(1) Summary: Massively parallel DNA sequencing continues to provide astonishing insights into the regulatory complexity of the mammalian genome, revealing heritable and de novo occurring risk factors predisposing humans to a wide variety of diseases and disorders. Notably, members of the ENCyclopedia of DNA Elements (ENCODE) Consortium, including Co-Investigators Drs. Mason and Snyder, have recently mapped thousands of non-coding, regulatory elements in the human genome that specify risk and protective sites for many diseases. These noncoding, genetic variations also show differential impact depending on the epigenetic changes of the genome, including methylation of DNA and chromatin states inside cells. Yet, these questions have not been comprehensively examined in human astronauts, due to the fact that the very first data sets that can delineate the putative impact of genome-wide noncoding variation and epigenetic changes from space travel are only now being generated in the NASA Twins Study. However, with this data from NASA Twins study, there is a unique opportunity to both generate new computational tools for the integration of the data from these samples and also to utilize novel, single-cell approaches for epigenetic states in the human cells taken from space. We propose to expand and further integrate our tools for examining the regulatory changes to DNA and RNA, together called “EpiSuite,” which will provide long-sought solutions to problems in integrative genomics and establish a more systems-level view of human physiology during space travel. Specifically, we will optimize and integrate open-source and published bioinformatics tools between both laboratories (HUGE-Seq, methylKit, eDMR, r-make), expand our code base with two new tools (methClone and CemBase), and then create a web-interface for users to upload, organize, and analyze these data relative to ENCODE public data. Then, we will use novel single-cell experimental methods (scATAC-seq) to confirm and characterize the impact and scope of such noncoding and epigenetic changes. Together, the data and tools in EpiSuite, concomitant with the new single-cell chromatin data, will enable discovery and validation of specific loci that represent the genetic and epigenetic risks faced by astronauts and also an improved understanding of the regulatory and genetic impact (BMED5) of these molecular changes. (2) Hypothesis: We hypothesize that specific areas of the genome are epigenetically dysregulated as a result of space travel, and that some of these areas differ between astronauts. Moreover, we hypothesize that the enrichment of non-coding variants in distinct biological pathways will also differ between astronauts, and analysis and integration of these data will reveal the first regions of “epigenetic space risk” for the astronauts, delineated in BMED5. To validate these predictions, we will perform single-cell ATAC-seq (Assay for Transposase Accessible Chromatin) on cell fractions from Earth-based control samples and those taken from space. (3) Specific Aims Aim 1. Create a web-based framework for integrating non-coding genomic variation and differential epigenetic states. This EpiSuite interface and set of machine-learning algorithms will help ongoing work with the NASA Twins study and aid future investigations that leverage multi-omics data sets from astronauts. These data will also be linked to gene expression data to gauge the impact of genetic and epigenetic changes on specific biological networks. Aim 2. Validate the altered chromatin states in cells from space travel and measure the proportion of cells changed with single-cell ATAC-seq. We will use an assay called single cell (scATAC-seq), which can measure the sites of open and closed chromatin on a cell-by-cell basis. This will validate the sites where we observe epigenetic changes and also provide the first estimate of the proportion of cells epigenetically impacted by space travel.