Figure 2.

Sodium bisulfite conversion rates. (A) Using a kit for sodium bisulfite conversion (Materials and Methods), the standard protocol (cyan) involves a step with a 45-min incubation at 80°C. This converts (on average) 79% of cytosines, consistent with expected rates of genomic cytosine methylation. Reducing the temperature to 73°C and the time to 3, 6 and 9 min results in mean conversions of 19%, 41% and 55%, respectively. (B) A high-depth sequencing library from the 6-min conversion shows a mean conversion rate 42% per cytosine position with near zero conversion of cytosines in the CpG context. (C) The per-template cluster mutation rate is shown as a histogram. The majority of template clusters are consistent with independent conversion at a fixed rate, whether or not we exclude CpG from the count. However, there are some templates—about 0.5%—that largely escape conversion.