Project description:Single-cell RNA-sequencing (scRNA-seq) is a powerful technique for describing cell states. Unfortunately, identifying the spatial arrangement of these states in tissues remains challenging. Here, we describe SEgmentation by Exogenous Perfusion (SEEP), a rapid and integrated method to link positional location to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of 3D disease models. Classification of sample layers based on dye accessibility permits dissociated and sorted cells to be retrospectively characterized by transcriptomic and regional identity. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs regarding the relationship between cell state and position while also revealing new concepts on how the spatially unique microenvironment of individual cells within tumors influences cell identity.
Project description:The use of yeast as a delivery system is an attractive option for the oral administration of therapeutic agents. We recently developed mutants of Saccharomyces cerevisiae capable of lysis upon conditional down-regulation of the expression of the cell wall genes PKC1 and SRB1. The lysis mechanism of the mutant is based on the use of the MET3 promoter, which, upon addition of methionine and cysteine, blocks transcription of SRB1 and PKC1. This strain has the potential to be an integral part of an oral yeast delivery system, in which there is lysis of yeasts in the human gut, followed by release of recombinant proteins for therapeutic use. In order to provide proof-of-principle, the system was evaluated testing the cells viability and lysis performance under conditions, which simulate those found in the human stomach and the duodenum. Upon incubation of yeast cells in these conditions, lysis could be induced and was accompanied by release of GFP reporter protein into the medium. However, the conditional lysis mechanism based on the MET3 promoter is not applicable in vivo. Therefore, alternative promoters suitable for in-vivo down-regulation of SRB1 and PKC1 were identified by a microarray experiments. The transcripts of genes ANB1, TIR1, and MF(ALPHA)2 were significantly reduced upon exposure of the yeast cells to conditions of the two gut compartments. Their promoters could be used to down-regulate SRB1/VIG9 and PKC1 in vivo to achieve lysis of the yeast in the gut to release cargo therapeutic proteins.
Project description:Background Trombidid mites have a unique lifecycle in which only the larval stage is ectoparasitic. In the superfamily Trombiculoidea (“chiggers”), the larvae feed preferentially on vertebrates, including humans. Species in the genus Leptotrombidium are vectors of a potentially fatal bacterial infection, scrub typhus, which affects 1 million people annually. Moreover, chiggers can cause pruritic dermatitis (trombiculiasis) in humans and domesticated animals. In the Trombidioidea (velvet mites), the larvae feed on other arthropods and are potential biological control agents for agricultural pests. Here, we present the first trombidid mites genomes, obtained both for a chigger, Leptotrombidium deliense, and for a velvet mite, Dinothrombium tinctorium. Results Sequencing was performed on the Illumina MiSeq platform. A 180 Mb draft assembly for D. tinctorium was generated from two paired-end and one mate-pair library using a single adult specimen. For L. deliense, a lower-coverage draft assembly (117 Mb) was obtained using pooled, engorged larvae with a single paired-end library. Remarkably, both genomes exhibited evidence of ancient lateral gene transfer from soil-derived bacteria or fungi. The transferred genes confer functions that are rare in animals, including terpene and carotenoid synthesis. Thirty-seven allergenic protein families were predicted in the L. deliense genome, of which nine were unique. Preliminary proteomic analyses identified several of these putative allergens in larvae. Conclusions Trombidid mite genomes appear to be more dynamic than those of other acariform mites. A priority for future research is to determine the biological function of terpene synthesis in this taxon and its potential for exploitation in disease control. Project was jointly supervised by Stuart Armstrong and Ben Makepeace.
Project description:The deep marine subsurface is one of the largest unexplored biospheres on Earth, where members of the phylum Chloroflexi are abundant and globally distributed. However, the deep-sea Chloroflexi have remained elusive to cultivation, hampering a more thorough understanding of their metabolisms. In this work, we have successfully isolated a representative of the phylum Chloroflexi, designated strain ZRK33, from deep-sea cold seep sediments. Phylogenetic analyses based on 16S rRNA genes, genomes, RpoB and EF-tu proteins indicated that strain ZRK33 represents a novel class within the phylum Chloroflexi, designated Sulfochloroflexia. We present a detailed description of the phenotypic traits, complete genome sequence and central metabolisms of the novel strain ZRK33. Notably, sulfate and thiosulfate could significantly promote the growth of the new isolate, possibly through accelerating the hydrolysis and uptake of saccharides. Thus, this result reveals that strain ZRK33 may play a crucial part in sulfur cycling in the deep-sea environments. Moreover, the putative genes associated with assimilatory and dissimilatory sulfate reduction are broadly distributed in the genomes of 27 metagenome-assembled genomes (MAGs) from deep-sea cold seep and hydrothermal vents sediments. Together, we propose that the deep marine subsurface Chloroflexi play key roles in sulfur cycling for the first time. This may concomitantly suggest an unsuspected availability of sulfur-containing compounds to allow for the high abundance of Chloroflexi in the deep sea.
Project description:Uncultivated Actinobacteria genomes recovered from three hot spring sediment samples
| PRJNA511649 | ENA
Project description:Pseudomonas aeruginosa, Echerichia coli, Mycobacterium tuberculosis, and uncultivated soil bacterium Raw sequence reads and de novo assembled draft genomes
Project description:Genome graphs, including the recently released draft human pangenome graph, can represent the breadth of genetic diversity and thus transcend the limits of traditional linear reference genomes. However, there are no genome-graph-compatible tools for analyzing whole genome bisulfite sequencing (WGBS) data. To close this gap, we introduce methylGrapher, a tool tailored for accurate DNA methylation analysis by mapping WGBS data to a genome graph. Notably, methylGrapher can reconstruct methylation patterns along haplotype paths precisely and efficiently. To demonstrate the utility of methylGrapher, we analyzed the WGBS data derived from five individuals whose genomes were included in the first Human Pangenome draft as well as WGBS data from ENCODE (EN-TEx). Along with standard performance benchmarking, we show that methylGrapher fully recapitulates DNA methylation patterns defined by classic linear genome analysis approaches. Importantly, methylGrapher captures a substantial number of CpG sites that are missed by linear methods, and improves overall genome coverage while reducing alignment reference bias. Thus, methylGrapher is a first step towards unlocking the full potential of Human Pangenome graphs in genomic DNA methylation analysis.