Project description:Part of a set of highly integrated epigenome maps for Arabidopsis thaliana. Keywords: Illumina high-throughput bisulfite sequencing Whole genome shotgun bisulfite sequencing of wildtype Arabidopsis plants (Columbia-0), and met1, drm1 drm2 cmt3, and ros1 dml2 dml3 null mutants using the Illumina Genetic Analyzer.
Project description:Whole genome shotgun bisulfite sequencing, small RNA sequencing and transcriptome sequencing of wildtype Arabidopsis plants (Col-0), and met1, drm1 drm2 cmt3, and ros1 dml2 dml3 null mutants using the Illumina Genetic Analyzer. A comparison was performed with regions of the genome containing cytosine DNA methylation identified by methylcytosine immunoprecipitation and whole-genome oligonucleotide tiling microarrays, for wildtype Col-0. Understanding the epigenetic regulatory mechanisms that mediate control of transcription at multiple levels is critical to understanding how plants develop and respond to their environment. We combined next-generation sequencing by synthesis (SBS) technology with novel methods for direct sequencing of the entire cytosine methylome (methylC-seq), transcriptome (RNA-seq), and the small RNA component of the transcriptome (smRNA-seq) to create a set of highly integrated epigenome maps for Arabidopsis thaliana, in conjunction with a set of informative mutants defective in DNA methyltransferase and DNA demethylase activity. At single-base resolution we discovered extensive, previously undetected, DNA methylation, identified the context and level of methylation at each site, and found that local composition has effects upon DNA methylation state. Deep sequencing of the smRNAome exposed a direct relationship between the location and abundance of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in the region of RNA-DNA homology. Finally, strand-specific RNA-seq revealed changes in the transcript abundance of hundreds of genes upon alteration of the DNA methylation state, and enabled the identification of numerous previously unidentified genes regulated by DNA methylation. Keywords: Whole genome shotgun bisulfite sequencing, small RNA sequencing, transcriptome sequencing, methylcytosine immunoprecipitation, whole-genome oligonucleotide tiling microarrays Whole genome shotgun bisulfite sequencing, small RNA sequencing and transcriptome sequencing of wildtype Arabidopsis plants (Col-0), and met1, drm1 drm2 cmt3, and ros1 dml2 dml3 null mutants using the Illumina Genetic Analyzer. A comparison was performed with regions of the genome containing cytosine DNA methylation identified by methylcytosine immunoprecipitation and whole-genome oligonucleotide tiling microarrays, for wildtype Col-0.
Project description:This study examined the functional response of a host (zebrafish) to implantation of a conspecific or allospecific (goldfish) gastrointestinal (GIT) microbiome followed by diet manipulation and the repercussions of these manipulations on host GIT physiology. Implantation of a native zebrafish biome successfully reintroduced wildtype (WT) communities with the exception of several rare, phylogenetically distant species. Implantation of a foreign goldfish biome created communities that were distinct from WT, suggesting that the seeding community created substantial differences from the native host communities. A mismatched ?natural? diet and an implanted allospecific biome enriched for rarer and more phylogenetically diverse bacteria. Transcriptional changes within the GIT clustered in relationship to biome treatments, mirroring clustering of biome implants. Implantation of an allospecific biome along with an altered diet markedly down-regulated approximately 70% of the transcripts involved in cholesterol biosynthesis, while tissue content analysis revealed an increase in total tissue cholesterol. Furthermore, transcripts involved in lipogenesis pathways were significantly downregulated and correlated with a striking decrease in intestinal lipase activity driven by both biome and diet. Glucose-6P dehydrogenase (G6PD) activities increased during dietary manipulations regardless of biome, while the allospecific biome down-regulated transcripts involved in gluconeogenesis and altered glucokinase (GK) and hexokinase (HK) activities regardless of diet. However, growth rates did not reveal an impact of these responses. Adult zebrafish are unable to reform proportional representation within bacterial communities following transplantation of an allospecific biome resulting in transcriptional and enzymatic alterations for lipid and carbohydrate metabolism that did not affect overall animal homeostasis.
Project description:Background: Skin biopsies represent a gold standard in skin immunology and pathology but can cause pain and induce scarring. Non-invasive techniques will facilitate study recruitment of e.g. patients with pediatric atopic dermatitis (AD), hand eczema or facial dermatitis. Objective: By RNA sequencing, we examined whether the stratum corneum transcriptome in AD skin can be assessed by tape stripping, as compared to the epidermal transcriptome of AD in skin biopsies. To make the procedure clinically relevant tape strips were stored and shipped at room temperature for up to 3 days. Methods: Nine adult Caucasian AD patients and three healthy volunteers were included. Tape samples were collected from non-lesional and lesional skin. Biopsies were collected from lesional skin and were split into epidermis and dermis. Total RNA was extracted, and shotgun sequencing was performed. Results: Shotgun sequencing could be performed on skin cells obtained from two consecutive tape strips which had been stored and shipped at room temperature for up to three days. The most prominent differences between the tape strip and biopsy derived transcriptome were due to structural genes, while established molecular markers of AD, including CCL17, CCL22, IL17A and S100A7-S100A9, were also identified in tape strip samples. Furthermore, the tape strip derived transcriptome showed promise in also analysing the skin microbiome. Conclusion: Our study shows that the stratum corneum (SC) transcriptome of AD can be assessed by tape stripping the skin, supporting that this method may be central in future skin biomarker research.
Project description:Whole-genome sequencing is an important way to understand the genetic information, gene function, biological characteristics, and living mechanisms of organisms. There is no difficulty to have mega-level genomes sequenced at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. The shotgun sequencing method failed to dissect this genome. After insisting for 10 years and going over 3 generations of sequencing techniques, we successfully dissected the PaP1 genome with 91,715 bp in length. Single-molecule sequencing revealed that this genome contains lots of modified bases, including 51 N6-methyladenines (m6A) and 152 N4-methylcytosines (m4C). At the same time, further investigations revealed a novel immune mechanism of bacteria, by which the host bacteria can recognize and repel the modified bases containing inserts in large scale, and this led to the failure of the shotgun method in PaP1 genome sequencing. Strategy of resolving this problem is use of non-library dependent sequencing techniques or use of the nfi- mutant of E. coli DH5M-NM-1 as the host bacteria to construct the shotgun library. In conclusion, we unlock the mystery of phage PaP1 genome hard to be sequenced, and discover a new mechanism of bacterial immunity in present study. Methylation profiling of Pseudomonas aeruginosa phage PaP1 using kinetic data generated by single-molecule, real-time (SMRT) sequencing on the PacBio RS.