Grimes Susan M, Kim Heon Seok, Roy Sharmili, Sathe Anuja, Ayala Carlos I, Bai Xiangqi, Almeda-Notestine Alison F, Haebe Sarah, Shree Tanaya, Levy Ronald, Lau Billy T, Ji Hanlee P
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA.
NAR Cancer. 2023 Jul 10;5(3):zcad034. doi: 10.1093/narcan/zcad034. eCollection 2023 Sep.
In this proof-of-concept study, we developed a single-cell method that provides genotypes of somatic alterations found in coding regions of messenger RNAs and integrates these transcript-based variants with their matching cell transcriptomes. We used nanopore adaptive sampling on single-cell complementary DNA libraries to validate coding variants in target gene transcripts, and short-read sequencing to characterize cell types harboring the mutations. CRISPR edits for 16 targets were identified using a cancer cell line, and known variants in the cell line were validated using a 352-gene panel. Variants in primary cancer samples were validated using target gene panels ranging from 161 to 529 genes. A gene rearrangement was also identified in one patient, with the rearrangement occurring in two distinct tumor sites.
在这项概念验证研究中，我们开发了一种单细胞方法，该方法可提供信使核糖核酸编码区中发现的体细胞改变的基因型，并将这些基于转录本的变异与其匹配的细胞转录组整合起来。我们对单细胞互补脱氧核糖核酸文库进行纳米孔自适应采样，以验证靶基因转录本中的编码变异，并使用短读长测序来表征携带这些突变的细胞类型。使用一种癌细胞系鉴定了针对16个靶点的CRISPR编辑，并使用一个352基因的检测板验证了该细胞系中的已知变异。使用包含161至529个基因的靶基因检测板验证了原发性癌症样本中的变异。在一名患者中还发现了一种基因重排，该重排在两个不同的肿瘤部位发生。
