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:An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient’s gene, it has been challenging to determine which two of the variants are responsible for the disease phenotype. To decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5’ and 3’ variants was around 19 kb at the level of genomic DNA. nCATS successfully delineated that the most 5’ and 3’ variants were located in one allele while the variant in between was in the other allele. Intriguingly, the proband’s mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study illuminates nCATS as a useful tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can tell which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.
Project description:To compare the impact of CRISPR-egineered R175 TP53 mutant variants in HCT116 and H460 cells, mutations at the amino acid position 175 were generated systematically by CRISP/Cas9 editing. Here, genomic amplicon regions covering the TP53 Exons 5 were sequenced via targeted sequencing.
Project description:We used targeted long-read Oxford Nanopore Technologies sequencing enriching for a panel of 1036 pharmacogenes extracted from the PharmGKB database. The enrichment was performed using ONT's adaptive sampling feature, enabling in silico enrichment without physically capturing the fragments of interest using hybridization.
Project description:Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, although genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. We introduced Nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin.
Project description:We used bs-ATLAS-seq to comprehensively map the genomic location and assess the DNA methylation status of human full-length LINE-1 elements (L1) in the genome of 2102Ep cells (E-MTAB-10895). We also achieved targeted nanopore sequencing to assay DNA methylation over a subset of loci (E-MTAB-12247). To further study the link between L1 DNA methylation and expression, we performed, in the same cell line, RNA-seq (E-MTAB-12246), as well as YY1 and H3K4me3 ChIP-seq (this dataset).
