Project description:We performed shallow whole genome sequencing (WGS) on circulating free (cf)DNA extracted from plasma or cerebrospinal fluid (CSF), and shallow WGS on the tissue DNA extracted from the biopsy in order to evaluate the correlation between the two biomaterials. After library construction and sequencing (Hiseq3000 or Ion Proton), copy number variations were called with WisecondorX.
Project description:Rapid advances in high-throughput DNA sequencing technologies are accelerating the pace of research into personalized medicine. While methods for variant discovery and genotyping from whole genome sequencing (WGS) datasets have been well established, linking variants together into a single haplotype remains a challenge. An understanding of complete haplotypes of an individual will help clarify the consequences of inheriting multiple alleles in combination, identify novel disease associations, and augment studies of gene regulation. Although numerous methods have been developed to reconstruct haplotypes from WGS data, chromosome-span haplotypes at high resolution have been difficult to obtain. Here we present a novel method to accurately reconstruct chromosome-span haplotypes from proximity-ligation and DNA shotgun sequencing. We demonstrate the utility of this approach in producing high-resolution chromosome-span haplotype phasing in mouse and human. While proximity-ligation based methods were originally designed to investigate spatial organization of the genome, our results lend support for their use as a general tool for haplotyping in the future. Hi-C experiments in two replicates of Human GM12878 Lymphoblastoid cells and two replicates of F123 mouse ES cells (4 total samples)
Project description:Whole genome sequencing (WGS) of tongue cancer samples and cell line was performed to identify the fusion gene translocation breakpoint. WGS raw data was aligned to human reference genome (GRCh38.p12) using BWA-MEM (v0.7.17). The BAM files generated were further analysed using SvABA (v1.1.3) tool to identify translocation breakpoints. The translocation breakpoints were annotated using custom scripts, using the reference GENCODE GTF (v30). The fusion breakpoints identified in the SvABA analysis were additionally confirmed using MANTA tool (v1.6.0).
Project description:Rapid advances in high-throughput DNA sequencing technologies are accelerating the pace of research into personalized medicine. While methods for variant discovery and genotyping from whole genome sequencing (WGS) datasets have been well established, linking variants together into a single haplotype remains a challenge. An understanding of complete haplotypes of an individual will help clarify the consequences of inheriting multiple alleles in combination, identify novel disease associations, and augment studies of gene regulation. Although numerous methods have been developed to reconstruct haplotypes from WGS data, chromosome-span haplotypes at high resolution have been difficult to obtain. Here we present a novel method to accurately reconstruct chromosome-span haplotypes from proximity-ligation and DNA shotgun sequencing. We demonstrate the utility of this approach in producing high-resolution chromosome-span haplotype phasing in mouse and human. While proximity-ligation based methods were originally designed to investigate spatial organization of the genome, our results lend support for their use as a general tool for haplotyping in the future.
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 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:Low coverage whole genome sequencing (lc-WGS) from inducible Tet TKO (Tet iTKO) and control (Ctrl) mouse ESCs (mESC), as well as for germline Dnmt TKO mESCs. mESCs were sorted to isolate the Live/Dead dye and Thy1.2 negative CD326+GFP+ population representing the mESCs populations responsive to the tamoxifen treatment. The cells were resuspended in FACS buffer and filtered with a 70 µM filter before sorting. These bulk-population samples were analyzed by using low coverage Whole Genome Sequencing (lc-WGS).
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 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.