to generate additional coverage using long-read data, and add that to the previously assembled contigs. SAMBA is a tool that is designed to scaffold and gap-fill existing genome assemblies with additional long-read data, resulting in substantially greater contiguity. SAMBA is the only tool of its kind that also computes and fills in the sequence for all spanned gaps in the scaffolds, yielding much longer contigs. Here we compare SAMBA to several similar tools capable of re-scaffolding assemblies using long-read data, and we show that SAMBA yields better contiguity and introduces fewer errors than competing methods. SAMBA is open-source software that is distributed at https://github.com/alekseyzimin/masurca . 

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.