Project Summary Human cancers are highly heterogeneous. However, due to the limit of technologies, the intercellular heterogeneity was not detectable genome-wide at single-cell level until recently. New technologies such as single-cell RNA-Seq and exome-Seq have revealed new insights and more profound complexity than what was previously thought. In collaboration with Dr. Weissman's group in the Genetics Department at Yale University, it is now feasible to perform multiple integrative assays from the same single tumor cell. However, rigorous computational methods are still lagging behind, in order to solve the computational challenge and determine true heterogeneity rather than noise. Here we propose a user-friendly bioinformatics platform that is optimized to integrative multiple types of single-cell NGS data, in particular, transcriptome, exome and CpG methylome data that are all obtained from the same cell. Specifically, in this study we will first construct and validate in parallel three new NGS bioinformatics pipelines to enable single-cell based RNA-Seq, exome- Seq, and CpG methylome data. We will then develop and validate an integration pipeline to analyze multiple types of high- throughput data, exemplified by the RNA-Seq, exome-Seq and CpG methylome single-cell data. To test the software suite, we will first obtain data sets from erythroleukemia cell line K562. We will then utilize this bioinformatics suite to investigate heterogeneity in Myeloid Leukemia patient samples provided by hematologist Dr. Stephanie Halene at Yale Stem Cell Center. Beyond deciphering tumor heterogeneity, this user-friendly bioinformatics platform is expected to be used widely by the single-cell sequencing community.