Summary A major unanswered question in neuroscience is whether there exists genomic variability between individual neurons of the brain, contributing to functional diversity or to an unexplained burden of neurological disease. To address this question, we developed a method to amplify genomes of single neurons from human brains. Since recent reports suggest frequent LINE-1 (L1) retrotransposition in human brains, we performed genome-wide L1 insertion profiling of 300 single neurons from cerebral cortex and caudate nucleus of 3 normal individuals, recovering >80% of germline insertions from single neurons. While we find somatic L1 insertions, we estimate <0.6 unique somatic insertions per neuron and most neurons lack detectable somatic insertions, suggesting that L1 is not a major generator of neuronal diversity in cortex and caudate. We then genotyped single cortical cells to characterize the mosaicism of a somatic AKT3 mutation identified in a child with hemimegalencephaly. Single-neuron sequencing allows systematic assessment of genomic diversity in the human brain.

The human cerebral cortex depends for its normal development and size on a precisely controlled balance between self-renewal and differentiation of diverse neural progenitor cells. Specialized progenitors that are common in humans, but virtually absent in rodents, called ‘outer radial glia’ (ORG), have been suggested to be crucial to the evolutionary expansion of the human cortex. We combined progenitor subtype-specific sorting with transcriptome-wide RNA-sequencing to identify genes enriched in human ORG, which included targets of the transcription factor Neurogenin and previously uncharacterized, evolutionarily dynamic long noncoding RNAs. We show that activating the Neurogenin pathway in ferret progenitors promotes delamination and outward migration. Finally, single-cell transcriptional profiling in human, ferret, and mouse revealed more cells co-expressing proneural Neurogenin targets in human compared to other species, suggesting greater neuronal lineage commitment and differentiation of self-renewing progenitors. Thus, we find that the abundance of human ORG is paralleled by increased transcriptional heterogeneity of cortical progenitors.