NGS参考标准品遗传学与表观遗传学内容的表征

Here, we provide a deeper genetic and epigenetic analysis of cell lines used in reference standards. Blends of tumor and donor-matched normal cell lines can be used to create reference standards for the development and analytical validation of diagnostics in oncology. The normal component is often used as the source of germline single nucleotide polymorphisms (SNPs) to assess copy number variation (CNV), allelic ratios, and loss of heterozygosity (LOH) in the tumor component, to identify additional somatic mutations, and to enumerate these somatic mutations to determine tumor mutational burden (TMB). By adjusting the ratio of tumor and normal components, it is possible to simulate different tumor fractions to establish limits of detection. In order to better characterize the cell lines that are used in TMB and homologous recombination deficiency (HRD) reference standards, we carried out shallow whole genome shotgun (WGS) sequencing to determine CNV and supplemented that with whole exome sequencing to assess allelic ratios and LOH. Epigenetic analyses were carried out to evaluate CpG methylation. Through this characterization, several of the cell lines used for TMB were found to harbor deletions in the region containing the genes MTAP, CDKN2A, and CDKN2B. In a HRD low-positive cell line, PTEN was found to be fully deleted and surrounded by a larger LOH region. In a HRD high-positive cell line, BRCA1 showed signs of CpG methylation in its promoter. In conclusion, these characterizations provide additional utility to existing reference standards.