Project description:Aim: We aim to compare current (MeDIP-seq), new (Illumina Infinium 450K BeadChip) and future (PacBio) methods for whole genome DNA methylation analysis. As the interest in determination of disease methylation profiles increases, the scope, advantages and limitations of these methods requires assessment. There are key questions to answer and specific challenges to overcome. For example, how much detail/resolution is sufficient to identify regions of differential methylation and regions of biological/medical significance within a sample? How much coverage of the genome is required for accurate methylation analysis? Is it important to confirm which regions of the genome are unmethylated in addition to focusing on those that are methylated? Loss of methylation may be of equal importance within the cell since this may also contribute to disease pathogenesis. A multi-method (affinity enrichment/bisulphite-conversion based/direct sequencing of methyl-cytosine) and technology platform (Illumina HiSeq/PacBio/Illumina Infinium BeadChip) comparison will enable us to determine the strengths and weakness of each method. We propose to compare four methods using two DNA samples from the Coriell Institute for Cell Repository to assess both current and future capabilities for whole genome methylation analysis in parallel: A) MeDIP-seq using Illumina HiSeq B) Illumina Infinium HumanMethylation 450K BeadChip and C) whole genome methylation sequencing using PacBio. Existing single molecule deep bisulphite sequencing data generated previously from these same samples at the WTSI for targeted regions (30-40 genes) on the human X chromosome will be used to assess performance of each method. The methods selected for this study will generate data covering a range of resolutions from a whole genome scan to array (target defined) resolution and up to single base pair, single molecule resolution; the highest level of detail possible with methods currently available.Samples: DNA from sibling pair GM01240 (female) and GM01240 (male).Requirements: Both samples will be analysed using;A.MeDIP-seq using Illumina HiSeq (one HiSeq lane, 75bp paired end, per sample) B.Illumina Infinium HumanMethylation 450K BeadChipWe are expecting a potentially unnecessary high coverage using one HiSeq lane per sample. However, for the MeDIP procedure we do not have a multiplexing procedure in place. Our requirements for PacBio sequencing have been discussed with and will be supported by the Sequencing Technology Development group.
Project description:Whole-genome sequencing on PacBio of laboratory mouse strains. See http://www.sanger.ac.uk/resources/mouse/genomes/ for more details. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:Iso-Seq (PacBio) sequencing was performed to generate a reference library of H. perforatum. We generated genome-wide transcriptome data from in vitro cell suspensions and shoot cultures of H. perforatum.
Project description:Bacteria belonging to phylum Gemmatimonadetes are found in a wide variety of environments and are particularly abundant in soils. To date, only two Gemmatimonadetes strains have been characterized. Here we report the complete genome sequence and methylation pattern of Gemmatirosa kalamazoonensis KBS708 (ATCC BAA-2150; NCCB 100411), the first characterized Gemmatimondetes strain isolated from soil. Examination of the methylome of Gemmatirosa kalamazoonenis KBS708 using kinetic data from single-molecule, real-time (SMRT) sequencing on the PacBio RS
Project description:Chromatin immunoprecipitation analysis of CENH3 in the Arabidopsis thaliana accessions Col-0, Ler-0, Cvi-0 and Tanz-1 was performed in order to align reads to PacBio HiFi genome assemblies which contain complete centromere repeat arrays.
Project description:Primary objectives: The primary objective is to investigate circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Primary endpoints: circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
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.