Recently, several more techniques have been developed using primers based on the same principles (32). One variant of the direct-BSP method uses two rounds of nested PCR with primers designed by standard methods for the first round and primers with a GC-rich tag at the 5′ end for the second round. This primer modification is intended to reduce non-specific amplification during direct-BSP and compensate for the frequent artifacts seen in direct-BSP results (33,34).
Bias in BSP: Several studies using MIP primers have shown bias toward unmethylated or methylated alleles (8,20), likely due to sequence differences between methylated and unmethylated alleles (34). For example, Warnecke et al. found a 33-fold amplification bias toward the unmethylated allele when assaying a region of the RB1 gene promoter (20). In some cases, adjusting MgCl2 concentrations or redesigning the primers to bind the opposite DNA strand may be sufficient to resolve this bias (20,34). Shen et al. found that, in some instances, adjusting the annealing temperature may correct this bias (35). Wojdacz et al. developed a new approach to primer design that allows the use of annealing temperature changes to adjust for amplification bias (36,37). These new guidelines for bias compensation are described in detail in the MS-HRM section of this article.
Another potential source of error occurs in cloning-based BSP methods. Cloning biases may skew the reliability of results generated from BSP assays (22,34). There is evidence that amplicons without cytosines may be more difficult to clone efficiently (8). Although direct-BSP has low sensitivity, it provides more accurate detection of differences as low as 20% in methylation status in a single CpG (29).
Data analysis: Quantification of methylation levels is determined by comparing the relative peak heights of cytosine and thymine (or adenine and guanine in cases of complementary strand sequences) (25) in each CpG position in the electropherogram (Figure 2B). A qualitative analysis of bisulfite sequencing results can be performed if a clear single peak is present for each CpG cytosine position. In that case, a thymine peak would be interpreted as a non-methylated CpG, and a cytosine peak would represent a methylated CpG. Analysis of raw sequence data from direct-BSP is often difficult, but correction algorithms aid data interpretation. The Epigenetic Sequencing Methylation Software (ESME) program includes an algorithm to analyze direct-BSP sequencing results and provides a quality control filter (Table 4) (29).Table 4.
ESME may also be used to analyze cloning-based BSP electropherograms (29). In cloning-BSP or digital-BSP, satisfactory sequencing results belonging to the same sample should be averaged to determine the level of methylation for each CpG position. This task is facilitated by BiQ-Analyzer and BISMA (38,39) (Table 4).
BSP selection: Different BSP methodologies are optimal for different methylation studies, depending on the particular conditions of a study and other parameters, including cost, research question, and available samples. For example, the study initially validating digital MethyLight is a case where digital-BSP was the best choice, since it allowed accurate validation of another single-molecule technique and the use of automated PCR-processing for a large number of sequencing reactions (30). In many other cases, cloning-BSP is preferred because it is the only option for determining DNA-methylation haplotypes in general laboratories (34). Direct-BSP was selected to assess DNA methylation in BDNF to complement initial results from MSP screening experiments (40). Before deciding between direct-BSP and cloning-BSP for a particular application, we recommend testing and comparing previously validated primers and strategies (26,27,33).Methylation Specific PCR
Methylation specific PCR (MSP), first described by Herman et al. in 1996, determines the methylation status of an ROI through selective amplification of methylated and unmethylated alleles. The two-tube approach employs two primer sets: one binding specifically to the methylated sequence and another binding to the unmethylated sequence (11,41) (Figure 2C). A two-round variant of MSP, referred to as nested-MSP (N-MSP), has been described and can be used in special cases (42).