The cane toad genome project

What are we doing? The Edwards Lab is part of an Australian, Portuguese and Brazilian consortium led by Peter White to sequence and assemble the genome of the cane toad (Rhinella marina). We are leading the bioinformatics component of the assembly effort.

How are we doing it? We are using a combination of Illumina (HiSeq X and NovaSeq) short read sequencing, PacBio (RS II) long read and sequence and 10x Genomics Chromium linked reads.

Details to follow. Please get in touch if you are interested in the project.

Opportunities

Honours and postgraduate* projects are available to work on the assembly and annotation. (*PhD students should have their own scholarship.)

Consortium members

Miguel Carneiro (CIBIO-InBIO), Richard Edwards (UNSW), Nuno Ferrand (CIBIO-InBIO), Eddie Holmes (U Sydney), Craig Moritz (ANU), Lee Ann Rollins (Deakin), Fernando Sequeira (CIBIO-InBIO), Rick Shine (U Sydney), Marcelo Vallinoto de Souza (Federal University of Pará & CIBIO-InBIO), Peter White (UNSW), Marc Wilkins (UNSW).

BABS is recruiting - academic positions available in bioinformatics, systems biology, medical genomics and biotechnology

The School of Biotechnology and Biomolecular Sciences (BABS) at the University of New South Wales, Sydney, Australia, is seeking to recruit world class scholars with outstanding and recognised research records in Biotechnology, Bioinformatics, Systems Biology or Medical Genomics.

Our School, located at the Kensington campus in Sydney, is part of the Faculty of Science. We have an international reputation in biotechnology, molecular and cellular biology, and microbiology. Particular areas of research strength within these fields include proteomics and genomics, gene regulation, metabolism,environmental microbiology and infectious diseases.

We currently have combined track (research and teaching) opportunities to join BABS as part of a strategic initiative in Systems level Biology and Genomics in the School. BABS hosts the Ramaciotti Centre for Genomics, which has world class facilities for sequencing and is arguably Australia’s premier facility for research genomics. BABS is also home to the Systems Biology Initiative, which is supported by high performance computing facilities, including national supercomputers. Our researchers also have access to outstanding proteomics and metabolomics facilities at the UNSW Bioanalytical Mass Spectrometry Facility and the Mark Wainwright Analytical Centre which provide state-of-the-art facilities and services for animal studies; in vivo and ex vivo preclinical imaging; flow cytometry; biochemical, biophysical and chemical analyses; and electron microscopy. We have collaborative links with the Garvan Institute, the Lowy Cancer Research Centre and other biomedical research organisations within and outside Sydney.

For more information about the School, please visit: www.babs.unsw.edu.au/

About UNSW Australia

• UNSW Australia is a research-intensive university ranked in the world’s top 50 (QS 2016)
• Strong regional and global engagement improving and transforming lives through excellence in research, outstanding education and a commitment to advancing a just society
• The top university in the state of New South Wales in the 2015 Excellence in Research for Australia

UNSW is at the cutting edge of academia with a strong and growing international reputation. A global leader in discovery, innovation, impact, education and thought leadership, can make an enormous difference to the lives of people in Australia and around the world. The recently launched UNSW 2025 Strategy is an innovative, ambitious and altruistic agenda, reflecting a conviction across our University to achieve great things for society during the next decade.

Further details

Further details can be found here:

• Bioinformatics, Systems Biology or Medical Genomics.
• Biotechnology.

Any enquiries contact: Associate Professor Mark Tanaka

Applications close: Sunday 13 November 2016 (AEST)

Predicting Motif Mimicry in Viruses

Sobia will be presenting the initial work from her PhD project today at the EMBO Workshop, The modularity of signalling proteins and networks at Seefeld in Tirol, Austria. Her talk is on:

Predicting Motif Mimicry in Viruses

Viruses mimic host motifs to hijack the host cellular machinery. Their interaction with host protein domains is through Short Linear Motifs (SLiMs). SLiMs are short stretches of amino acids (~3-10) which are involved in post translational modifications (PTMs), protein-protein Interactions (PPIs), cell regulation and cell compartment targeting. To date, several studies have been conducted to identify PPIs, but no specific study to see how well different PPI capturing methods capture SLiMs-mediated interactions. The main objectives of this study are 1) to predict Domain Motif Interactions (DMIs) among viral and host proteins 2) to find whether virhostome (virus-human interaction) data is enriched for DMIs and, 3) to see which PPI method is better for studying DMIs. Results have shown that virhostome data is enriched for DMIs and can be a good source to study motif mimicry in viruses. The permutation test showed more enrichment for TAP data as compared to the Y2H data. Moreover, novel candidate DMIs have been discovered which need further validations. The outcome of this study will be helpful in uncovering unique strategies of viruses to interact with human proteins which will eventually be significant for pathogen research.

Poster to follow.

Honours and undergrad research opportunities

Honours

BABS are currently recruiting the next cohort of Honours students for Semester 1 2017. As usual, the EdwardsLab is looking to recruit enthusiastic students in two main areas:

1. Functional genomics using long-read PacBio sequencing. We are particularly keen to get a student to work on either (a) aspects of our ARC Linkage grant, investigating the evolution of a novel biochemical pathway in yeast, or (b) de novo whole genome sequencing of the cane toad. We also have a number of projects with bacteria for those with a keen interest in microbiology. In each case, the lab is collaborating with experts in the relevant organisms.

2. Applying biological sequence analysis and molecular evolution to study the molecular basis of protein-protein interactions. The main lab software, SLiMSuite has a number of improvements and developments that would benefit from some dedicated attention from a research student. We are also looking for someone who might want to help develop the lab servers.

More details of honours can be found on the BABS website, or please get in touch if you have questions about specific projects. Applications from non-UNSW students are also encouraged.

* BABS are also running an Honours information and networking night on 16^th September.*

Summer Vacation Research Scholarships

BABS is once again running its highly successful Summer Vacation Research Scholarship (SVRS) scheme and the EdwardsLab are looking to take on one or two students in the same areas as indicated above.

How to apply

We do not yet have a specific undergraduate application form but it is helpful if you can follow the PhD application process and just make it clear that you are interested in Honours or SVRS. As well as helping select between applicants, this form is also useful for me to make sure that students are assigned an appropriate project.

Plant adaptation or acclimation to rising CO2? Insight from first multigenerational RNA-Seq transcriptome

Watson-Lazowski A, Lin Y, Miglietta F, Edwards RJ, Chapman MA & Taylor G (2016): Plant adaptation or acclimation to rising CO₂? Insight from first multigenerational RNA-Seq transcriptome. Glob Chang Biol. Adv. access. doi: 10.1111/gcb.13322

Abstract

Atmospheric carbon dioxide (CO2 ) directly determines the rate of plant photosynthesis and indirectly effects plant productivity and fitness and may therefore act as a selective pressure driving evolution, but evidence to support this contention is sparse. Using Plantago lanceolata L. seed collected from a naturally high CO2 spring and adjacent ambient CO2 control site, we investigated multigenerational response to future, elevated atmospheric CO2 . Plants were grown in either ambient or elevated CO2 (700 μmol mol-1 ), enabling for the first time, characterization of the functional and population genomics of plant acclimation and adaptation to elevated CO2 . This revealed that spring and control plants differed significantly in phenotypic plasticity for traits underpinning fitness including above-ground biomass, leaf size, epidermal cell size and number and stomatal density and index. Gene expression responses to elevated CO2 (acclimation) were modest [33-131 genes differentially expressed (DE)], whilst those between control and spring plants (adaptation) were considerably larger (689-853 DE genes). In contrast, population genomic analysis showed that genetic differentiation between spring and control plants was close to zero, with no fixed differences, suggesting that plants are adapted to their native CO2 environment at the level of gene expression. An unusual phenotype of increased stomatal index in spring but not control plants in elevated CO2 correlated with altered expression of stomatal patterning genes between spring and control plants for three loci (YODA, CDKB1;1 and SCRM2) and between ambient and elevated CO2 for four loci (ER, YODA, MYB88 and BCA1). We propose that the two positive regulators of stomatal number (SCRM2) and CDKB1;1 when upregulated act as key controllers of stomatal adaptation to elevated CO2 . Combined with significant transcriptome reprogramming of photosynthetic and dark respiration and enhanced growth in spring plants, we have identified the potential basis of plant adaptation to high CO2 likely to occur over coming decades.

PMID: 27539677

SMBE2016: Investigating the evolution of new biochemical pathways in baker’s yeast Saccharomyces cerevisiae

Well done to Åsa for her oral presentation at SMBE2016 (#204):

Investigating the evolution of new biochemical pathways in baker’s yeast Saccharomyces cerevisiae

Åsa Pérez-Bercoff, Tonia L. Russell, Philip J. L. Bell, Paul V. Attfield & Richard J. Edwards

Abstract

Understanding how new biochemical pathways evolve in a sexually reproducing population is a complex and largely unanswered question. We have successfully evolved a novel biochemical pathway in yeast using a sex based population approach.

For over 30 years, wild type Saccharomyces has been widely reported to not grow on xylose at all, but we discovered that most strains can grow, albeit at almost undetectable rates. A mass mated starting population of Saccharomyces cerevisiae strains was evolved under selection on Xylose Minimal Media (XMM) with forced sexual mating every ~two months for 1463 days. This produced a population that could grow on xylose as a sole carbon source. Initial studies show the xylose growth trait is quantitative and presumably governed by many genes. To investigate the evolution of the xylose phenotype, a xylose utilising strain MBG11a was isolated. MBG11a was sequenced with PacBio RSII long read sequencing at the Ramaciotti Centre for Genomics. A high quality complete genome was assembled de novo using the hierarchical genome-assembly process (HGAP3) using only PacBio non-hybrid long-read SMRT sequencing data, corrected using Quiver, and compared to the genome of the S. cerevisiae S288C reference genome.

Approximately 98.5% of the MBG11a genome could be aligned to S288C at 99.5% sequence identity, with over 15,000 non-synonymous and 200 nonsense SNP differences. We have crossed MBG11a with a reference wild type yeast strain (X2180 gal2, Xyl-) and are testing offspring on different minimal media in an attempt to identify MBG11a variants responsible for the novel growth phenotype.

Understanding what has occurred in the evolving yeast population, and how the yeast genome adapted under the selection pressures is of broad interest as it allows experimental analysis of how novel complex biological functions can evolve in an organism.

Transcriptome analysis of human brain tissue identifies reduced expression of complement complex C1Q Genes in Rett syndrome

Lin P, Nicholls L, Assareh H, Fang Z, Amos TG, Edwards RJ, Assareh AA, Voineagu I (2016): Transcriptome analysis of human brain tissue identifies reduced expression of complement complex C1Q Genes in Rett syndrome. BMC Genomics 17(1):427. doi: 10.1186/s12864-016-2746-7.

Abstract

BACKGROUND: MECP2, the gene mutated in the majority of Rett syndrome cases, is a transcriptional regulator that can activate or repress transcription. Although the transcription regulatory function of MECP2 has been known for over a decade, it remains unclear how transcriptional dysregulation leads to the neurodevelopmental disorder. Notably, little convergence was previously observed between the genes abnormally expressed in the brain of Rett syndrome mouse models and those identified in human studies.

METHODS: Here we carried out a comprehensive transcriptome analysis of human brain tissue from Rett syndrome brain using both RNA-seq and microarrays.

RESULTS: We identified over two hundred differentially expressed genes, and identified the complement C1Q complex genes (C1QA, C1QB and C1QC) as a point of convergence between gene expression changes in human and mouse Rett syndrome brain.

CONCLUSIONS: The results of our study support a role for alterations in the expression level of C1Q complex genes in RTT pathogenesis.

PMID: 27267200

Research snapshot: June 2016

Research interests in the Edwards lab stem from a fascination with the molecular basis of evolutionary change and how we can harness the genetic sequence patterns left behind to make useful predictions about contemporary biological systems.

The core research in the lab is the study of Short Linear Motifs (SLiMs), which are short regions of proteins that mediate interactions with other proteins. This research originated with Rich’s postdoctoral research, during which he developed a bioinformatics (sequence analysis) method for rational design of biologically active short peptides. He subsequently developed SLiMDisc, one of the first algorithms for successfully predicting novel SLiMs from sequence data - and coined the term “SLiM” into the bargain - before developing the first SLiM prediction algorithm able to estimate the statistical significance of motif predictions (SLiMFinder), which greatly increased the reliability of predictions. SLiMFinder has since spawned a number of motif discovery tools and webservers and is still arguably the most successful SLiM prediction tool on benchmarking data.

Current research is looking to develop these SLiM prediction tools further and apply them to important biological questions. Of particular interest is the molecular mimicry employed by viruses to interact with host proteins and the role of SLiMs in other diseases, such as cancer. Other work is concerned with the evolutionary dynamics of SLiMs within protein interaction networks.

A new and exciting area of research in the lab is functional genomics with PacBio long-read sequencing. We are collaborating with industrial and academic partners to de novo sequence, assemble, annotate and interrogate the genomes of a selection of microbes with interesting metabolic abilities.

Finally, the lab has a number of interdisciplinary collaborative projects applying bioinformatics tools and molecular evolution theory to experimental biology, often using large genomic, transcriptomic and/or proteomic datasets. These projects often involve the development of bespoke bioinformatics pipelines and a number of open source bioinformatics tools have been generated as a result.

In Conversation With Dr Rich Edwards, CI On 2016 ARC Linkage Grant

The latest UNSW School of Biotechnology and Biolomolecular Sciences new article is a brief interview about our recent ARC Linkage success. You can read it here.

Honours applications close next Friday (3 June)

We still have space in the group for one more Honours student, with a variety of projects available to work on PacBio SMRT data for bacteria, yeast or cane toad de novo genome assembly. Please get in touch if you are interested. (Better still, fill in the lab application form, making it clear it’s for Honours, not PhD!) The deadline is Friday, 3rd of June. See the BABS website for more details.

Congratulations, Dr Joe Jenkins!

Congratulations to Joe Jenkins, who successfully defended his PhD on 12th May and was awarded his doctorate following his viva, with Professor Gary Bending as external examiner. It’s been a busy week for Joe, with the main paper from his PhD out this week too!

Biochar alters the soil microbiome and soil function: results of next generation amplicon sequencing across Europe

Jenkins JR, Viger M, Arnold EC, Harris ZM, Ventura M, Miglietta F, Girardin C, Edwards RJ, Rumpel C, Fornasier F, Zavalloni C, Tonon G, Alberti G & Taylor G (2016): Biochar alters the soil microbiome and soil function: results of next generation amplicon sequencing across Europe. GCB Bioenergy Adv. Access DOI: 10.1111/gcbb.12371

Abstract

Wide scale application of biochar to soil has been suggested as a mechanism to offset increases in CO2 emissions through the long-term sequestration of a carbon rich and inert substance to the soil, but the implications of this for soil diversity and function remain to be determined. Biochar is capable of inducing changes in soil bacterial communities, but the exact impacts of its application are poorly understood. Using three European sites (UK SRC, short rotation coppice, French grassland (FR) and Italian SRF, short rotation forestry (IT)) treated with identical biochar applications; we undertook 16S and ITS amplicon DNA sequencing. In addition, we carried out assessments of community change over time and N and P mobilisation in the UK.

Significant changes in bacterial and community structure occurred due to treatment, although the nature of the changes varied by site. STAMP differential abundance analysis showed enrichment of Gemmatimonadete and Acidobacteria in UK biochar plots one year after application, whilst control plots exhibited enriched Gemmataceae, Isosphaeraceae and Koribacteraceae. Increased mobility of ammonium and phosphates were also detected after one year, coupled with a shift from acid to alkaline phophomonoesterase activity, which may suggest an ecological and functional shift towards a more copiotrophic ecology. Italy also exhibited enrichments, in both the Proteobacteria (driven by an increase in the order Rhizobiales) and the Gemmatimonadetes. No significant change in the abundance of individual taxa were noted in FR, although a small significant change in unweighted UNIFRAC occurred, indicating variation in the identities of taxa present due to treatment. Fungal β diversity was affected by treatment in IT and FR, but was unaffected in UK samples. The effects of time and site were greater than that of biochar application in UK samples. Overall, this report gives a tantalising view of the soil microbiome at several sites across Europe, and suggests that although application of biochar has significant effects on microbial communities, these may be small compared with the highly variable soil microbiome that is found in different soils and changes with time.

ARC Linkage Success! - Elucidating the genetic basis of newly evolved metabolic functions in yeast

We are very happy to report a successful ARC Linkage Projects 2016 grant application:

LP160100610: Elucidating the genetic basis of newly evolved metabolic functions in yeast

Dr Richard Edwards; Professor Marc Wilkins; Associate Professor Mark Tanaka; Dr Paul Attfield; Dr Phillip Bell

This project intends to research how complex metabolic pathways originate and evolve. This project will use cutting edge genome sequencing and molecular techniques to elucidate the heritable genetic basis of Baker’s yeast, which has been the selectively evolved to use xylose as a sole carbon source: something vital for second generation biofuel production that wild yeast cannot do. This project will combine detailed molecular characterisation of highly adapted yeast strains with a novel “molecular palaeontology” approach to trace the evolutionary process and identify functionally significant loci under selection. Detailed characterisation of this trait will accelerate the development of future yeast strains and test fundamental evolutionary theories.

This will continue the work we have been doing on PacBio sequencing and yeast genomics in collaboration with our industrial partners, Microbiogen Pty Ltd.

There will be job and studentship opportunities associated with this grant, so watch this space! (Or get in touch!)

Parallel evolution in Streptococcus pneumoniae biofilms

Churton NWV, Misra RV, Howlin RP, Allan RN, Jefferies J, Faust SN, Gharbia SE, Edwards RJ, Clarke SC & Webb JS (2016): Parallel evolution in Streptococcus pneumoniae biofilms. Genome Biology and Evolution Adv. Access doi: 10.1093/gbe/evw072

Abstract

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and non-invasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51-264 bp, one insertion resulting in a coding frameshift, and seven nonsense single nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae, and therefore may have important implications for colonization, carriage and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.

Lorne #Genome2016 poster 132: PacBio sequencing and comparative genomics of three Saccharomyces cerevisiae strains

Richard J. Edwards, Åsa Pérez-Bercoff, Tonia L. Russell, Zhiliang Chen , Marc R. Wilkins, Paul V. Attfield & Philip J.L. Bell. F1000Research 2016, 5:172 (poster) (doi: 10.7490/f1000research.1111305.1).

Abstract

PacBio Single Molecule Real Time (SMRT™) sequencing is rapidly becoming the technology of choice for de novo whole genome sequencing. The long read lengths and random error of PacBio data make genome assembly considerably easier and more accurate than short read data. Here, we report on de novo genome sequencing and assembly of three Saccharomyces cerevisiae genomes using the PacBio RSII at the UNSW Ramaciotti Centre for Genomics. A haploid reference yeast genome strain, S288C, and two novel diploid strains were sequenced as part of a larger functional genomics project. For each strain, 20kb SMRT Bell library preps were performed and sequenced on two SMRT Cells using the P6-C4 chemistry with read lengths of up to 53.3 kb. Whole genome de novo assemblies are then generated through the PacBio SMRT Portal.

We are using the S288C data to explore performance in comparison to the published genome as a reference. An initial assembly of S288C yielded over 99.97% genome coverage at 99.99% accuracy on only 26 contigs, with 16/17 reference chromosomes (16 nuclear chromosomes plus mitochondrion) essentially returned as a single, complete contig. The long reads enable accurate reconstruction of tandemly repeated genes (except >900kb of rRNA repeats), transposition and chromosomal translocations. We are now using the S288C data to optimise the assembly process and derive assembly settings for the two novel diploid strains. To this end, we have developed a new pipeline for the comparative assessment of high quality whole genomes against a reference, which we are now adapting for the additional challenge of appropriately handling diploid data.

Quantitative Proteomics of the Infectious and Replicative Forms of Chlamydia trachomatis

Skipp PJS, Hughes C, McKenna T, Edwards R, Langridge J, Thomson NR & Clarke IN (2016): Quantitative Proteomics of the Infectious and Replicative Forms of Chlamydia trachomatis. PLoS ONE 11(2): e0149011. doi:10.1371/journal.pone.0149011

Abstract

The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR, from which the concentrations of chlamydial proteins per bacterial cell/genome were determined. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology. EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins. The ability to express the data as molecules per cell gave ranking in both abundance and energy requirements for synthesis, allowing meaningful identification of rate-limiting components. The study assigned 562 proteins with high confidence and provided absolute estimates of protein concentration for 489 proteins. Interestingly, the data showed an increase in TTS capacity at 15h PI. Most of the enzymes involved in peptidoglycan biosynthesis were detected along with high levels of muramidase (in EBs) suggesting breakdown of peptidoglycan occurs in the non-dividing form of the microorganism. All the genome-encoded enzymes for glycolysis, pentose phosphate pathway and tricarboxylic acid cycle were identified and quantified; these data supported the observation that the EB is metabolically active. The availability of detailed, accurate quantitative proteomic data will be invaluable for investigations into gene regulation and function.

Tara McDonnell (SVRS Student)

Tara McDonnell is a second year undergraduate at UNSW.

For her Summer Vacation Research Studentship project, Tara is looking at SLiM enrichment in the protein-protein interaction (PPI) partners of 14-3-3 proteins. This is part of ongoing work assessing how well our current PPI data captures SLiM-mediated interactions. She is also looking to see whether there is any evidence of phosphomimicry in 14-3-3 ligand binding.

Sobia Idrees (PhD student)

Sobia Idrees has a Virology and Bioinformatics background. She completed her MPhil in Biotechnology and BS in Bioinformatics from GC University, Faisalabad, Pakistan. During her MPhil, she worked on various Virology and Bioinformatics projects which helped her in learning Molecular biology as well as Computational biology techniques. She joined UNSW in October 2015 with a particular interest in learning new Bioinformatics approaches especially to understand host-pathogen interactions, joining the Edwards Lab in January 2016. Her PhD is focused on the bioinformatic prediction of molecular mimicry in human viruses.

SUMMARY OF ACADEMIC QUALIFICATIONS

1. MPhil. IN BIOTECHNOLOGY (Virology) — GC University, Faisalabad, Pakistan
2. BS (Hons. ) IN BIOINFORMATICS — GC University, Faisalabad, Pakistan

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