The sequencing results from WGBS can undergo several types of analysis. These include mC calling, sequence depth and coverage analysis, methylation level analysis, global trends of methylome, and alignment against reference genome.
The sequencing reads are aligned to a reference genome using tools like SOAP software, allowing for C-C matches and C-T mismatches. Only aligned reads are used for further methylation analysis.
Methylated cytosine (mC) positions across the genome are determined by computing mC ratios, this is done with the read quality and the probability of mapping to multiple loci. Alignments with low reliability are removed.
The methylation level of each methylated C base is calculated as a ratio of reads to total reads, expressed as a percentage (e.g., 100*reads/total reads). This provides information about the methylation status of individual bases.
Global trends of the methylome are examined by analyzing the distribution ratio of methylated C bases in different sequence contexts (e.g., CG, CHGG, and CHH).
Sequence depth and coverage analysis is then performed to understand the correlation between gene coverage and sequencing depth. This analysis helps determine the confidence level of methylation discovery at certain base positions.