#ABACBS2024 Poster 102: Improving phased Hifiasm assemblies with 20 kb ONT reads

After a great presentation this morning by Emma de Jong on our High-Quality Genomes for Australian Lutjanidae Species (abstract below), if you’re at ABACBS2024 then please drop by Poster #102 to find out about some of the work we’re doing with ONT data.

Abstracts

Improving phased Hifiasm assemblies with 20 kb ONT reads

Richard J Edwards, Adrianne Doran, Emma de Jong, Lara Parata, Shannon Corrigan

The quality and quantity of genome assembly has improved dramatically over recent years. Many large-scale genome projects combine assembly of HiFi and HiC reads using Hifiasm to produce contiguous phased assemblies, scaffolded to chromosome-level. Nevertheless, HiFi reads are typically under 25 kb and can still struggle to assemble long, low diversity repeat regions. Obtaining ultra-long (100 kb or longer) ONT reads to solve this problem remains a significant challenge due to technical constraints and DNA sample requirements. Here, we explore the utility of using standard ONT long reads (20 kb or more) as “ultra-long” input to improve phased Hifiasm assemblies for 22 species of bony fish (Genome Size, 627 Mb 1.54 Gb). We also explore whether the new --telo-m mode in Hifiasm v0.9.0 improves telomere prediction. Incorporating 20kb+ ONT reads (7.8X 93.5X) significantly increased assembly contiguity. BUSCO Completeness was not significantly altered, although there was some re-partitioning of BUSCO genes between phased haplotypes for some species. Improvement did not strongly correlate with read depth (either HiFi or ONT), suggesting that the underlying read length distributions and/or specific genome features are more important for determining the outcome. Hifiasm --telo-m mode significantly increased telomere recovery, assembling over six times the number of gapless telomere-to-telomere chromosomes when combined with 20kb+ ONT reads. Verification of how these results translate to the ease of curation and/or quality of final HiC-scaffolded chromosome-level assemblies is ongoing, with a goal to determine whether the additional sample preparation and sequencing in the lab is cost-effective.

High-Quality Genomes for Australian Lutjanidae Species

Emma de Jong, Lara Parata, Philipp E Bayer, Shannon Corrigan, Richard J Edwards

Lutjanidae (snappers) are highly valued in commercial and recreational fisheries worldwide and serve as indicator species of the health of marine environments and fishery bioregions in Western Australia. Comprehensive genomic mapping of immune gene families of Lutjanidae species are lacking, but this information is critical for understanding disease vulnerability, the impact of environmental stress, improving aquaculture efforts and to provide insights into the health of wild populations. Despite their importance, only 3 out of 113 Lutjanid species currently have available reference genomes, two of which are highly fragmented (>11,000 and >200,000 contigs), impacting studies on gene families relevant to aquaculture. In this study, we present high-quality chromosome-level reference genomes for 14 Australian Lutjanidae species across seven genera, generated using HiFi and HiC data. We present initial comparative genomic analyses, including immune gene content and chromosomal synteny analyses across species. These analyses provide insights into the genomic architecture and evolutionary relationships within Lutjanidae. Ongoing work aims to comprehensively map and compare the immune gene family repertoire across Lutjanidae genera, as well as Lethrinidae species as an outgroup, to determine genus-specific changes in genes (e.g. loss, selection, duplication) important for pathogen detection, antigen presentation, inflammation, and immune memory. These genome assemblies will serve as a foundational resource to the wider scientific community interested in Lutjanidae

#PAGAustralia Poster 14: Synteny-Guided Semi-Automated Curation of Chromosome-Level Genome Assemblies

If you are attending PAG Australia 2024, come and have a chat at Poster 14 about easing the burden of chromosome-level assembly curation.

Abstract Text

Reference genomes are fundamental resources that underpin research across most aspects of modern biology. Technological improvements in the length and accuracy of long-read sequencing platforms, combined with Hi-C proximity ligation sequencing, has enabled the routine generation of highly contiguous phased assemblies, scaffolded to chromosome-level. Nevertheless, automated generation of perfect gapless “telomere-to-telomere” assemblies remains out of reach for most eukaryotic organisms. Scaffolding errors and false duplications can still occur, and assembly curation is now the main bottleneck for large-scale assembly projects. Here, I present a streamlined data workflow and scaffolding assessment for high-throughput manual curation of chromosome-level genome assemblies. Synteny between haplotypes, or closely related species, is combined with read mapping to orient, pair and visualise assembled chromosomes. Assembly gaps are classified according to scaffolding confidence, highlighting candidates for simple scaffolding corrections, such as inversions. Synteny visualisation, gap classification, and HiC contact maps are then combined to identify and document scaffolding edits with increased speed, precision and confidence. This accelerates the production of curated chromosome-level assemblies, and enables the identification of regions of the assembly that may require further attention. Individual tools used in the workflow (ChromSyn, Telociraptor, SynBad, PAFScaff, DepthKopy and DepthCharge) are available at https://github.com/slimsuite/.

Origin and maintenance of large ribosomal RNA gene repeat size in mammals

Our latest paper is out as a Featured Article in the journal Genetics, featuring ONT from both the cane toad and BABS Genome snake genomes. This paper looks at how ribosomal RNA gene repeats (a.k.a. rDNA repeats) have evolved in vertebrates to expand in size in mammals. For something so fundamental to the function of an organism - literally every process of every cell ultimately relies on rRNA - there is surprising diversity. These regions are traditionally hard to assemble with short reads, and still provide challenges for long-read assemblies, so the new era of high-quality long-read assemblies is likely to reveal a lot about their evolution.

• Macdonald E, Whibley A, Waters PD, Patel H, Edwards RJ & Ganley ARD (2024): Origin and maintenance of large ribosomal RNA gene repeat size in mammals. Genetics 228(1): iyae121 [Genetics] [PubMed]

Abstract

The genes encoding ribosomal RNA are highly conserved across life and in almost all eukaryotes are present in large tandem repeat arrays called the rDNA. rDNA repeat unit size is conserved across most eukaryotes but has expanded dramatically in mammals, principally through the expansion of the intergenic spacer region that separates adjacent rRNA coding regions. Here, we used long-read sequence data from representatives of the major amniote lineages to determine where in amniote evolution rDNA unit size increased. We find that amniote rDNA unit sizes fall into two narrow size classes: “normal” (∼11–20 kb) in all amniotes except monotreme, marsupial, and eutherian mammals, which have “large” (∼35–45 kb) sizes. We confirm that increases in intergenic spacer length explain much of this mammalian size increase. However, in stark contrast to the uniformity of mammalian rDNA unit size, mammalian intergenic spacers differ greatly in sequence. These results suggest a large increase in intergenic spacer size occurred in a mammalian ancestor and has been maintained despite substantial sequence changes over the course of mammalian evolution. This points to a previously unrecognized constraint on the length of the intergenic spacer, a region that was thought to be largely neutral. We finish by speculating on possible causes of this constraint.

New pre-print: The Genomics for Australian Plants (GAP) framework initiative – developing genomic resources for understanding the evolution and conservation of the Australian flora

The Bioplatforms Australia Genomics for Australian Plants (GAP) initiative aims to sequence and assemble representative genomes of Australia’s unique flora, which boasts over 24,000 native vascular plant species evolved over millions of years. The program brings together academic groups, herbaria and botanic gardens from across the country to build genomic capacity and create valuable resources for the classification, conservation and utilisation of Australian plants. We were lucky enough to sequence one of the first GAP species, the NSW Waratah. Now, the capstone paper outlining the project and its key findings from multiple species is out as a pre-print at EcoEvoRxiv:

Simpson L, Cantrill DJ, Byrne M, Allnutt TR, King GJ, Lum M, Al Bkhetan Z, Andrew R, Baker WJ, Barrett MD, Batley J, Berry O, Binks RM, Bragg JG, Broadhurst L, Brown G, Bruhl J, Edwards RJ, Ferguson S, Forest F, Gustafsson J, Hammer TA, Holmes GD, Jackson CJ, James EA, Jones A, Kersey PJ, Leitch IJ, Maurin O, McLay TGB, Murphy DJ, Nargar K, Nauheimer L, Sauquet H, Schmidt-Lebuhn AN, Shepherd KA, Syme AE, Waycott M, Wilson TC, Crayn DM (preprint): The Genomics for Australian Plants (GAP) framework initiative – developing genomic resources for understanding the evolution and conservation of the Australian flora. EcoEvoRxiv DOI: https://doi.org/10.32942/X2RP70

The generation and analysis of genome-scale data—genomics—is driving a rapid increase in plant biodiversity knowledge. However, the speed and complexity of technological advance in genomics presents challenges for its widescale use in evolutionary and conservation biology. Here, we introduce and describe a national-scale collaboration conceived to build genomic resources and capability for understanding the Australian flora: the Genomics for Australian Plants (GAP) Framework Initiative. We outline (a) the history of the project including the collaborative framework, partners, and funding; (b) GAP principles such as rigour in design, sample verification and documentation, data management, and data accessibility; and (c) the structure of the consortium and its four activity streams (reference genomes, phylogenomics, conservation genomics, and training), with the rationale and aims for each of them. We show, through discussion of its successes and challenges, the value of this multi-institutional consortium approach and the enablers, such as well-curated collections and national collaborative research infrastructure, all of which have led to a substantial increase in capacity and delivery of biodiversity knowledge outcomes.

The initiative is about more than just reference genomes, with core activity in phylogenomics, conservation genomics and training too. For more information on the project and the resources generated (with more to come), read the paper and/or visit the GAP website.

Towards telomere-to-telomere fish genomes with Oxford Nanopore Technologies gap-filling

It was a pleasure to be invited to the “What You’re Missing Matters” tour at Perth, and present some ongoing work investigating the best way to incorporate Oxford Nanopore Technologies data into our high-quality HiFi+HiC fish genomes. The results are too preliminary to share here (and will soon be superseded) but do get in touch if it sounds interesting to you.

Is developmental plasticity triggered by DNA methylation changes in the invasive cane toad (Rhinella marina)?

Second cane toad paper of the week! This time, we're revisiting invasive epigenomics.

Yagound B, Sarma RR, Edwards RJ, Richardson MF, Rodriguez Lopez CM, Crossland MR, Brown GP, DeVore JL, Shine R & Rollins LA (2024): Is developmental plasticity triggered by DNA methylation changes in the invasive cane toad (Rhinella marina)? Ecology and Evolution 14:e11127. [Ecol Evol] [PubMed] [bioRxiv]

Many organisms can adjust their development according to environmental conditions, including the presence of conspecifics. Although this developmental plasticity is common in amphibians, its underlying molecular mechanisms remain largely unknown. Exposure during development to either ‘cannibal cues’ from older conspecifics, or ‘alarm cues’ from injured conspecifics, causes reduced growth and survival in cane toad (Rhinella marina) tadpoles. Epigenetic modifications, such as changes in DNA methylation patterns, are a plausible mechanism underlying these developmental plastic responses. Here we tested this hypothesis, and asked whether cannibal cues and alarm cues trigger the same DNA methylation changes in developing cane toads. We found that exposure to both cannibal cues and alarm cues was associated with local changes in DNA methylation patterns. These DNA methylation changes affected genes putatively involved in developmental processes, but in different genomic regions for different conspecific-derived cues. Genetic background explains most of the epigenetic variation among individuals. Overall, the molecular mechanisms triggered by exposure to cannibal cues seem to differ from those triggered by alarm cues. Studies linking epigenetic modifications to transcriptional activity are needed to clarify the proximate mechanisms that regulate developmental plasticity in cane toads.

Whole-mitogenome analysis unveils previously undescribed genetic diversity in cane toads across their invasion trajectory

Congratulations to Kelton Cheung for getting her first PhD paper out. This one has been a long time brewing and involved quite a lot of data wrangling, but we got there in the end. Invasive cane toads might be a little more complex than we thought.

Cheung K, Amos TG, Shine R, DeVore JL, S Ducatez S, Edwards RJ & Rollins LA (2024): Whole-mitogenome analysis unveils previously undescribed genetic diversity in cane toads across their invasion trajectory. Ecology and Evolution 14:e11115. [Ecol Evol] [PubMed] [bioRxiv]

Invasive species offer insights into rapid adaptation to novel environments. The iconic cane toad (Rhinella marina) is an excellent model for studying rapid adaptation during invasion. Previous research using the mitochondrial NADH dehydrogenase 3 (ND3) gene in Hawai’ian and Australian invasive populations found a single haplotype, indicating an extreme genetic bottleneck following introduction. Nuclear genetic diversity also exhibited reductions across the genome in these two populations. Here, we investigated the mitochondrial genomics of cane toads across this invasion trajectory. We created the first reference mitochondrial genome for this species using long-read sequence data. We combined whole-genome resequencing data of 15 toads with published transcriptomic data of 125 individuals to construct nearly complete mitochondrial genomes from the native (French Guiana) and introduced (Hawai’i and Australia) ranges for population genomic analyses. In agreement with previous investigations of these populations, we identified genetic bottlenecks in both Hawai’ian and Australian introduced populations, alongside evidence of population expansion in the invasive ranges. Although mitochondrial genetic diversity in introduced populations was reduced, our results revealed that it had been underestimated: we identified 45 mitochondrial haplotypes in Hawai’ian and Australian samples, none of which were found in the native range. Additionally, we identified two distinct groups of haplotypes from the native range, separated by a minimum of 110 base pairs (0.6%). These findings enhance our understanding of how invasion has shaped the genetic landscape of this species.