Today, the 25th of April 2013, marks the 60th anniversary since the groundbreaking publication on DNA structure by James Watson and Francis Crick. In recent years we are witnessing another revolution in biology; epigenetics. And, like figuring out the structure of DNA required the development of X-ray technology, so epigenetics is intimately linked with next generation sequencing.
Genome-wide by nature
Histone and DNA modifications (as well as histone variants) are the main tools in the cell’s epigenetic toolbox. Depending on the cell type and the environment, a medley of these chemical modifications decorates chromatin in specific patterns. As scientists embarked in early attempts to decode the meaning of these markings, it was obvious that this genome-wide phenomenon required genome-wide approaches.
A power boost!
Microarrays enabled the creation of a first ‘dictionary’ of epigenetic modifications. However, it was the adoption of NGS that truly boosted the power of epigenetic research and changed the way we view and study the genome.
Chromatin immunoprecipitation (ChIP) is the powerhouse of epigenetic research. In its earlier incarnation, the immunoprecipitated DNA was hybridized to microarrays (ChIP-chip) to obtain a read-out. This meant that the aspiring epigeneticist had to indulge in a series of hybridizations on multiple tilling arrays that covered the genome one unique 50-mer at a time.
Time and money
A lot of work and money was required for the identification of a relatively small number of locations. Researchers needed to have a gut feeling for epigenetic hot-spots and pick a small number of interesting genomic loci- as many as would fit within a single microarray, or, literally, pay a premium on their scientific meticulousness.
An old hero gets new powers
ChIP-seq took epigenetics research out of specialized laboratories and made it accessible to all (you can read James Hadfield’s Introduction here). The initial reason for combining ChIP with the emerging NGS technology was probably the ease of adapting the original DNA-seq protocols to immunoprecipitated DNA. When the first data started emerging, ChIP-seq pioneers were making a strong case for the new technology.
The start of the love affair
ChIP-seq eliminated a lot of the labor attached to microarray hybridization. It allowed the detection of previously unresolvable, low complexity regions. It had lower background, higher sensitivity and increased resolution. Even the false discovery rates where better! A love affair between epigenetics and NGS was born.
This-seq, that-seq, everything-seq
Soon after the world of epigenetics embraced ChIP-seq, many laboratories started adapting established epigenetics techniques to the new sequencing technology. Old protocols were taken out of the drawer and a whole generation of ‘-seq’ methodologies emerged, bringing with it new and exciting epigenetic information. MNase-seq mapped precisely all the nucleosomes on the genome. DNAse-seq probed into the level of chromatin compaction. RNA-seq found a new application in the identification of imprinted genes. And, as in the case of ChIP-seq, the new methodologies exceeded their microarray cousins in every metric.
A problem of resolution
In an analogy to ChIP-seq, DNA methylation was initially probed genome-wide using affinity purification of methylated DNA (MeDIP and MAP). However, the resolution of this approach was limited by the fragment size of the purified DNA. This was a far cry from the single-nucleotide resolution which is desirable in DNA methylation mapping.
A specific problem requires a specific solution
Detection of individual methylcytosines was only possible with the low-throughput methodology of bisulfite DNA treatment. The researcher had to decide between breadth and resolution. The marriage of bisulfite DNA treatment with NGS seemed inevitable and transformed epigenetics. It provided a unique method that combined accuracy and genome-wide power in DNA methylation mapping. But epigeneticists were already used to expecting good performances from NGS. What they were not expecting was that already, the first two reports on bisulfite-seq would be changing the textbooks on DNA methylation.
A growing legacy
In the short time which has passed since the first ChIP-seq experiments, the combination of NGS with epigenetics has already bestowed its legacy to humankind. The ambitious ENCODE project aims to decrypt the function of the non-coding 98% of our genome. The first epigenetic drugs are already making a shy appearance. With the third generation of sequencing technologies just around the corner, it is hard to imagine what the future holds. Oxford’s Nanopore sequencer for example is able to discriminate between different base modifications. This could prove paramount in understanding the role of hydroxymethylcytosine, the mysterious, newly discovered base that embellishes our genome.
A story in the making
It is hard not to look 60 years back and be amazed by how far molecular biology has come. The story of epigenetics and NGS illustrates the way towards the future: the creative combination of basic research and applied technologies.