Monday, 19 April 2021

Intergenerational effects of manipulating DNA methylation in the early life of an iconic invader

Another cane toad paper has hit the shelves! This is another paper from our ongoing collaboration with Lee Ann Rollins and her great team of invasion biologists and molecular ecologists. This paper once again uses our draft cane toad genome* and builds on the previous cane toad methylation analysis by PhD student Roshmi Sarma to look at some really interesting intergenerational effects. [*Genome update coming soon - watch this space!]

Could this be epigenetic inheritance? Maybe. But it could also be some kind of parental germline thing. Either way, it’s a fascinating result and further evidence to support the fact that whilst our genome may establish our genetic potential, it does not control our destiny.

And if you want to know what it takes to identify effects like this, check out the mind-boggling experiment design Figure! (PDF available on request!)

This article is part of the theme issue ‘How does epigenetics influence the course of evolution?’

Sarma RR, Crossland MR, Eyck HJF, Edwards RJ, DeVore JL, Cocomazzo M, Zhou J, Brown GP, Shine R & Rollins LA (2021): Intergenerational effects of manipulating DNA methylation in the early life of an iconic invader. Philosophical Transactions of the Royal Society B 376:20200125. [Phil Trans Roy Soc B] [PubMed]


In response to novel environments, invasive populations often evolve rapidly. Standing genetic variation is an important predictor of evolutionary response but epigenetic variation may also play a role. Here, we use an iconic invader, the cane toad (Rhinella marina), to investigate how manipulating epigenetic status affects phenotypic traits. We collected wild toads from across Australia, bred them, and experimentally manipulated DNA methylation of the subsequent two generations (G1, G2) through exposure to the DNA methylation inhibitor zebularine and/or conspecific tadpole alarm cues. Direct exposure to alarm cues (an indicator of predation risk) increased the potency of G2 tadpole chemical cues, but this was accompanied by reductions in survival. Exposure to alarm cues during G1 also increased the potency of G2 tadpole cues, indicating intergenerational plasticity in this inducible defence. In addition, the negative effects of alarm cues on tadpole viability (i.e. the costs of producing the inducible defence) were minimized in the second generation. Exposure to zebularine during G1 induced similar intergenerational effects, suggesting a role for alteration in DNA methylation. Accordingly, we identified intergenerational shifts in DNA methylation at some loci in response to alarm cue exposure. Substantial demethylation occurred within the sodium channel epithelial 1 subunit gamma gene (SCNN1G) in alarm cue exposed individuals and their offspring. This gene is a key to the regulation of sodium in epithelial cells and may help to maintain the protective epidermal barrier. These data suggest that early life experiences of tadpoles induce intergenerational effects through epigenetic mechanisms, which enhance larval fitness.

Thursday, 8 April 2021

Transcript- and annotation-guided genome assembly of the European starling

Our starling genome paper is now available as a pre-print on bioRxiv! This was some great work by PhD student, Kat Stuart. Kat assembled a new Australian starling genome, using a combination of linked reads, low coverage long reads, and long-read PacBio iso-seq transcriptomics data. A second Illumina assembly of a North American group is also presented. As we saw with our Basenji genome paper, having two (or more) genomes from a species can be really useful for disentangling real difference from assembly artefacts. (No assembly is perfect!)

This paper is a great example of how a bit of TLC and imagination can get the most out of data produced with a limited budget. We were unable to get deep long-read sequencing this time, but instead show the additional power that long-read full-length transcriptome data can provide in assembling a genome - above and beyond the annotation.

This paper also officially describes a couple of genomics tools from the lab BUSCOMP has been in the works for some time, and this paper updates previous results to BUSCO v5 analysis and confirms our previous snake results using starling-derived test data. BUSCO is a powerful and popular tool that estimates genome completeness using gene prediction and curated models of single-copy protein orthologues. However, we demonstrate how results can be counterintuitive: adding/removing scaffolds can alter BUSCO predictions elsewhere in the assembly, while low sequence quality may reduce “completeness” scores and miss genes that are present in the assembly. BUSCOMP (BUSCO Compilation and Comparison) ( complements BUSCO to identify/overcome these issues by compiling a non-redundant set of the highest-scoring single-copy BUSCO complete sequences and re-searching these against assemblies for consistent completness scoring. SAAGA ( is a new tool for annotation versus reference proteome comparisons. SAAGA can compare different annotations of the same assembly, or be combined with a lightweight annotation tool like GeMoMa to compare different assemblies of the same organism.

Stuart KC, Edwards RJ, Cheng Y, Warren WC, Burt DW, Sherwin WB, Hofmeister NR, Werner SJ, Ball GF, Bateson M, Brandley MC, Buchanan KL, Cassey P, Clayton DF, De Meyer T, Meddle SL & Rollins LA (preprint): Transcript- and annotation-guided genome assembly of the European starling. bioRxiv 2021.04.07.438753; doi: 10.1101/2021.04.07.438753. [*Joint first authors] [bioRxiv]


The European starling, Sturnus vulgaris, is an ecologically significant, globally invasive avian species that is also suffering from a major decline in its native range. Here, we present the genome assembly and long-read transcriptome of an Australian-sourced European starling (S. vulgaris vAU), and a second North American genome (S. vulgaris vNA), as complementary reference genomes for population genetic and evolutionary characterisation. S. vulgaris vAU combined 10x Genomics linked-reads, low-coverage Nanopore sequencing, and PacBio Iso-Seq full-length transcript scaffolding to generate a 1050 Mb assembly on 1,628 scaffolds (72.5 Mb scaffold N50). Species-specific transcript mapping and gene annotation revealed high structural and functional completeness (94.6% BUSCO completeness). Further scaffolding against the high-quality zebra finch (Taeniopygia guttata) genome assigned 98.6% of the assembly to 32 putative nuclear chromosome scaffolds. Rapid, recent advances in sequencing technologies and bioinformatics software have highlighted the need for evidence-based assessment of assembly decisions on a case-by-case basis. Using S. vulgaris vAU, we demonstrate how the multifunctional use of PacBio Iso-Seq transcript data and complementary homology-based annotation of sequential assembly steps (assessed using a new tool, SAAGA) can be used to assess, inform, and validate assembly workflow decisions. We also highlight some counter-intuitive behaviour in traditional BUSCO metrics, and present BUSCOMP, a complementary tool for assembly comparison designed to be robust to differences in assembly size and base-calling quality. Finally, we present a second starling assembly, S. vulgaris vNA, to facilitate comparative analysis and global genomic research on this ecologically important species.