Studying the epigenome using next generation sequencing

Abstract

The advances in next generation sequencing (NGS) technologies have had a significant impact on epigenomic research. The arrival of NGS technologies has enabled a more powerful sequencing based method--that is, ChIP-Seq--to interrogate whole genome histone modifications, improving on the conventional microarray based method (ChIP-chip). Similarly, the first human DNA methylome was mapped using NGS technologies. More importantly, studies of DNA methylation and histone modification using NGS technologies have yielded new discoveries and improved our knowledge of human biology and diseases. The concept that cytosine methylation was restricted to CpG dinucleotides has only been recently challenged by new data generated from sequencing the DNA methylome. Approximately 25% of all cytosine methylation identified in stem cells was in a non-CG context. The non-CG methylation was more enriched in gene bodies and depleted in protein binding sites and enhancers. The recent developments of third generation sequencing technologies have shown promising results of directly sequencing methylated nucleotides and having the ability to differentiate between 5-methylcytosine and 5-hydroxymethylcytosine. The importance of 5-hydroxymethylcytosine remains largely unknown, but it has been found in various tissues. 5-hydroxymethylcytosine was particularly enriched at promoters and in intragenic regions (gene bodies) but was largely absent from non-gene regions in DNA from human brain frontal lobe tissue. The presence of 5-hydroxymethylcytosine in gene bodies was more positively correlated with gene expression levels. The importance of studying 5-methylcytosine and 5-hydroxymethylcytosine separately for their biological roles will become clearer when more efficient methods to distinguish them are available.

The Importance of Cancer Cell Lines as in vitro Models in Cancer Methylome Analysis and Anticancer Drugs Testing

“…These are based in Next Generation Sequencing (NGS), which allows the sequencing of almost all coding regions (and at a low-extension, non-coding sequences) of both the genome and the methylome [42,43]. The NGS platforms have the power of sequencing massively-parallel short-read DNA [42] with a high-throughput at a low cost [44], substituting some techniques as the Sanger traditional sequencing [45] and microarrays [42].…”

“…NGS is responsible for the recent increase of epigenetic studies, transforming the resolution of the characterization at the epigenetic level [42,43,46], and have allowed the construction of the first map of the human methylome [43]. The genome-wide DNA methylation profiling can be done by array-based or sequencing-based (NGS) with the combination of bisulfite conversion (that transforms the unmethylated cytosines into uracil, preserving the methylated cytosines) or immunoprecipitation of the methylated DNA (MeDIP) [42,47].…”

“…For the study of epigenetics genome-wide, a technology that combines chromatin-immunoprecipitation (ChIP) and NGS technologies has been used [42]. The ChIP methodology is based on DNA and proteins interactions and together with NGS platforms (ChIP-Seq) is used to analyse histones' modifications genome-wide, as methylation [42,43].…”

The Contribution of Next Generation Sequencing Technologies to Epigenome Research of Stem Cell and Tumorigenesis

“…As described above, advances have been achieved in recent years. However, there are still two major challenges in epigenome research, respectively referring to sampling and integrated analysis of various epigenetic modifications [10]. Next, the review will discuss the two aspects in detail.…”

“…Multilevel epigenetic mechanisms constitute the system of regulating gene expression in cells. By cell specific regulations, those mechanisms are crucial for cellular developments, such as embryogenesis, cell differentiation [10]. Thus, the aberration in the epigenetic regulation system is reported to be associated with a wide range of diseases [11,14].…”