Parallel RNA and DNA analysis after deep sequencing (PRDD-seq) reveals cell type -specific lineage patterns in human brain

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we de- veloped parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single -cell and bulk DNA sequencing by single -cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation ( ?birthdate ?) in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate muta- tion mosaic frequency to clonal patterns, confirming an early di- vergence of precursors for excitatory and inhibitory neurons, and an ?inside -out ? layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excit- atory neuron -restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype -specific patterns of neurogenesis, in- cluding some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron de- velopment not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single -cell resolution and in potentially any developmental or disease condition.