Sunday, 13 February 2022

Edwards Lab at #LorneGenome 2022

Lorne Genome 2022 (the 43rd Annual Lorne Genome Conference 2022) kicks off today in Lorne and online. I wasn’t able to make it in person this year due to Omicron and teaching commitments, but happily the lab is still well represented. As well as an online talk, we have two in-person posters, so please check these out if you are lucky enough to be attending in the flesh.

Details below.


A chromosome-level reference genome for Telopea speciosissima (New South Wales waratah) provides insight into waratah evolution (#138)

Stephanie H Chen, Jason G Bragg, Richard J Edwards

Telopea is an eastern Australian genus of five species of long-lived shrubs in the family Proteaceae. Previous work has characterised population structure and patterns of introgression between Telopea species. These studies were performed using a limited set of genetic markers, but point to the great potential of waratah as a model clade for understanding the processes of divergence, environmental adaptation and speciation, when enhanced by a genome-wide perspective enabled by a reference genome. However, few Proteaceae genomes and no waratah genomes are available.

We assembled the first chromosome-level reference genome for T. speciosissima (New South Wales waratah; 2n = 22) using Nanopore long-reads, 10x Chromium linked-reads and Hi-C data. The assembly spans 823 Mb (scaffold N50 of 69.0 Mb) with 97.8 % of Embryophyta universal single-copy orthologues (BUSCOs; n = 1,614) complete. Read depth analysis of 140 ‘Duplicated’ BUSCO genes reveals that almost all are real duplications, increasing confidence in protein family analysis using annotated protein-coding genes, highlighting a possible need to revise the BUSCO set for this lineage. Genome annotation predicted 34,706 genes and pseudogenes, including 27,481 protein-coding genes. We examined the evolutionary dynamics of Telopea using the reference genome in conjunction with DArTseq (n = 244) and whole genome shotgun sequencing (n = 14) of each of the seven lineages; there are three lineages of T. speciosissima – coastal, upland and southern.

Here, I will discuss the population structure and demographic history of the genus. We also examined phylogenomic relationships and developed a scalable method of rapidly generating species trees from short-read data to maximise the recovery of informative data from genomic datasets. The waratah reference genome represents an important new genomic resource in Proteaceae to accelerate our understanding of the origins and evolutionary dynamics of the Australian flora.

[Read more about the waratah genome, here.]


Small but mitey: high-quality long-read assembly of a streamlined mite genome from contaminated sequencing data (#17)

Richard J Edwards, Stephanie H Chen, Jason G Bragg.

As pilot data for project on myrtle rust resistance, we previously assembled two Myrtaceae genomes using 10x Chromium linked reads: Rhodamnia argentea (silver malletwood) and Syzygium oleosum (blue lilly pilly). Both draft genomes achieved scaffolding (N50 > 850 kb) and completeness (BUSCOv3 embryophyta_odb9 > 90 %) of sufficient quality to be annotated by NCBI RefSeq. However, signs of arthropod sequence contamination were subsequently found in the Rhodamnia argentea assembly. We therefore sought to identify and eliminate this contamination during improvement and curation of the genome for publication.

A risk-averse analysis highlighted 49.6 Mb (11.95%) on 2,996 of 15,781 scaffolds of possible arthropod origin. An improved assembly of the same tree, incorporating ~50X long-read (ONT) sequencing, has confirmed this contamination as 11 scaffolds (34.6 Mb) that are distinct from 75 R. argentea assembly scaffolds (346.7 Mb), increasing the likelihood of contamination over the integration of horizontally transferred genes. Taxonomic analysis of predicted protein-coding genes using Taxolotl (https://github.com/slimsuite/taxolotl) suggested that the contamination most likely originates from some form of mite (Order: Trombidiformes), but limited NCBInr mite sequences precluded better taxonomic resolution. Curiously, these contamination scaffolds showed a high depth of coverage (~36X), but a fairly low BUSCO completeness of 58.1% (v5 Augustus, metazoa_odb10 n=954), apparently inconsistent with typical mite genomes.

Phylogenomic analysis with available mite genomes identified the closest relative as Aculops lycopersici, a microscopic (0.2 mm long) eriophyoid mite with a heavily streamlined 32.5 Mb genome. Original low completeness appears to be from a combination of genome reduction and poor performance of that BUSCO version; BUSCO v5 MetaEuk eukaryota_odb10 (n=255) reports 82.8% completeness, which is approaching the 86.3% of A. lycopersici. Here, we discuss the evidence that we have assembled a highly complete but streamlined genome from an unknown eriophyoid mite, plus the need to improve genomic representation of contaminating pest species.


A genetic perspective on rapid adaptation in the globally invasive European starling (Sturnus vulgaris) (#255)

Katarina C Stuart, Richard J Edwards, William (Bill) B Sherwin, Lee Ann Rollins.

Few invasive birds are as globally successful or as well-studied as the common starling (Sturnus vulgaris). Native to the Palaearctic, the starling has been a prolific invader in North and South America, southern Africa, Australia, and The Pacific Islands, while facing declines in excess of 50% in in some native regions. Starlings present an invaluable opportunity to test predictions about the evolutionary trajectory of invasive populations, and gain insight into genetic shifts in response to anthropogenic alteration and climate change.

My research focuses primarily on the invasive European starling population in Australia and aims to investigate the genetics underlying their evolution, using a range of genomic approaches. Through historic museum sample sequencing, I examine single nucleotide polymorphism variations shifts between the native range and Australia, and find parallel selection on both continents, possibly resulting from common global selective forces such as exposure to pollutants and carbohydrate exposure. I further examine matched genetic, morphological, and environmental data to reveal patterns of heritability and plasticity across ecologically significant phenotypic traits, revealing that elevation, as well as rainfall and temperature variability plays an important role in shaping morphology and genetics. Finally, I investigated patterns of structural variants, to uncover evolutionarily significant large-scale genetic variants across a global data set, and more specifically characterise their role in rapid starling adaptation across the entirety of the Australian range. Overall, my research seeks to better understand mechanisms and patterns of genetic change within this species, which may be used to inform invasion or native range management. More broadly, this evolutionary research into the starling provide an important perspective on the role of rapid evolution in invasive species persistence, and the global pressures that may shape range shifts and evolution across many similar avian taxa.

Tuesday, 25 January 2022

Horizontal transposon transfer and its implications for the ancestral ecology of hydrophiine snakes

The first of the BABS Genome papers has finally arrived, featuring our two 10x Genomics Supernova snake genomes. Such is the speed that genomics is moving, the snake assemblies themselves have moved on quite a bit since then and we hope to release chromosome-level versions soon. (The goalposts for a genome paper moved faster than they could be written up - always a challenge without dedicated researchers working on assemblies! Do get in touch if they’d be useful and we can collaborate.)

Rather than a pure genome paper, this paper makes use of our two elapid genomes to ask some interesting questions about possible horizontal transfer of transposable (mobile genetic) elements during the evolution of sea snakes - our two elapids provided good sister (mainland tiger snake) and outgroup (eastern brown snake) taxa for the olive sea snake, which was the focus of the study. It was doubly pleasing to collaborate on a transposable elements paper, as they were the subject of my PhD (albeit in bacteria, see here and here).

This paper is part of a special issue, Mobile Elements in Phylogenomic Reconstructions, and features some interesting examples of probable horiztonal transfer of mobile elements that provide insights into the evolutionary history of these species.


Galbraith JD, Ludington AJ, Sanders KL, Amos TG, Thomson VA, Enosi Tuipulotu D, Dunstan N, Edwards RJ, Suh A, Adelson DL (2022): Horizontal transposon transfer and its implications for the ancestral ecology of hydrophiine snakes. Genes 13(2):217. [Genes] [PDF] [bioRxiv]

Abstract

Transposable elements (TEs), also known as jumping genes, are sequences able to move or copy themselves within a genome. As TEs move throughout genomes they often act as a source of genetic novelty, hence understanding TE evolution within lineages may help in understanding environmental adaptation. Studies into the TE content of lineages of mammals such as bats have uncovered horizontal transposon transfer (HTT) into these lineages, with squamates often also containing the same TEs. Despite the repeated finding of HTT into squamates, little comparative research has examined the evolution of TEs within squamates. Here we examine a diverse family of Australo–Melanesian snakes (Hydrophiinae) to examine if the previously identified, order-wide pattern of variable TE content and activity holds true on a smaller scale. Hydrophiinae diverged from Asian elapids ~30 Mya and have since rapidly diversified into six amphibious, ~60 marine and ~100 terrestrial species that fill a broad range of ecological niches. We find TE diversity and expansion differs between hydrophiines and their Asian relatives and identify multiple HTTs into Hydrophiinae, including three likely transferred into the ancestral hydrophiine from fish. These HTT events provide the first tangible evidence that Hydrophiinae reached Australia from Asia via a marine route.

Friday, 14 January 2022

The Waratah genome paper is out!

The final version of the waratah genome paper now out in Molecular Ecology Resources. This was a fun collaboration with the Royal Botanic Gardens and Domain Trust as one of the pilot genomes for BioPlatforms Australia’s Genomics for Australian Plants (GAP) initiative.

You can read the press release here, or our piece in the Conversation, We’ve unveiled the waratah’s genetic secrets, helping preserve this Australian icon for the future.

In this paper, we present a chromosome-level assembly for the NSW State Floral Emblem, the New South Wales waratah, Telopea speciosissima. This joins macadamia as the 2nd reference genome for the Proteaceae family & should help future studies for the remaining ca. 1700 species.

The genome was assembled from a ONT chassis, scaffolded with 10x Genomics linked reads and Phase Genomics HiC - made possible thanks to quality data from AGRF and the Ramaciotti Centre for Genomics. The final assembly was chromosome-level, with 94.1% on the 11 chromosomes (2n = 22).

As well as the assembly itself, the paper presents a three genomics tools that we hope will be helpful for other assemblies:

1. DepthSizer uses long-read depths and BUSCO predictions to estimate genome size. We estimated the waratah genome to be ca. 900 Mbp - bigger than kmer estimates, but smaller than flow cytometry of Tasmanian waratah.

2. Diploidocus builds on Purge Haplotigs, combining read depths, kmer frequencies & BUSCO predictions to classify and curate/filter assembly scaffolds. This decreases false duplications & contamination, and flags collapsed repeats for closer inspection.

3. DepthKopy uses BUSCO Complete genes to establish sequencing depth (like DepthSizer) and then estimates copy number for regions (e.g. genes), scaffolds & sliding windows of the assembly. This showed that most “Duplicated” BUSCOs are real duplicates.


Chen SH, Rossetto M, van der Merwe M, Lu-Irving P, Yap JS, Sauquet H, Bourke G, Amos TG, Bragg JG & Edwards RJ (accepted): Chromosome-level de novo genome assembly of Telopea speciosissima (New South Wales waratah) using long-reads, linked-reads and Hi-C. Molecular Ecology Resources.
[Mol Ecol Res] [bioRxiv]

Abstract

Telopea speciosissima, the New South Wales waratah, is an Australian endemic woody shrub in the family Proteaceae. Waratahs have great potential as a model clade to better understand processes of speciation, introgression and adaptation, and are significant from a horticultural perspective. Here, we report the first chromosome-level genome for T. speciosissima. Combining Oxford Nanopore long-reads, 10x Genomics Chromium linked-reads and Hi-C data, the assembly spans 823 Mb (scaffold N50 of 69.0 Mb) with 97.8% of Embryophyta BUSCOs “Complete”. We present a new method in Diploidocus (https://github.com/slimsuite/diploidocus) for classifying, curating and QC-filtering scaffolds, which combines read depths, k-mer frequencies and BUSCO predictions. We also present a new tool, DepthSizer (https://github.com/slimsuite/depthsizer), for genome size estimation from the read depth of single-copy orthologues and estimate the genome size to be approximately 900 Mb. The largest 11 scaffolds contained 94.1% of the assembly, conforming to the expected number of chromosomes (2n = 22). Genome annotation predicted 40,158 protein-coding genes, 351 rRNAs and 728 tRNAs. We investigated CYCLOIDEA (CYC) genes, which have a role in determination of floral symmetry, and confirm the presence of two copies in the genome. Read depth analysis of 180 “Duplicated” BUSCO genes using a new tool, DepthKopy (https://github.com/slimsuite/depthkopy), suggests almost all are real duplications, increasing confidence in the annotation and highlighting a possible need to revise the BUSCO set for this lineage. The chromosome-level T. speciosissima reference genome (Tspe_v1) provides an important new genomic resource of Proteaceae to support the conservation of flora in Australia and further afield.

If you want a read and don’t have access, please get it touch or check out the bioRxiv preprint.

Friday, 3 December 2021

Limited Introgression between Rock-Wallabies with Extensive Chromosomal Rearrangements

When we were assembling the 10x Genomics linked read assemblies of several rock wallabies, one of them broke the lab record for Supernova assembly quality.

10x linked reads are a bit of a dead technology now, but genomes made with them are still useful and generating insights. One example is the first rock wallaby we assembled, as a (very small) part of a team that used the draft 10x genome assembly for Petrogale penicillata as a reference to investigate chromosomal rearrangements, published online today in Molecular Biology and Evolution, one of my favourite journals.


Potter P, Bragg JG, Turakulov R, Eldridge MDB, Deakin J, Kirkpatrick M, Edwards RJ & Moritz C (2022): Limited introgression between rock-wallabies with extensive chromosomal rearrangements. Molecular Biology and Evolution 39(1):msab333 https://doi.org/10.1093/molbev/msab333

Abstract

Chromosome rearrangements can result in the rapid evolution of hybrid incompatibilities. Robertsonian fusions, particularly those with monobrachial homology, can drive reproductive isolation amongst recently diverged taxa. The recent radiation of rock-wallabies (genus Petrogale) is an important model to explore the role of Robertsonian fusions in speciation. Here, we pursue that goal using an extensive sampling of populations and genomes of Petrogale from north-eastern Australia. In contrast to previous assessments using mitochondrial DNA or nuclear microsatellite loci, genomic data are able to separate the most closely related species and to resolve their divergence histories. Both phylogenetic and population genetic analyses indicate introgression between two species that differ by a single Robertsonian fusion. Based on the available data, there is also evidence for introgression between two species which share complex chromosomal rearrangements. However, the remaining results show no consistent signature of introgression amongst species pairs and where evident, indicate generally low introgression overall. X-linked loci have elevated divergence compared with autosomal loci indicating a potential role for genic evolution to produce reproductive isolation in concert with chromosome change. Our results highlight the value of genome scale data in evaluating the role of Robertsonian fusions and structural variation in divergence, speciation, and patterns of molecular evolution.

Thursday, 2 December 2021

EdwardsLab at Australasian Evolution Society #AUSEVO2021

Look out for some interesting talks by Edwards Lab members at this year’s Australasian Evolution Society 2021 conference, starting today:

Thursday 2nd December: 3-minute talks | 1130-1230

Kelton Cheung - Analysis of mitochondrial DNA reveals significant genetic diversity in invasive Australian cane toads

Kelton Cheung, Mark Richardson, Richard Edwards & Lee Ann Rollins

Mitochondrial DNA haplotype patterns across the native and invaded ranges can reveal the history and evolutionary trajectory of invasions. The invasion of Australia by the cane toad (Rhinella marina) has accelerated as it expanded westward. Despite this success, previous studies reported no mitochondrial genetic diversity in Australia. Here, we assembled a complete mitochondrial reference genome and haplotyped toads (N=119) from the native range and two introduced populations (Hawai’i and Australia), using whole genome and RNA-seq data. The complete R. marina mitochondrial genome consists of 18,152 base pairs with no significant gene arrangement as compared to other bufonid species. Although native range genetic diversity was much higher than that of the introduced ranges, we identified 29 haplotypes in Australia. While we did find evidence of founder effects following introduction, our results suggest there is significant genetic diversity within Australia, which may assist adaptation and invasion success in this species.


Thursday 2nd December: Selection 1 | 1415-1430

Katarina Stuart - A genetic perspective on rapid adaptation in the globally invasive European starling (Sturnus vulgaris)

Abstract coming soon…


Thursday 2nd December: Biogeography and phylogenetics | 1530-1545

Richard Edwards - DepthKopy: copy number prediction using single-copy long-read depth profiles

Richard J Edwards, Stephanie H Chen, Katarina C Stuart, Mark M Tanaka & Jason G Bragg

Gene duplication, followed by functional divergence of gene copies, is a fundamental component of genetic adaption and the evolution of novelty. Increasingly, evolutionary studies make use of the ever-expanding number of high-quality genome assemblies to characterise patterns of gene gain and loss. However, even reference genomes of the highest quality can experience assembly errors at tandemly repeated gene loci, resulting in incorrect inference of gene duplication patterns. Here, we present DepthKopy (https://github.com/slimsuite/depthkopy), which estimates the copy number for a gene, region or sequence of interest, using an estimate of single-copy sequencing depth derived from complete BUSCO genes. This is useful for identifying haplotigs, and collapsed repeat regions during genome assembly curation. Critically, for evolutionary studies of gene families, DepthKopy can identify genes for which the number of genes in the assembly does not seem to match the genome.


Friday 3rd December: Climate change and temperature | 1330-1345

Collin Ahrens - Genomic constraints of drought adaptation

Abstract coming soon…

Wednesday, 24 November 2021

#ABACBS2021 Lightning talk - DepthSizer and DepthKopy: genome size and copy number prediction using single-copy long-read depth profiles

Tune in for the ABACBS 2021 lightning talks this morning to hear about our applications of long reads to genome size and copy number prediction. For more information, you can read the NSW Waratah genome paper pre-print or visit the GitHub pages for DepthSizer and DepthKopy.

Richard J Edwards, Stephanie H Chen, Katarina C Stuart, Mark M Tanaka, Jason G Bragg.

DepthSizer and DepthKopy: genome size and copy number prediction using single-copy long-read depth profiles

A fundamental part of any genome project is establishing the genome size of the organism being sequenced. The gold standard for genome size measurement is flow cytometry, but this is not available to all groups and can give surprisingly variable results. Popular bioinformatic approaches predict genome size using kmer frequency profiles from high-accuracy (e.g. illumina or hifi) sequencing reads, or the mean depth of coverage reads mapped to an assembly. Both of these approaches can be adversely affected by repetitive regions of the genome. Mean sequencing depth is also highly reliant on assembly completeness.

Here, we present DepthSizer (https://github.com/slimsuite/depthsizer), which refines this approach by estimating sequencing depth based on single-copy complete BUSCO genes. DepthSizer works on the principle that genuine single-copy regions will tend towards the same, true, single-copy read depth. In contrast, assembly errors, collapsed repeats within those genes, or incorrect BUSCO predictions, will give inconsistent read depth deviations. The modal read depth across single-copy BUSCO genes, calculated from a depth density profile of these regions, should therefore provide a good estimate of the true depth of coverage. The method is benchmarked on model organism data and corrections for possible contamination, biases/inconsistencies in read mapping and/or raw read insertion/deletion error profiles are discussed. We also present DepthKopy (https://github.com/slimsuite/depthkopy), which uses the same read depth approach to estimate the copy number of assembly regions. This can be useful for identifying haplotigs, and collapsed repeat regions.

Keywords: BUSCO, Genome Assembly, Genomics, ONT, PacBio, copy number variants