Bisulfite genomic sequencing: systematic investigation of critical experimental parameters

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 degrees C (4-18 h) and 95 degrees 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.

Figure 1

Alignment of model DNA used in this study. Primers art-1 and art-2 are given in lower case. ARTα and ARTβ are identical with the exception of the primer binding sites and six more mutations, one of them an A to G transition in position 92 which allows identification of the templates after bisulfite treatment.

Figure 2

Part of an autoradiogram for four sequencing reactions. The unconverted native sequence is GAC TCC GGG AAC GCC TAC CTG ATA AGT GCT A (positions 48–64 of ARTα). Arrowheads mark the cytosine positions. Plasmid DNA was treated with sodium bisulfite solution for 4 h at 0, 15, 35 and 55°C, PCR amplified and sequenced. Only at 55°C did complete cytosine conversion occur. The low PCR yield for DNA treated at 0°C thwarts accurate sequencing of the PCR product.

Figure 3

Degradation of single-stranded M13mp18 DNA during bisulfite treatment as a function of time: M13mp18 DNA was treated with sodium bisulfite for 5, 15 and 60 min at 55°C. After the treatment the DNA was incubated with nuclease P1 to generate 5′ monophosphate nucleotides. The amount of these dNMPs was quantified on a Smart HPLC system with MiniQ PC 3.2/3 column (Pharmacia). DNA degradation proceeds very fast and after 60 min only 4.2 ± 0.1 mass % of the initial DNA amount is detectable. At this time the dCMP concentration is below the detection limit. dUMP can be found after 15 min.