GeDI-MeDIP sequencing: A novel method for parallel identification of genetic and epigenetic variations in a same reduced fraction of large genomes. GeDI-MeDIP sequencing

Epigenetic modifications involve chemical interactions between DNA and other molecules such as histones, methyl groups or small RNAs 1. These interactions influence gene expression, allowing the genome to be differentially active depending on the stage of development, the type of tissue, or the existence of a disease 2. DNA methylation involves the addition of methyl groups to DNA bases, mainly cytosines. This interaction may remain stable throughout multiple cell divisions 1.Cancer as a heterogeneous disease occurs by a combination of genetic mutations and epigenetic abnormalities 3. The identification of diagnostic and prognostic molecular biomarkers for early and as accurate as possible detection remains a promising approach to improve the survival of cancer patients. Owning a combination of various kinds of biomarkers including genetic, epigenetic and molecular markers like Prostate Specific Antigen (as a distinguished prostate cancer signature), could have positive influences on the credibility of the diagnosis 4. During last years, aiming in the improvement of cancer screening approaches and decreasing the mortality rate of cancer patients, have engaged scientists' attentions to discover robust sensitive diagnostic and prognostic biomarkers rather than treatments approaches. Meanwhile, DNA methylation based signatures are among valuable epigenetic detection biomarkers because of their capability in early awarding of tumorigenesis events, identifying the type of the malignancies as well as in tracking of tumors recurrence after surgery 5,6.DNA methylation as an essential epigenetic modification can affect gene expression 7. Evidently, DNA hypo- methylation may leads to the gene reactivation, chromosomal instability, upregulation of proto- oncogenes, increasing of the mutation and recombination, suppression of X- chromosome inactivation and elimination of genomic imprinting 8. On the other hand, hyper- methylation of the DNA is often associated with genomic instability (through the extinction of the DNA repairing genes) and gene suppression resulting in down-regulation of tumor suppressor genes and chromatin condensation 9. The investigations offered hypermethylated variants such as MGMT 10 (a potential therapy choice marker in glioma), MLH1 (a forecaster marker for cisplatin therapy outcome in case of colon cancer) 4, GSTP1 11 (a diagnostic element in prostate cancer) and a number of others 4 as potential epigenetic markers. Nonetheless, research is ongoing to find specific and sensitive DNA methylation based cancer detection panels, and recently, SEPT9 gene methylation biomarker has passed the process of Food and Drug Administration (FDA) certificate approval to consider as a clinical test for screening of the colon cancer 12.On the other hand, genetic variants like single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) may cause susceptibility to cancer progression and even change the efficiency of drugs for cancer treatments 13-15. There is a list of genetic mutations used in cancer screening like BRCA1 and BRCA2 gene mutations for breast, ovarian and prostate cancers detection 16,17 and mutations in MLH1 and MSH2 genes, as risk factors for colon cancer development 18. Additionally, copy number variation analyses reported amplified variants in oncogenes like MITF in case of melanoma malignancy and NKX2-1 in lung cancers 17. At the moment, there are a number of personalized medicine based tests commercially accessible e.g, for breast cancer, colorectal cancer, lung cancer and chronic myelogenous leukemia 19. Although, the implement of pharmacogenomics into routine clinical care is still in its infancy, it is promising a tremendous transformation toward more reliable precise treatments 20.With the advent of next generation sequencing (NGS), different high- throughput techniques have been evolved with the aim of DNA methylation status profiling in genome- wide scales including reduced representation bisulfite sequencing (RRBS), whole-genome bisulfite sequencing (WGBS) and methylated DNA immunoprecipitation (MeDIP) 21. Each of these methods represent its own strengths and weaknesses 22. Quantitative comparison of different sequencing based DNA methylation approaches, for example, shows that these methods are different regards to CpG coverage, resolution, quantitative accuracy, efficiency and cost 23. However, all methods yielded comparable methylation calls 23. The best strategy selection depends on the experiment aim, the DNA input available, the multiplex flexibility of the method, the resolution scale to receive the desired purpose 22 and the cost efficiency of the profiling per sample. It is suggested that the exploiting of innovative DNA barcoding system and pooling the samples before directing the sequencing procedure in order to reduce the complicity of whole genome may offer more facilities to ever decreasing the cost and efficiency of parallel whole genome sequencing 17. Although methods mentioned above have been applied in the high- throughput analysis research, none of them proposes a cost-efficient method that allows the sequencing of a unique reduced portion of the genome for parallel identification of genetic and epigenetic markers on a whole genome level without methylation bias towards any CpG related motif. The development of "omics" approaches not only help in understanding the complexity of the genetic and epigenetic molecular events involved in malignancies but also can help in conducting the improvement of cancer treatments 22. Both the genome and epigenome sequencing of the countless individuals is very expensive. Therefore, the development of a cost-efficient sequencing technique that provides a parallel-reduced representation of the genome and epigenome, resulting in ever increasing the samples included in the assessment, which, in turn, yields a more accurate statistical analysis 24, is necessary. In the way to the implementation of more efficient cure to overcome the smart intrinsic nature of cancer cells, development of the decreased cost genome wide parallel genetic and epigenetic approach will be inevitable. In current paper, we are studying cancer as the model in which we applied our innovative cost effective tool integrating genetic and epigenetic evaluations in parallel. This new approach was named GeDI MeDIP -Seq (Genotyping Digested and Methylated DNA Immunoprecipitation Sequencing).