Project description:The Zika outbreak, spread by the Aedes aegypti mosquito, highlights the need to create high-quality assemblies of large genomes in a rapid and cost-effective fashion. Here, we combine Hi-C data with existing draft assemblies to generate chromosome-length scaffolds. We validate this method by assembling a human genome, de novo, from short reads alone (67X coverage, Sample GSM1551550). We then combine our method with draft sequences to create genome assemblies of the mosquito disease vectors Aedes aegypti and Culex quinquefasciatus, each consisting of three scaffolds corresponding to the three chromosomes in each species. These assemblies indicate that virtually all genomic rearrangements among these species occur within, rather than between, chromosome arms. The genome assembly procedure we describe is fast, inexpensive, accurate, and can be applied to many species. Overall design: We use DNA proximity ligation (Hi-C) to create a genome assembly with chromosome-length scaffolds for the mosquito Aedes aegypti, principal vector of the Zika virus.
Project description:Nicotiana benthamiana is an important model organism and representative of the Solanaceae (Nightshade) family. N. benthamiana has a complex ancient allopolyploid genome with 19 chromosomes, and an estimated genome size of 3.1Gb. Several draft assemblies of the N. benthamiana genome have been generated, however, many of the gene-models in these draft assemblies appear incorrect. Here we present a nearly non-redundant database of improved N. benthamiana gene-models based on gene annotations from well-annotated genomes in the Nicotiana genus. We show that the new predicted proteome is more complete than the previous proteomes and more sensitive and accurate in proteomics applications, while maintaining a reasonable low gene number (~43,000). As a proof-of-concept we use this proteome to compare the leaf extracellular (apoplastic) proteome to a total extract of leaves. Several gene families are more abundant in the apoplast. For one of these apoplastic protein families, the subtilases, we present a phylogenetic analysis illustrating the utility of this database. Besides proteome annotation, this database will aid the research community with improved target gene selection for genome editing and off-target prediction for gene silencing.
Project description:Cyflumetofen is a novel acaricide on the international market with an unknown mode of action. Under laboratory conditions, we selected for high levels of cyflumetofen resistance in a susceptible mite strain and performed genome-wide gene-expression analysis. Differential expression between the resistant and susceptible strain was identified to study on the molecular level how mites develop resistance to this novel acaricide. Our results show that in T. urticae, the selection for cyflumetofen resistance resulted in a differential expression in only a limited number of genes. This transcriptomic study provides an unbiased look how cyflumetofen resistance triggers selection on the transcriptional level in T. urticae. 4 replicates for one comparison; per replicate 150 mites were pooled. Mites of the derived cyflumetofen resistant spider mite (cy5-labelled) were directly compared to mites of the ancestral susceptible strain (cy3-labelled).
Project description:Cyenopyrafen is a novel acaricide with a currently unknown mode of action. We selected for high levels of cyenopyrafen resistance in a susceptible spider mite strain and previously published patterns of cross-resistance to other well-described acaricides (Khalighi et al., 2014). In addition, we also included an independent cyenopyrafen-susceptible mite strain (referred to as Akita) in this study. We performed transcriptomic analysis to identify on the molecular level how T. urticae develops resistance to this novel acaricide. Using this dataset, we show that the selection for cyenopyrafen resistance resulted in massive transcriptomic responses in T. urticae. The multi-gene family of cytochrome P450 monooxygenases (CYPs) exhibited a drastic differential expression with a significant transcriptional induction in the acaricide resistant strain. This transcriptomic study highlights the potential importance of CYPs in mite resistance to the cyenopyrafen acaricide. Overall design: 8 replicates for two comparisons using an Agilent dual color GE microarray design; per replicate 150 mites were pooled. Mites of the cyenopyrafen resistant spider mite (cy5) were directly compared to mites of a susceptible strain (cy3). In a second set of samples, mites of the cyenopyrafen susceptible Akita strain (cy5) and of a reference strain (cy3) were included.
Project description:House dust mite/HDM atopy patch test/APT elicits positive reactions in the majority of atopic dermatitis/AD and healthy individuals. Experimental systems for new-onset/chronic AD are needed to support rapid therapeutic development, particularly since animal models representing AD pathology in humans are lacking. HDM APT historically simulated AD, but its suitability to model the emerging AD skin phenotype as Th2/Th22 polarized with Th1 and Th17 components is unknown. To assess whether HDM APT tissues reproduce acute or chronic AD, positive HDM APT (n=14) were compared with nonlesional, acute (<72hrs; n=10), and chronic phase AD biopsies (n=8), allergic contact reactions (to nickel [n=10] and fragrance [n=3]) using arrays. Overall design: 14 patients with positive dust mite reactions, were compared to 8 AD patients (acute and chronic lesions). 8 patients out of 14 patients with positive dust mite reactions did not have positive reactions to nickel or fragrance. 5 patients out of 14 patients with positive dust mite reactions had positive reactions to nickel. 1 patient out of 14 patients with positive dust mite reactions had positive reaction to fragrance. 5 other patients with positive nickel reactions and 2 other patients with fragrance reaction but not dustmite reactions were included in the study. The study considered a total of 29 patients (8 Atopic Dermatitis and 21 dust mite/nickel/fragrance positive reactions). Lesional and Non Lesional skin for AD patients were analyzed. Atopic patch skin and non lesional skin for dust mite/nickel/fragrance patients were analyzed. For patients with more than one positive patch reaction a sample from each atopy patch test reaction was obtained. We are submiting here only the samples from dust mite atopic patch skin (14) and the non lesional skin samples related to patients with positive dust mite reaction only (8). The other samples have been submited in: series accession no. GSM815426, GSM815427, GSE36842 and GSE36842.
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 PI3K-AKT pathway is known to regulate cytokines in dust mite-induced pediatric asthma. However, the underlying molecular steps involved are not clear. In order to clarify further the molecular steps, this study investigated the expression of certain genes and the involvement of miRNAs in the PI3K-AKT pathway, which might affect the resultant cytokine-secretion. In-vivo and in-vitro ELISA, qRT-PCR, western-blot and microarrays analyses were used in this study. A down-expression of miRNA-27b-3p in dust mite induced asthma group (group D) was found by microarray analysis. This was confirmed by qRT-PCR that found the miRNA-27b-3p transcripts that regulated the expression of SYK and EGFR were also significantly decreased (p < 0.01) in group D. The transcript levels of the SYK and PI3K genes were higher, while those of EGFR were lower in the former group. Meanwhile, we found significant differences in plasma concentrations of some cytokines between the dust mite-induced asthma subjects and the healthy controls. On the other hand, this correlated with the finding that the transcripts of SYK and its downstream PI3K were decreased in HBE transfected with miRNA-27b-3p, but were increased in HBE transfected with the inhibitor in vitro. Our results indicate that the differential expression of the miRNAs in dust mite-induced pediatric asthma may regulate their target gene SYK and may have an impact on the PI3K-AKT pathway associated with the production of cytokines. These findings should add new insight into the pathogenesis of pediatric asthma. Overall design: Twelve pairs of gender- and age-matched Group D dust mite-induced asthma children and Group N normal control children were included in the initial microarray-based discovery analysis.