A probabilistic framework for cellular lineage reconstruction using integrated single-cell 5-hydroxymethylcytosine and genomic DNA sequencing

Lineage reconstruction is central to understanding tissue development and maintenance. To overcome the limitations of current techniques that typically reconstruct clonal trees using genetically encoded reporters, we report scPECLR, a probabilistic algorithm to endogenously infer lineage trees at a single-cell-division resolution by using 5-hydroxymethylcytosine (5hmC). When applied to 8-cell pre-implantation mouse embryos, scPECLR predicts the full lineage tree with greater than 95% accuracy. In addition, we developed scH&G-seq to sequence both 5hmC and genomic DNA from the same cell. Given that genomic DNA sequencing yields information on both copy number variations and single-nucleotide polymorphisms, when combined with scPECLR it enables more accurate lineage reconstruction of larger trees. Finally, we show that scPECLR can also be used to map chromosome strand segregation patterns during cell division, thereby providing a strategy to test the “immortal strand” hypothesis. Thus, scPECLR provides a generalized method to endogenously reconstruct lineage trees at an individual-cell-division resolution.