**Acknowledgements**

enrichment based technique, what the specificity of your antibody is. Different antibodies appear to show differing levels of repeat enrichment when performing MeDIP[67]. It would be of benefit to standardize these analyses. Similarly, different bisulphite conversion protocols may lead to differing conversion success. Global CpG methylation levels obtained from WGBS for 3 human embryonic stem cell (HESC) lines showed surprising variability (72% - 85%)[68]. This could be due to unstable gain and loss of methylation as previously reported in embryonic stem cells (ESCs)[69, 70], but it could also be a result of pre-analysis protocol and lab specific differences in sample preparation. Equally, it will be interesting to discover more about the biological roles and genomic location of the different cytosine modifications (5-hydroxyme‐ thylcytosine[47], 5-Formylcytosine and 5-Carboxylcytosine[71]) and also non-CpG methyla‐

162 Next Generation Sequencing - Advances, Applications and Challenges

New technologies with the potential for adaption for the analysis of DNA methylation are being developed constantly. For example, improved methods of methylation validation would be highly beneficial. Often hundreds or thousands of potential candidate regions are generated from a multi-sample MeDIP-seq comparison, and similar numbers could be produced by future EWAS (Epigenome-Wide Association Studies)[72]. Ideally, many of these regions would be validated using a different technology. Targeted bisulphite sequencing is often used, however this can often be laborious and time-consuming. Combining new technologies such as microdroplet-based PCR target enrichment (e.g. RainDance Technologies) with 2GS has recently been developed into a high-throughput platform termed RainDropBS-seq [73], providing an excellent option to remove the validation bottle-neck. There is also the emergence of third generation sequencing on the horizon. Third generation sequencing (3GS) theoretically promises many advantages over existing 2GS methods including higher throughput, longer read lengths, improved accuracy and requiring smaller amounts of starting material[74], indeed some companies e.g. Oxford Nanopore Technologies, are promising single molecule sequencing[75, 76]. The potential of single molecule nanopore sequencing is particularly exciting for researchers working in the field of DNA methylation. Theoretically, it should be possible to sequence complex mammalian genomes and determine any base modifications such as methylation[77], potentially including hitherto undiscovered modifications, without

As the large-scale projects, such as IHEC, BLUEPRINT and increasingly clinically oriented projects such as OncoTrack progress, it is expected that many methods and tools will become standardized. This will be an important step in translating epigenomic knowledge from the bench to the clinic[78, 79]. In the future, it is hoped that a patient will be treated with drugs tailored to their particular condition – this is of particular relevance for cancer patients. Preliminary work using whole genome, exome and RNA-seq has demonstrated the potential for treating a real patient in a relatively short time period (24 days) and a relatively low cost (~\$3600)[80]. Adding reliable epigenetic information, utilising the IHEC reference genomes, to this diagnostic toolbox is a logical next step. Extrapolating from these advances, it is quite clear that the bottleneck is shifting from logistics and data generation to computational

the need for any of the treatments or enrichments discussed above.

tion.

analysis.

Research in the Beck lab was supported by: Wellcome Trust (99148), Royal Society Wolfson Research Merit Award (WM100023), IMI-JU OncoTrack (115234) and EU-FP7 projects, EPIGENESYS (257082) and BLUEPRINT (282510).
