RETrace: simultaneous retrospective lineage tracing and methylation profiling of single cells

Retrospective lineage tracing harnesses naturally occurring mutations in cells to elucidate single cell development. Common single cell phylogenetic fate mapping methods have utilized highly mutable microsatellite loci found within the human genome. Such methods were limited by the introduction of in vitro noise through polymerase slippage inherent in DNA amplification, which we characterized to be approximately 10-100? higher than in vivo replication mutation rate. Here, we present RETrace, a method for simultaneously capturing both microsatellites and methylation-informative cytosines to characterize both lineage and cell type, respectively, from the same single cell. An important unique feature of RETrace was the introduction of linear amplification of microsatellites in order to reduce in vitro amplification noise. We further coupled microsatellite capture with single-cell reduced representation bisulfite sequencing (scRRBS), to measure the CpG methylation status on the same cell for cell type inference. When compared to existing retrospective lineage tracing methods, RETrace achieved higher accuracy (88% triplet accuracy from an ex vivo HCT116 tree) at a higher cell division resolution (lowering the required number of cell division difference between single cells by approximately 100 divisions). Simultaneously, RETrace demonstrated the ability to capture on average 150,000 unique CpGs per single cell in order to accurately determine cell type. We further formulated additional developments that would allow high-resolution mapping on microsatellite stable cells or tissues with RETrace. Overall, we present RETrace as a foundation for multi-omics lineage mapping and cell typing of single cells. Overall design: 138 HCT116, 67 U87MG, and 137 adult post-mortem single cells/nuclei were processed along with respective bulk

回顾性谱系示踪（Retrospective lineage tracing）借助细胞内自然发生的突变，阐明单细胞发育机制。常见的单细胞谱系命运作图方法多利用人类基因组中的高度可变微卫星（microsatellite）位点。此类方法的局限性在于DNA扩增过程中固有的聚合酶滑移会引入体外噪声，经本研究表征，该噪声水平约较体内复制突变率高10~100倍。本研究报道了RETrace技术，该方法可同时捕获微卫星与甲基化信息胞嘧啶，分别用于表征同一单细胞的谱系特征与细胞类型。RETrace的一项核心独特优势在于引入了微卫星线性扩增策略，以降低体外扩增带来的噪声。本研究进一步将微卫星捕获技术与单细胞简化代表性亚硫酸氢盐测序（single-cell reduced representation bisulfite sequencing, scRRBS）相结合，可在同一细胞中检测CpG甲基化状态，进而实现细胞类型推断。相较于现有回顾性谱系示踪方法，RETrace在更高的细胞分裂分辨率下实现了更优异的准确率（来自体外HCT116细胞系谱系的三重准确率达88%），可将区分单细胞所需的最小细胞分裂差异数降低约100个分裂事件。与此同时，RETrace可在每个单细胞中平均捕获15万个独特CpG位点，从而实现精准的细胞类型判定。本研究还开发了适配方案，使RETrace可应用于微卫星稳定的细胞或组织的高分辨率谱系作图。综上，本研究推出RETrace技术，可作为单细胞多组学谱系作图与细胞分型的基础工具。实验整体设计：共处理138株HCT116细胞、67株U87MG细胞与137例成人死后单细胞/细胞核样本，并同步配套处理对应的bulk样本。