Next Generation Sequencing (NGS)/Epigenetics
Epigenetics is the science that studies inheritable traits not transmitted by plain sequence information. NGS can assess a particular non-standard epigenetics effect, which is the amount of methylation occurring on cytosines. This methylation is important biologically because it may influence the level of packing of chromatin and therefore affect efficiency of transcription in entire genomic areas. Cytosine methylation is reversible but inheritable somatically and germinally.
Genomic DNA can be treated with bisulfite, protocol that will transform only non-methylated cytosines into thymidines. Methylated cytosines are not affected, and will still be sequenced as such. A common NGS application in epigenetics is to align bisulfite-treated reads from a known organism on a reference genome, to assess the degree of methylation in particular areas. However, the complexity of the alignment will be higher, alongside with the reduced complexity of the reads (with several Cs being turned into Ts). Therefore, genomic DNA samples are sequenced both with and without bisulfite treatment, operation which allow to assess and normalize for the initial of reads aligning on specific regions.
Specific short read aligners exist for this task, just to name a few:
Chip-seq (Chromatin immunoprecipitation sequencing)
Chip-Sequencing is an effective technology that uses chromatin immunoprecipitation to DNA-protein or protein- protein interaction in the genome and it uses the more accurate, higher throughput method of sequencing.Even though ChIP-chip can be used to determine protein-DNA interaction, ChIP-seq is rapidly becoming the method of choice for the genome-wide localization of epigentics, transcription regulation and post-transcription regulation.
The goal of ChIP-Seq method is to identify genome-wide binding patterns of a protein of interest. The major step involving ChIP-seq pipeline would be crosslinking, shearing, immunoprecipitaion and sequencing. Formaldehyde is a widely used in DNA-protein crosslinking agent in ChIP methods. To perform ChIP, cells are treated with formaldehyde resulting in the covalent crosslinking of proteins to the DNA sequences which they are associated with. Then, shearing of DNA is done either by sonication or by MNase digestion. The DNA is broken into pieces of about 0.2 to 1.0 kb in length. The fragmented DNA is purified using immunoprecipitation which is the process of binding antibody that are specific to the protein that is associated to DNA. The purified DNA-protein complexes are heated which separates protein from DNA. The fragmented DNAs are isolated and are sequenced by using next generation sequencing methods. Either 454, Solexa or Solid can be used to sequence based on the convenience.
Creating a dataset
- Grunau, C.; Clark, S.J.; Rosenthal, A. (2001). "Bisulfite genomic sequencing: Systematic investigation of critical experimental parameters". Nucleic Acids Research 29 (13): e65. doi:10.1093/nar/29.13.e65. PMID 11433041.
- Barker, S.; Weinfeld, M.; Murray, D. (2005). "DNA–protein crosslinks: Their induction, repair, and biological consequences". Mutation Research 589 (2): 111–135. doi:10.1016/j.mrrev.2004.11.003. PMID 11433041.