Department of Oncology-Pathology Karolinska Institutet, 171 64 Stockholm, Sweden.
Department of Oncology-Pathology Karolinska Institutet, 171 64 Stockholm, Sweden.
Mol Cell. 2020 Nov 5;80(3):541-553.e5. doi: 10.1016/j.molcel.2020.09.025. Epub 2020 Oct 16.
To address how genetic variation alters gene expression in complex cell mixtures, we developed direct nuclear tagmentation and RNA sequencing (DNTR-seq), which enables whole-genome and mRNA sequencing jointly in single cells. DNTR-seq readily identified minor subclones within leukemia patients. In a large-scale DNA damage screen, DNTR-seq was used to detect regions under purifying selection and identified genes where mRNA abundance was resistant to copy-number alteration, suggesting strong genetic compensation. mRNA sequencing (mRNA-seq) quality equals RNA-only methods, and the low positional bias of genomic libraries allowed detection of sub-megabase aberrations at ultra-low coverage. Each cell library is individually addressable and can be re-sequenced at increased depth, allowing multi-tiered study designs. Additionally, the direct tagmentation protocol enables coverage-independent estimation of ploidy, which can be used to identify cell singlets. Thus, DNTR-seq directly links each cell's state to its corresponding genome at scale, enabling routine analysis of heterogeneous tumors and other complex tissues.
为了解决遗传变异如何在复杂的细胞混合物中改变基因表达的问题，我们开发了直接核片段化和 RNA 测序（DNTR-seq）技术，该技术可以在单个细胞中同时进行全基因组和 mRNA 测序。DNTR-seq 很容易在白血病患者中识别出次要亚克隆。在大规模的 DNA 损伤筛选中，DNTR-seq 用于检测受净化选择的区域，并鉴定出那些 mRNA 丰度不受拷贝数改变影响的基因，这表明存在强烈的遗传补偿。mRNA 测序（mRNA-seq）的质量与仅 RNA 方法相当，基因组文库的低位置偏差允许在超低覆盖度下检测亚兆碱基的异常。每个细胞文库都可以单独寻址，并可以在增加的深度上重新测序，从而实现多层次的研究设计。此外，直接片段化协议允许独立于覆盖度来估计倍性，这可用于识别单细胞。因此，DNTR-seq 直接将每个细胞的状态与其相应的基因组在大规模上联系起来，使对异质肿瘤和其他复杂组织的常规分析成为可能。
