Project description:We used the nanopore Cas9 targeted sequencing (nCATS) strategy to specifically sequence 125 L1HS-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. The genome-wide profile of L1 methylation was also assessed by bs-ATLAS-seq in the same cell lines (E-MTAB-10895).
Project description:We present a draft genome assembly that includes 200 Gb of Illumina reads, 4 Gb of Moleculo synthetic long-reads and 108 Gb of Chicago libraries, with a final size matching the estimated genome size of 2.7 Gb, and a scaffold N50 of 4.8 Mb. We also present an alternative assembly including 27 Gb raw reads generated using the Pacific Biosciences platform. In addition, we sequenced the proteome of the same individual and RNA from three different tissue types from three other species of squid species (Onychoteuthis banksii, Dosidicus gigas, and Sthenoteuthis oualaniensis) to assist genome annotation. We annotated 33,406 protein coding genes supported by evidence and the genome completeness estimated by BUSCO reached 92%. Repetitive regions cover 49.17% of the genome.
Project description:Based on the hypothesis that, enhancing the local concentration of donor oligos could increase the correction rates, we generated and tested novel CRISPR-Cas9 systems, in which the DNA repair template is covalently conjugated to Cas9 (RNPD system). To validate our results from the HEK293T reporter cells, we here tested our approach at different endogenous genomic loci and in different cell types. We first targeted the human beta globin (HBB) locus in the K562 cell line, and analyzed correction- and editing frequencies using next generation sequencing (NGS). Next we targeted the Rosa26 and proprotein convertase subtilisin/kexin type 9 (Pcsk9) locus in mouse embryonic stem cells (mESCs). Here, RNPD system was always compared to Cas9 SNAP-tag fusion proteins with uncoupled donor oligos. To also directly compare the engineered RNPD system to the classical CRISPR-Cas9 system, we performed experiments where we used wild-type Cas9 with the uncoupled donor oligos as a control. We therefore targeted the fluorescent reporter locus as well as the endogenous loci HBB, empty spiracles homeobox 1 (EMX1), and C-X-C chemokine receptor type 4 (CXCR4) in HEK293T cells. Finally, we performed the analysis of three computationally predicted off-target sites of the reporter locus.
Project description:This experiment was conducted to generate targeted resequencing data covering a region associated with osteosarcoma in greyhounds. 8 greyhounds diagnosed with osteosarcoma and 7 greyhounds without tumors were sequenced. DNA from the 15 dogs was used to prepare libraries and hybrid capture performed to enrich the region of interest prior to paired-end sequencing using Illumina Genome Analyzer II. The reads were aligned to the dog-genome CanFam2.0 using bwa and pre-processed using Picard and GATK. Variant discovery was performed using GATK. The resulting list of variants were used in the study to finemap the associated region and look for causal variants. We submit the preprocessed BAM-files that still have all reads although some reads are flagged. We also submit the resulting vcf-file with called and filtered variants in all individuals.