Extended Data Fig. 3: NA is not a general mutagen.

Towards assessing whether NA-treatment acted as a general mutagen to sequences other than CAG slip-outs, we harnessed the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS). Single-molecule sequencing was done on the HPRT1 gene – widely used as a surrogate indicator of the global effect of induced genetic variation. For each replicate, we calculated the relative mutation rate between NA- and saline-treated cells as the mutation rate for NA-treated cells minus the rate for saline-treated cells and identified excess mutation rates based on an absolute relative rate >0.5%. a, Schematic of HPRT1 sequencing for mutation detection. Briefly, cells were grown under identical conditions differing only by the addition of NA (50 μM) or saline, DNAs were isolated, HPRT1 exons 2 and 3 PCR amplified and sequenced. b, Quality control for our analysis. c,d, Comparison of sequence variations between NA-treated and saline treated is presented. We chose to compare the single-molecule sequence reads of individual X chromosome-linked HPRT1 alleles (exons 2 and 3) from our male HD patient-derived cells (c), and our male R6/2 mice (d), that had been NA- or saline-treated. Each read represents a single cell (Supplementary Note). Graphs show the distribution of sequence variants by relative mutation rate between three experimental replicates of NA-treated and saline-treated cells sequenced with PacBio single-molecule long reads.