Bisulfite genomic sequencing: systematic investigation of critical experimental parameters

TLDR

Bisulfite genomic sequencing generates single‑base resolution methylation maps by selectively deaminating cytosine to uracil while leaving 5‑methylcytosine unchanged, enabling its distinction during PCR‑based sequencing. The study aimed to identify optimal incubation time and temperature for bisulfite deamination and assess DNA degradation. The authors varied incubation time and temperature, measured deamination efficiency and DNA degradation, and constructed homologous templates with cytosine‑containing or cytosine‑free primer sites to evaluate primer effects. Maximum cytosine conversion was achieved at 55 °C (4–18 h) and 95 °C (1 h), but these conditions degraded 84–96 % of DNA, while primer‑binding site composition did not affect cytosine or 5‑methylcytosine recognition rates (≥97 % and ≥94 %, respectively).

Abstract

Bisulfite genomic sequencing is the method of choice for the generation of methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil by treatment with bisulfite and the sequencing of subsequently generated PCR products. In contrast to cytosine, 5-methylcytosine does not react with bisulfite and can therefore be distinguished. In order to investigate the potential for optimization of the method and to determine the critical experimental parameters, we determined the influence of incubation time and incubation temperature on the deamination efficiency and measured the degree of DNA degradation during the bisulfite treatment. We found that maximum conversion rates of cytosine occurred at 55°C (4–18 h) and 95°C (1 h). Under these conditions at least 84–96% of the DNA is degraded. To study the impact of primer selection, homologous DNA templates were constructed possessing cytosine-containing and cytosine-free primer binding sites, respectively. The recognition rates for cytosine (≥97%) and 5-methylcytosine (≥94%) were found to be identical for both templates.