Primary template-directed amplification (PTA) is an improved amplification technique for single-cell DNA sequencing. We generated whole-genome analysis of 76 single neurons and developed SCAN2, a computational method to accurately identify both clonal and non-clonal somatic (i.e., limited to a single neuron) single nucleotide variants (SNVs) and small insertions and deletions (indels) using PTA data. Our analysis confirms an increase in non-clonal somatic mutation in single neurons with age, but revises estimates for the rate of this accumulation to be 15 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels also increase by at least 2 indels per year per neuron and that indels may have a larger impact on gene function than somatic SNVs in human neurons.

Cancer immunotherapy by immune checkpoint blockade (ICB) is effective for several cancer types 1, however, its clinical use is encumbered by a high variability in patient response. Several studies have suggested that Tumor Mutational Burden (TMB) correlates with patient response to ICB treatments 2–6, likely due to immunogenic neoantigens generated by novel mutations accumulated during cancer progression 7. Association of TMB and response to checkpoint inhibitors has become widespread in the oncoimmunology field, within and across cancer types 7–11, and has led to the development of commercial TMB-based biomarker platforms. As a result, patient prioritization for ICB based on individual TMB level was recently approved by the FDA 12. Here we revisit the association of mutational burden with response to checkpoint inhibitors by aggregating pan-cancer data of ICB-treated patients with whole-exome sequencing and clinical annotation. Surprisingly, we find little evidence that TMB is predictive of patient response to immunotherapy. Our analysis suggests that previously reported associations arise from a combination of confounding disease subtypes and incorrect statistical testing. We show that using a TMB threshold for clinical decisions regarding immunotherapy could deprive potentially responding patients of receiving efficacious and life-extending treatment. Finally, we present a simple mathematical model that extends the neoantigen theory, is consistent with the lack of association between TMB and response to ICB and highlights the role of immunodominance. Our analysis calls for caution in the use of TMB as a biomarker and emphasizes the necessity of continuing the search for other genetic and non-genetic determinants of response to immunotherapy.