skip to main content

Title: High-quality chromosome-scale assembly of the walnut (Juglans regia L.) reference genome
Abstract Background The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements in the characterization of walnut genetic and functional variation. However, it is highly fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to fully elucidate walnut biological processes. Findings Here, we report the new chromosome-scale assembly of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the previous reference genome, the new assembly features an 84.4-fold increase in N50 size, with the 16 chromosomal pseudomolecules assembled and representing 95% of its total length. Using full-length transcripts from single-molecule real-time sequencing, we predicted 37,554 gene models, with a mean gene length higher than the previous gene annotations. Most of the new protein-coding genes (90%) present both start and stop codons, which represents a significant improvement compared with Chandler v1.0 (only 48%). We then tested the potential impact of the new chromosome-level genome on different areas of walnut research. By studying the proteome changes occurring during male flower development, we observed that the virtual proteome obtained from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic regions highly differentiated between Western and Eastern walnut cultivars. Conclusion Overall, Chandler v2.0 will serve as a valuable resource to better understand and explore walnut biology.  more » « less
Award ID(s):
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ; ;  ;  ;  ;  ;  ;  ;  ;  
Date Published:
Journal Name:
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. INTRODUCTION Transposable elements (TEs), repeat expansions, and repeat-mediated structural rearrangements play key roles in chromosome structure and species evolution, contribute to human genetic variation, and substantially influence human health through copy number variants, structural variants, insertions, deletions, and alterations to gene transcription and splicing. Despite their formative role in genome stability, repetitive regions have been relegated to gaps and collapsed regions in human genome reference GRCh38 owing to the technological limitations during its development. The lack of linear sequence in these regions, particularly in centromeres, resulted in the inability to fully explore the repeat content of the human genome in the context of both local and regional chromosomal environments. RATIONALE Long-read sequencing supported the complete, telomere-to-telomere (T2T) assembly of the pseudo-haploid human cell line CHM13. This resource affords a genome-scale assessment of all human repetitive sequences, including TEs and previously unknown repeats and satellites, both within and outside of gaps and collapsed regions. Additionally, a complete genome enables the opportunity to explore the epigenetic and transcriptional profiles of these elements that are fundamental to our understanding of chromosome structure, function, and evolution. Comparative analyses reveal modes of repeat divergence, evolution, and expansion or contraction with locus-level resolution. RESULTS We implemented a comprehensive repeat annotation workflow using previously known human repeats and de novo repeat modeling followed by manual curation, including assessing overlaps with gene annotations, segmental duplications, tandem repeats, and annotated repeats. Using this method, we developed an updated catalog of human repetitive sequences and refined previous repeat annotations. We discovered 43 previously unknown repeats and repeat variants and characterized 19 complex, composite repetitive structures, which often carry genes, across T2T-CHM13. Using precision nuclear run-on sequencing (PRO-seq) and CpG methylated sites generated from Oxford Nanopore Technologies long-read sequencing data, we assessed RNA polymerase engagement across retroelements genome-wide, revealing correlations between nascent transcription, sequence divergence, CpG density, and methylation. These analyses were extended to evaluate RNA polymerase occupancy for all repeats, including high-density satellite repeats that reside in previously inaccessible centromeric regions of all human chromosomes. Moreover, using both mapping-dependent and mapping-independent approaches across early developmental stages and a complete cell cycle time series, we found that engaged RNA polymerase across satellites is low; in contrast, TE transcription is abundant and serves as a boundary for changes in CpG methylation and centromere substructure. Together, these data reveal the dynamic relationship between transcriptionally active retroelement subclasses and DNA methylation, as well as potential mechanisms for the derivation and evolution of new repeat families and composite elements. Focusing on the emerging T2T-level assembly of the HG002 X chromosome, we reveal that a high level of repeat variation likely exists across the human population, including composite element copy numbers that affect gene copy number. Additionally, we highlight the impact of repeats on the structural diversity of the genome, revealing repeat expansions with extreme copy number differences between humans and primates while also providing high-confidence annotations of retroelement transduction events. CONCLUSION The comprehensive repeat annotations and updated repeat models described herein serve as a resource for expanding the compendium of human genome sequences and reveal the impact of specific repeats on the human genome. In developing this resource, we provide a methodological framework for assessing repeat variation within and between human genomes. The exhaustive assessment of the transcriptional landscape of repeats, at both the genome scale and locally, such as within centromeres, sets the stage for functional studies to disentangle the role transcription plays in the mechanisms essential for genome stability and chromosome segregation. Finally, our work demonstrates the need to increase efforts toward achieving T2T-level assemblies for nonhuman primates and other species to fully understand the complexity and impact of repeat-derived genomic innovations that define primate lineages, including humans. Telomere-to-telomere assembly of CHM13 supports repeat annotations and discoveries. The human reference T2T-CHM13 filled gaps and corrected collapsed regions (triangles) in GRCh38. Combining long read–based methylation calls, PRO-seq, and multilevel computational methods, we provide a compendium of human repeats, define retroelement expression and methylation profiles, and delineate locus-specific sites of nascent transcription genome-wide, including previously inaccessible centromeres. SINE, short interspersed element; SVA, SINE–variable number tandem repeat– Alu ; LINE, long interspersed element; LTR, long terminal repeat; TSS, transcription start site; pA, xxxxxxxxxxxxxxxx. 
    more » « less
  2. Abstract Background Gastrointestinal (GIT) helminthiasis is a global problem that affects livestock health, especially in small ruminants. One of the major helminth parasites of sheep and goats, Teladorsagia circumcincta , infects the abomasum and causes production losses, reductions in weight gain, diarrhoea and, in some cases, death in young animals. Control strategies have relied heavily on the use of anthelmintic medication but, unfortunately, T. circumcincta has developed resistance, as have many helminths. Vaccination offers a sustainable and practical solution, but there is no commercially available vaccine to prevent Teladorsagiosis. The discovery of new strategies for controlling T. circumcincta , such as novel vaccine targets and drug candidates, would be greatly accelerated by the availability of better quality, chromosome-length, genome assembly because it would allow the identification of key genetic determinants of the pathophysiology of infection and host-parasite interaction. The available draft genome assembly of T. circumcincta (GCA_002352805.1) is highly fragmented and thus impedes large-scale investigations of population and functional genomics. Results We have constructed a high-quality reference genome, with chromosome-length scaffolds, by purging alternative haplotypes from the existing draft genome assembly and scaffolding the result using chromosome conformation, capture-based, in situ Hi-C technique. The improved (Hi-C) assembly resulted in six chromosome-length scaffolds with length ranging from 66.6 Mbp to 49.6 Mbp, 35% fewer sequences and reduction in size. Substantial improvements were also achieved in both the values for N50 (57.1 Mbp) and L50 (5 Mbp). A higher and comparable level of genome and proteome completeness was achieved for Hi-C assembly on BUSCO parameters. The Hi-C assembly had a greater synteny and number of orthologs with a closely related nematode, Haemonchus contortus. Conclusion This improved genomic resource is suitable as a foundation for the identification of potential targets for vaccine and drug development. 
    more » « less
  3. Abstract Hares (genus Lepus) provide clear examples of repeated and often massive introgressive hybridization and striking local adaptations. Genomic studies on this group have so far relied on comparisons to the European rabbit (Oryctolagus cuniculus) reference genome. Here, we report the first de novo draft reference genome for a hare species, the mountain hare (Lepus timidus), and evaluate the efficacy of whole-genome re-sequencing analyses using the new reference versus using the rabbit reference genome. The genome was assembled using the ALLPATHS-LG protocol with a combination of overlapping pair and mate-pair Illumina sequencing (77x coverage). The assembly contained 32,294 scaffolds with a total length of 2.7 Gb and a scaffold N50 of 3.4 Mb. Re-scaffolding based on the rabbit reference reduced the total number of scaffolds to 4,205 with a scaffold N50 of 194 Mb. A correspondence was found between 22 of these hare scaffolds and the rabbit chromosomes, based on gene content and direct alignment. We annotated 24,578 protein coding genes by combining ab-initio predictions, homology search, and transcriptome data, of which 683 were solely derived from hare-specific transcriptome data. The hare reference genome is therefore a new resource to discover and investigate hare-specific variation. Similar estimates of heterozygosity and inferred demographic history profiles were obtained when mapping hare whole-genome re-sequencing data to the new hare draft genome or to alternative references based on the rabbit genome. Our results validate previous reference-based strategies and suggest that the chromosome-scale hare draft genome should enable chromosome-wide analyses and genome scans on hares. 
    more » « less
  4. null (Ed.)
    Legumes are of great interest for sustainable agricultural production as they fix atmospheric nitrogen to improve the soil. Medicago truncatula is a well-established model legume, and extensive studies in fundamental molecular, physiological, and developmental biology have been undertaken to translate into trait improvements in economically important legume crops worldwide. However, M. truncatula reference genome was generated in the accession Jemalong A17, which is highly recalcitrant to transformation. M. truncatula R108 is more attractive for genetic studies due to its high transformation efficiency and Tnt1-insertion population resource for functional genomics. The need to perform accurate synteny analysis and comprehensive genome-scale comparisons necessitates a chromosome-length genome assembly for M. truncatula cv. R108. Here, we performed in situ Hi-C (48×) to anchor, order, orient scaffolds, and correct misjoins of contigs in a previously published genome assembly (R108 v1.0), resulting in an improved genome assembly containing eight chromosome-length scaffolds that span 97.62% of the sequenced bases in the input assembly. The long-range physical information data generated using Hi-C allowed us to obtain a chromosome-length ordering of the genome assembly, better validate previous draft misjoins, and provide further insights accurately predicting synteny between A17 and R108 regions corresponding to the known chromosome 4/8 translocation. Furthermore, mapping the Tnt1 insertion landscape on this reference assembly presents an important resource for M. truncatula functional genomics by supporting efficient mutant gene identification in Tnt1 insertion lines. Our data provide a much-needed foundational resource that supports functional and molecular research into the Leguminosae for sustainable agriculture and feeding the future. 
    more » « less
  5. Abstract The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has benefitted society 1,2 . However, it still has many gaps and errors, and does not represent a biological genome as it is a blend of multiple individuals 3,4 . Recently, a high-quality telomere-to-telomere reference, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a nearly homozygous genome 5 . To address these limitations, the Human Pangenome Reference Consortium formed with the goal of creating high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity 6 . Here, in our first scientific report, we determined which combination of current genome sequencing and assembly approaches yield the most complete and accurate diploid genome assembly with minimal manual curation. Approaches that used highly accurate long reads and parent–child data with graph-based haplotype phasing during assembly outperformed those that did not. Developing a combination of the top-performing methods, we generated our first high-quality diploid reference assembly, containing only approximately four gaps per chromosome on average, with most chromosomes within ±1% of the length of CHM13. Nearly 48% of protein-coding genes have non-synonymous amino acid changes between haplotypes, and centromeric regions showed the highest diversity. Our findings serve as a foundation for assembling near-complete diploid human genomes at scale for a pangenome reference to capture global genetic variation from single nucleotides to structural rearrangements. 
    more » « less