Project description:To compare the performance of Illumina and BGI sequencing technologies for high-throughput single cell sequencing, four Chromium single cell libraries of the following human cell types: Induced Pluripotent Stem Cells (hIPSC), cultured Trabecular MeshWork Cells (TMWC) and Peripheral Blood Mononuclear Cells (PBMCs), were sequenced on Illumina sequencers (NextSeq 500, NovaSeq 6000) and a BGI sequencer (MGISEQ-2000). The technologies were benchmarked based on sequencing quality, characterisation of cell populations within samples and for specific single cell analyses such as variant calling and detection of guide RNAs from pooled CRISPR screens.
Project description:Single Gland Whole-exome sequencing: building on our prior description of multi-region WES of colorectal tumors and targeted single gland sequencing (E-MTAB-2247), we performed WES of multiple single glands from different sides (right: A and left: B) of two tumors in this study (tumor O and U) on the illumina platform using the Agilent SureSelect 2.0 or illumina Nextera Rapid Capture Exome kit (SureSelect or NRCE, as indicated in the naming of fastq files). Colorectal Cancer Xenograft Whole-exome sequencing: The HCT116 and LoVo Mismatch-Repair-deficient colorectal adenocarcinoma cell lines were obtained from the ATCC and cultured under standard conditions. For both cell lines, a single âfoundingâ cell was cloned and expanded in vitro to ~6M cells. Two aliquots of ~1M cells were subcutaneously injected into opposite flanks (right and left) of a nude mouse and tumors allowed to reach a size of ~1B cells (1cm3) before the animal was sacrificed. Tumor tissue was collected separately from the right and left lesions and DNA was extracted for WES using the illumina TruSeq Exome kit or Nextera Rapid Capture Exome expanded Kits (Truseq or NRCEe), as was DNA from the first passage population (a polyclonal tissue culture for HCT116 and a polyclonal xenograft sample for LoVo), which were employed as a control to study mutation accumulation in culture and post xenotransplantation.
Project description:Colorectal cancer is an epigenetically heterogeneous disease, the extent to which is unclear. In this study we interrogate the methylome of 216 unselected colorectal cancers using Illumina HumanMethylation450 arrays, and correlate these data with transcript profiles and exome sequencing analysis to elucidate the degree of epigenetic dysregulation in colorectal cancers.
Project description:Colorectal cancer is an epigenetically heterogeneous disease, the extent to which is unclear. In this study we interrogate the methylome of 216 unselected colorectal cancers using Illumina HumanMethylation450 arrays, and correlate these data with transcript profiles and exome sequencing analysis to elucidate the degree of epigenetic dysregulation in colorectal cancers.
Project description:We benchmark Illumina’s HiseqX10 instrument against Beijing Genomics Institute’s (BGI) DNBSEQ-G400 platform, a considerably cheaper sequencing alternative. For comparisons, the same bulk ATAC-seq libraries generated from pluripotent stem cells (PSCs) and fibroblasts were sequenced on both platforms. Both instruments generate sequencing reads with comparable mapping rates and genomic context. However DNBSEQ-G400 data contained a significantly higher number of small, sub-nucleosomal reads (>30% increase) and a reduced number of bi-nucleosomal reads (>75% decrease), which resulted in narrower peak-bases and improved peak calling, enabling the identification of 4% more differentially accessible regions between PSCs and fibroblasts. Ability to identify master TFs that underpin the PSC state relative to fibroblasts, including aggregate and de novo foot-printing capacity, were highly similar between data generated on both platforms.
Project description:Clinical exome sequencing of cells freshly isolated from 12 human colorectal carcinoma patients (tumor endothelial cells, normal colon endothelial cells, PBMCs, each n=12) in comparison to DNA isolated from microdissected tumor cells (n=11) from corresponding FFPE-tissue blocks
Project description:Agilent whole exome hybridisation capture was performed on genomic DNA derived from Chondrosarcoma cancer and matched normal DNA from the same patients. Next Generation sequencing performed on the resulting exome libraries and mapped to build 37 of the human reference genome to facilitate the identification of novel cancer genes. Now we aim to re find and validate the findings of those exome libraries using bespoke pulldown methods and sequencing the products.
