Whole genome bisulfite sequencing

Whole genome bisulfite sequencing is a next-generation sequencing technology used to determine the DNA methylation status of single cytosines by treating the DNA with sodium bisulfite before high-throughput DNA sequencing. The DNA methylation status at various genes can reveal information regarding gene regulation and transcriptional activities. This technique was developed in 2009 along with reduced representation bisulfite sequencing after bisulfite sequencing became the gold standard for DNA methylation analysis. Whole genome bisulfite sequencing measures single-cytosine methylation levels genome-wide and directly estimates the ratio of molecules methylated rather than enrichment levels. Currently, this technique has recognized and tested approximately 95% of all cytosines in known genomes. With the improvement of library preparation methods and next-generation sequencing technology over the past decade, whole genome bisulfite sequencing has become an increasingly widespread and informative method for analyzing DNA methylation in epigenomic-wide studies.

History Prior to the development of whole genome bisulfite sequencing, genome methylation analysis relied heavily on early non-specific and differential methods such as paper chromatography, high-performance liquid chromatography, and thin-layer chromatography to analyze methylation profiles. These methods were limited by the inability to amplify methylated DNA via polymerase chain reaction in vitro due to loss of methylation status. As a result, much of these early methods relied on detecting and analyzing naturally-manifested methylated cytosines in vivo rather than chemically methylated cytosines. In 1970, a breakthrough occurred when it was discovered that treating DNA with sodium bisulfite deaminated cytosine residues into uracil. In the following decade, this discovery led to the revelation that unmethylated cytosine reacted much faster to sodium bisulfite treatment than did 5-methylcytosine. This difference in reaction rates created the possibility of identifying chemical changes in DNA as an easily detectable genetic marker. Whole genome bisulfite sequencing was derived as a combination of this bisulfite treatment and next-generation sequencing technology, such as shotgun sequencing. The whole genome sequencing technique was first applied to the DNA methylation mapping at single nucleotide resolution to Arabidopsis thaliana in 2008, and shortly after in 2009, the first single-base-resolution DNA methylation map of the entire human genome was created using whole genome bisulfite sequencing. Since its development, many various protocols of whole genome bisulfite sequencing have been developed aiming to improve the efficiency and efficacy of its single-base mapping. As the costs of next-generation sequencing have decreased, whole genome bisulfite sequencing has become more widely used in clinical and experimental research. Currently, multiple public datasets of genomic data have been established, and this technique has recognized and tested approximately 95% of all cytosines in known genomes.

Method The following steps are derived from one potential workflow of conventional whole genome bisulfite sequencing: target DNA extraction, bisulfite conversion, library amplification, and bioinformatics analysis. However, various sequencing systems and analysis tools often adapt the technical parameters and order of the following step processes in order to optimize assay coverage and efficacy.

DNA extraction Library preparation protocols undergo DNA fragmentation, end repair, dA-tailing, and adapter ligation prior to bisulfite treatment and library amplification. Standard fragmentation under high-throughput technology such as Illumina Genome Analyser and Solexa requires nebulization to generate fragments that range from 0-1200 base pairs. After fragmentation, end repair enzymes and complementary adapters are then applied to the DNA in an end-prep polymerase chain reaction and adapter ligation reaction, respectively. Size selection occurs before the DNA is treated with sodium bisulfite. Conventional methods of eukaryotic DNA preparation during sequencing use a wide variety of DNA input amount, varying from as little as 10 ng for novel NGS library alternatives, such as the tagmentation approach, to as much as 500-1000 ng of DNA as sample input.

Bisulfite conversion The adapter-ligated DNA sample is treated with sodium bisulfite, a chemical compound that converts unmethylated cytosines into uracil, at low pH and high temperatures. The chemical reaction is depicted in Figure 1, where sulfonation occurs at the carbon-6 position of cytosine to produce the intermediate cytosine sulfonate. This intermediate then undergoes irreversible hydrolytic deamination to create uracil sulfonate. Under alkaline conditions, uracil sulfonate desulfonates to generate uracil. This enables methylation detection by distinguishing the methylated cytosines (5-methylcytosine), which resist bisulfite treatment, from uracil. During amplification by polymerase chain reaction, the uracils are converted into thymines. Methylated cytosines are then recognized as cytosines. Their locations are then identified by comparison of the bisulfite-treated and original DNA sequence. Following bisulfite treatment, purification of the sample is required to remove unwanted products including bisulfite salts.

Library amplification In order to amplify the epigenome library, bisulfite-treated DNA is primed to generate DNA with a specific tagging sequence. The 3' end of this sequence is then tagged again, creating DNA fragments with markers on either end. These fragments are amplified in a final polymerase chain reaction reaction, after which the library is prepped for sequencing-by-synthesis. This is demonstrated in Figure 2, in which high-throughput sequencing system developed by biotechnology company, Illumina, perform comprehensive assays based on sequencing-by-synthesis of base pairs.

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