Incorporating molecular data in fungal systematics: a guide for aspiring researchers

The last twenty years have witnessed molecular data emerge as a primary research instrument in most branches of mycology. Fungal systematics, taxonomy, and ecology have all seen tremendous progress and have undergone rapid, far-reaching changes as disciplines in the wake of continual improvement in DNA sequencing technology. A taxonomic study that draws from molecular data involves a long series of steps, ranging from taxon sampling through the various laboratory procedures and data analysis to the publication process. All steps are important and influence the results and the way they are perceived by the scientific community. The present paper provides a reflective overview of all major steps in such a project with the purpose to assist research students about to begin their first study using DNA-based methods. We also take the opportunity to discuss the role of taxonomy in biology and the life sciences in general in the light of molecular data. While the best way to learn molecular methods is to work side by side with someone experienced, we hope that the present paper will serve to lower the learning threshold for the reader.Comment: Submitted to Current Research in Environmental and Applied Mycology - comments most welcom

Work ow-based systematic design of high throughput genome annotation

The genus Eimeria belongs to the phylum Apicomplexa, which includes many obligate intra-cellular protozoan parasites of man and livestock. E. tenella is one of seven species that infect the domestic chicken and cause the intestinal disease coccidiosis which is economy important for poultry industry. E. tenella is highly pathogenic and is often used as a model species for the Eimeria biology studies. In this PhD thesis, a comprehensive annotation system named as \WAGA" (Workflow-based Automatically Genome Annotation) was built and applied to the E. tenella genome. InforSense KDE, and its BioSense plug-in (products of the InforSense Company), were the core softwares used to build the workflows. Workflows were made by integrating individual bioinformatics tools into a single platform. Each workflow was designed to provide a standalone service for a particular task. Three major workflows were developed based on the genomic resources currently available for E. tenella. These were of ESTs-based gene construction, HMM-based gene prediction and protein-based annotation. Finally, a combining workflow was built to sit above the individual ones to generate a set of automatic annotations using all of the available information. The overall system and its three major components were deployed as web servers that are fully tuneable and reusable for end users. WAGA does not require users to have programming skills or knowledge of the underlying algorithms or mechanisms of its low level components. E. tenella was the target genome here and all the results obtained were displayed by GBrowse. A sample of the results is selected for experimental validation. For evaluation purpose, WAGA was also applied to another Apicomplexa parasite, Plasmodium falciparum, the causative agent of human malaria, which has been extensively annotated. The results obtained were compared with gene predictions of PHAT, a gene finder designed for and used in the P. falciparum genome project

CorNet : assigning function to networks of co-evolving residues by automated literature mining

CorNet is a web-based tool for the analysis of co-evolving residue positions in protein superfamily sequence alignments. CorNet projects external information such as mutation data extracted from literature on interactively displayed groups of co-evolving residue positions to shed light on the functions associated with these groups and the residues in them. We used CorNet to analyse six enzyme super-families and found that groups of strongly co-evolving residues tend to consist of residues involved in a same function such as activity, specificity, co-factor binding, or enantioselectivity. This finding allows to assign a function to residues for which no data is available yet in the literature. A mutant library was designed to mutate residues observed in a group of co-evolving residues predicted to be involved in enantioselectivity, but for which no literature data is available yet. The resulting set of mutations indeed showed many instances of increased enantioselectivity

The impact of within-herd genetic variation upon inferred transmission trees for foot-and-mouth disease virus

Full-genome sequences have been used to monitor the fine-scale dynamics of epidemics caused by RNA viruses. However, the ability of this approach to confidently reconstruct transmission trees is limited by the knowledge of the genetic diversity of viruses that exist within different epidemiological units. In order to address this question, this study investigated the variability of 45 foot-and-mouth disease virus (FMDV) genome sequences (from 33 animals) that were collected during 2007 from eight premises (10 different herds) in the United Kingdom. Bayesian and statistical parsimony analysis demonstrated that these sequences exhibited clustering which was consistent with a transmission scenario describing herd-to-herd spread of the virus. As an alternative to analysing all of the available samples in future epidemics, the impact of randomly selecting one sequence from each of these herds was used to assess cost-effective methods that might be used to infer transmission trees during FMD outbreaks. Using these approaches, 85% and 91% of the resulting topologies were either identical or differed by only one edge from a reference tree comprising all of the sequences generated within the outbreak. The sequence distances that accrued during sequential transmission events between epidemiological units was estimated to be 4.6 nucleotides, although the genetic variability between viruses recovered from chronic carrier animals was higher than between viruses from animals with acute-stage infection: an observation which poses challenges for the use of simple approaches to infer transmission trees. This study helps to develop strategies for sampling during FMD outbreaks, and provides data that will guide the development of further models to support control policies in the event of virus incursions into FMD free countries

Mapping the transcriptional landscape of haematopoietic stem and progenitor cells

Maintenance of the blood system requires balanced cell-fate decisions of haematopoietic stem and progenitor cells (HSPCs). Individual haematopoietic stem cells (HSCs) decide between self-renewal and differentiation and can generate all mature cell types. Cell-fate decisions are made at the single-cell level and are governed by regulatory networks. Dysregulation in this balanced process could lead to serious blood disorders such as leukaemia; therefore, it is important to understand how individual cells make these cell-fate decisions. To investigate HSPC populations, 1,654 cells were profiled by single-cell RNA-sequencing. Index sorting made it possible to sort HSPCs using broad sorting gates and retrospectively assign them to common HSPC populations, retaining all information about specific functionally pure populations while also capturing any intermediate cells normally excluded by conventional gating. Reconstruction of differentiation trajectories revealed dynamic expression changes associated with early lineage differentiation from HSCs. This transcriptional atlas of HSPC differentiation was further used to identify candidate genes for a CRISPR screen investigating genes implicated in HSC biology. These candidate gene perturbations were interrogated for changes in the expression of the HSC marker EPCR, as well as changes in apoptosis and lineage output. Transcription factors play a key role in regulating cell-fate decisions and operate within organized regulatory programs. To study relationships between transcription factors in HSPC populations, qRT-PCR was used to profile the expression of 41 genes, including 31 transcription factors, in HSPCs at the single-cell level. This approach confirmed known aspects of haematopoiesis and made deeper investigation of HSPC heterogeneity possible. Regulatory networks were reconstructed using Boolean network inference models and recapitulated differentiation of HSCs towards megakaryocyte–erythrocyte progenitors and lymphoid-primed multipotent progenitors. By comparing these two models, a rule specific to the megakaryocyte-erythrocyte progenitor network was identified, in which GATA2 positively regulated Nfe2 and Cbfa2t3h. This was subsequently validated using transcription factor binding profiles and in vitro luciferase assays using a model cell line. Overall, the work presented in this thesis confirmed known aspects of HSPC biology using single-cell gene expression analysis and demonstrated how in silico approaches can be used to guide in vitro and in vivo investigations. In addition, the single-cell RNA-sequencing data was developed into an intuitive web interface that can be used to visualise the gene expression for any gene of choice at single-cell resolution across the HSPC atlas, providing a powerful resource for the haematopoietic community.My funding for the CIMR 4 year programme was provided by the Medical Research Council (MRC)

A versatile set of Lifeact-RFP expression plasmids for live-cell imaging of F-actin in filamentous fungi

Here we report the construction and application of a range of expression plasmids designed to facilitate live-cell imaging of F-actin dynamics in filamentous fungi simultaneously with other, preferably GFP-tagged fusion proteins. Pros and cons of the use of three different red fluorescent proteins (RFPs), two different promoters and three different selection markers are addressed

Identifying a role for WASH in the endocytic pathway of Dictyostelium discoideum

Members of the WASP protein family are direct activators of the arp2/3 complex, thereby regulating the nucleation of branched actin assemblies within the cell. Each sub-class possesses a unique N-terminal domain architecture allowing a division of labour between its members, each coupling different signal transduction pathways to the nucleation of specific actin structures. WASH (WASP and SCAR homologue) is a newly identified member of the WASP protein family. Due to its disruption in $Drosophila$ proving lethal (Linardopopoulou et al., 2007) little is know as to the functional role of WASH at the cellular level. Other than it is important in the development of multicellular organisms. Here we successfully disrupt WASH in the single celled amoebae $Dictyostelium$ $discoideum$ and discover a role for WASH in the endocytic pathway. WASH was shown to be essential for the trafficking of indigestible material through the endocytic pathway, with its disruption causing a complete bock in cellular defecation. This was shown to be due to a defect in lysosomal maturation into neutral post-lysosomes. Using fluorescently tagged fusion proteins we show that WASH recruitment coincides with removal of the Vacuolar H+ ATPase from lysosomal membranes, and suggests a possible role for WASH and actin in regulating the luminal pH of intracellular compartments

Processing, visualising and reconstructing network models from single-cell data.

New single-cell technologies readily permit gene expression profiling of thousands of cells at single-cell resolution. In this review, we will discuss methods for visualisation and interpretation of single-cell gene expression data, and the computational analysis needed to go from raw data to predictive executable models of gene regulatory network function. We will focus primarily on single-cell real-time quantitative PCR and RNA-sequencing data, but much of what we cover will also be relevant to other platforms, such as the mass cytometry technology for high-dimensional single-cell proteomics.S.W is supported by a Microsoft Research PhD Scholarship.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/icb.2015.10

Complex polar machinery required for proper chromosome segregation in vegetative and sporulating cells of Bacillus subtilis

Chromosome segregation is an essential process of cell multiplication. In prokaryotes, segregation starts with the newly replicated sister origins of replication, oriCs, which move apart to defined positions in the cell. We have developed a genetic screen to identify mutants defective in placement of oriC during spore development in the Gram-positive bacterium Bacillus subtilis. In addition to the previously identified proteins Soj and DivIVA, our screen identified several new factors involved in polar recruitment of oriC: a reported regulator of competence ComN, and the regulators of division site selection MinD and MinJ. Previous work implicated Soj as an important regulator of oriC positioning in the cell. Our results suggest a model in which the DivIVA-interacting proteins ComN and MinJ recruit MinD to the cell pole, and that these proteins work upstream of Soj to enable oriC placement. We show that these proteins form a polar complex, which acts in parallel with but distinct from the sporulation-specific RacA pathway of oriC placement, and also functions during vegetative growth. Our study further shows that MinD has two distinct cell cycle roles, in cell division and chromosome segregation, and highlights that cell probably use multiple parallel mechanisms to ensure accurate chromosome segregation.</p