? Core B: Epigenomics The epigenetic modifications found in chromatin (DNA methylation and post-translational modifications of histones) are involved in gene regulation during development and differentiation. Core B will generate epigenomics data from massive parallel, multi-omic sequencing from human and mouse and xenopus developing neural tube. The Core will identify gene regulatory elements and epigenomic variants having the potential to cause or influence phenotypes. The Program PI and Director of Core B have worked together extensively in the past with great success producing significant mouse neural tube epigenomic data. Dr. Ecker has worked broadly in the area of genomics and epigenomics and in the development methodologies employing multi-modal ?multi-omics? techniques for generation multiple types of NGS datasets from single cells. The data generated from Core B, as well as imported from Project I, II and III, will be delivered to Core C for extraction of results which will be delivered to each of the Projects for further validation. These goals will be accomplished by developing the key pipelines of Core B that involve data production, and analysis: 1] Bulk whole genome bisulfite sequencing (WGBS) Pipeline. The Ecker lab published the first human methylome and thus has significant expertise in the workflow for data production. Our data workflow was established, and standard operating procedures developed and adopted, by the ENCODE project and will take advantage of the same features of our WGBS workflow that has made it successful for that effort. 2] Single cell methylome sequencing (snmC-seq) pipeline. The methods for production and analysis of single cell methylome data snmC-seq2 and more recently snmC-seq3, were established and standard operating procedures develop for the NIH BRAIN initiative. 3] Multi-omics analysis combining two different NGS datasets measured from single cells. These include snMethyl-3C-seq, snPaired-seq and snMethyl-HiC. The Epigenomics Core and the Bioinformatics Core will work together to perform analysis of all epigenomic data produced by Core B, will work with existing pipelines or create new pipelines as need demands, and will share data the Projects I, II and III. We anticipate that 25,000 single cell methylomes/yr will be generated, along with detailed analyses. Data processing quantification of genome wide unmethylated and methylated cytosine base calls are generated using an algorithm that we previously developed called Methylpy. Clustering of snmC-seq2 data for cell type classification will utilize both mCG and mCH patterns to effectively classify both neuronal and non-neuronal cell types in the developing neural tube. These analyses will result in a prioritized list of candidate marker features (genes and predicted regulatory elements) that will be provided to the project PIs for further examination and validation.
