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 the 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.

中文翻译：

---

RETrace：同时对单细胞进行回顾性谱系追踪和甲基化分析。

回顾性谱系追踪利用细胞中自然发生的突变来阐明单细胞发育。常见的单细胞系统发育命运图谱方法利用了在人类基因组中发现的高度可变的微卫星位点。这些方法受到通过 DNA 扩增中固有的聚合酶滑动引入体外噪声的限制，我们将其表征为比体内复制突变率高约 10-100 倍。在这里，我们介绍了 RETrace，这是一种同时捕获微卫星和甲基化信息胞嘧啶的方法，以分别从同一单个细胞中表征谱系和细胞类型。RETrace 的一个重要独特功能是引入了微卫星的线性放大，以减少体外放大噪音。我们进一步将微卫星捕获与单细胞减少代表性亚硫酸氢盐测序 (scRRBS) 相结合，以测量同一细胞上的 CpG 甲基化状态以进行细胞类型推断。与现有的回顾性谱系追踪方法相比，RETrace 以更高的细胞分裂分辨率（将单个细胞之间所需的细胞分裂差异减少约 100 次分裂）实现了更高的准确度（来自离体 HCT116 树的三元组准确度为 88%）。同时，RETrace 展示了每个细胞平均捕获 150,000 个独特 CpG 以准确确定细胞类型的能力。我们进一步制定了额外的发展，允许使用 RETrace 在微卫星稳定细胞或组织上进行高分辨率映射。总体，