What are the basic steps of whole genome bisulfite sequencing (WGBS)?

The basic steps of whole genome bisulfite sequencing (WGBS) are DNA extraction, bisulfite conversion, library preparation, sequencing, and bioinformatic analysis. 

1. To prepare samples for whole-genome bisulfite sequencing, tissue samples (approximately 1-5 mg) from various sources like humans, animals, or plants, are first collected. These samples must be from eukaryotic organisms and exhibit hypomethylation. They also must have genome annotations that are relatively comprehensive and the reference genome of the organism should be made to at least the scaffold level.
2. DNA is extracted from the tissue samples using an appropriate kit. The extracted DNA must have the following characteristics: minimum mass of 5 μg concentration of at least 50 ng/μl, and an OD260/280 ratio between 1.8 and 2.0. 
3. Bisulfite then converts unmethylated cytosines to uracil while leaving methylated cytosines unaffected. 
4. In library preparation, DNA is randomly primed and the 3’ end of the newly synthesized DNA strand is selectively labeled with another specific sequence, resulting in a DNA molecule with known sequence tags at both the 5’ and 3’ ends. Subsequently, Illumina P7 and P5 adapters are added via PCR to the 5’ and 3’ ends of the DNA before sequencing. Illumina's HiSeq sequencing technology is a widely utilized method for WGBS. It functions on the sequencing-by-synthesis (SBS) principle. During bridge amplification on a flow cell, individual DNA molecules are immobilized and amplified into clusters. This amplification is facilitated by a single molecule array. This allows for the sequential addition of nucleotides, one at a time, and labeling with fluorophores. In general, WGBS uses a paired-end 150 base pair (bp) strategy, which sequences both ends of DNA fragments. 
5. Lastly, the sequencing results from WGBS can undergo several types of analysis. These include mC calling, sequence depth and coverage analysis, methylation level analysis, and alignment against reference genome.