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: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.
Project description:The genetic structure of the indigenous hunter-gatherer peoples of Southern Africa, the oldest known lineage of modern man, holds an important key to understanding humanity's early history. Previously sequenced human genomes have been limited to recently diverged populations. Here we present the first complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and of a Bantu from Southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, and 13,146 novel amino-acid variants. These data allow genetic relationships among Southern African foragers and neighboring agriculturalists to be traced more accurately than was previously possible. Adding the described variants to current databases will facilitate inclusion of Southern Africans in medical research efforts. Copy number differences between NA18507 and KB1 were predicted from the depth of whole-genome shotgun sequence reads. These predictions were then validated using array-CGH using a a genome-wide design as well as a custom design targeted at specific regions of copy number difference
Project description:Moniliophthora perniciosa is the causal agent of cacao Witche´s broom disease. This disease has been causing extensive damages to Brazilian cacao plantation, especially in Southern Bahia. Using glass slides microarrays, we analyzed the expression profile of 3872 whole genome shotgun reads from M. perniciosa genome, comparing two stages of development (Biotrophic-like mycelia and saprotrophic mycelia). Keywords: Moniliophthora perniciosa, Witches Broom Disease, Pathogenesis, Cacao