Abstract: The present invention relates to methods of detecting region(s) of interest in a gene comprising a polyA tail. The region(s) of interest can include gene(s), region(s), mutation(s), deletion(s), insertion(s), indel(s), and/or translocation(s). The region(s) can be greater than or less than 1 kilobases from the polyA tail. Methods can include forming a library of single cell transcripts comprising the region(s) in close proximity to a cell barcode and a unique molecular identifier (UMI). Methods for distinguishing cells by genotype can include amplifying the transcripts using PCR methods and detecting the cell barcode and UMI using single cell sequencing methods. Transcripts can be enriched using tagged region-specific PCR primers. Cell barcodes can be brought into close proximity to the region(s) by circularizing the transcripts. Sequencing of the transcripts can include using primer binding sites added during PCR amplification and library indexes for multiplexed sequencing.

Abstract: The present invention provides for single-molecule profiling of combinatorial protein modifications and single-molecule profiling of combinatorial protein modifications combined with single-molecule sequencing of protein/nucleic acids complexes. High-throughput single-molecule imaging was applied to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. Applicants identified bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. Applying genetic and chemical perturbations of chromatin enzymes show a preferential affect on nucleosomes harboring specific modification states. The present invention also combines this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes.

Abstract: The present invention relates to methods of detecting region(s) of interest in a gene comprising a polyA tail. The region(s) of interest can include gene(s), region(s), mutation(s), deletion(s), insertion(s), indel(s), and/or translocation(s). The region(s) can be greater than or less than 1 kilobases from the polyA tail. Methods can include forming a library of single cell transcripts comprising the region(s) in close proximity to a cell barcode and a unique molecular identifier (UMI). Methods for distinguishing cells by genotype can include amplifying the transcripts using PCR methods and detecting the cell barcode and UMI using single cell sequencing methods. Transcripts can be enriched using tagged region-specific PCR primers. Cell barcodes can be brought into close proximity to the region(s) by circularizing the transcripts. Sequencing of the transcripts can include using primer binding sites added during PCR amplification and library indexes for multiplexed sequencing.

Abstract: This invention relates generally to compositions and methods for identifying genes and gene networks that respond to, modulate, control or otherwise influence tumors and tissues, including cells and cell types of the tumors and tissues, and malignant, microenvironmental, or immunologic states of the tumor cells and tissues. The invention also relates to methods of diagnosing, prognosing and/or staging of tumors, tissues and cells, and provides compositions and methods of modulating expression of genes and gene networks of tumors, tissues and cells, as well as methods of identifying, designing and selecting appropriate treatment regimens.

Abstract: The present invention provides for single-molecule profiling of combinatorial protein modifications and single-molecule profiling of combinatorial protein modifications combined with single-molecule sequencing of protein/nucleic acids complexes. High-throughput single-molecule imaging was applied to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. Applicants identified bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. Applying genetic and chemical perturbations of chromatin enzymes show a preferential affect on nucleosomes harboring specific modification states. The present invention also combines this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes.

Abstract: The present invention features compositions and methods for assaying DNA methylation.

Abstract: The present application generally to the diagnosis and treatment of diseases resulting from the alteration of chromatin boundaries between topologically-associated domains. In particular, the present application relates to detection of mutations causing DNA hypermethylation phenotypes, CpG methylation within CTCF binding motifs, and aberrant gene expression caused by altered chromatin topology. Applicants show that IDH mutant gliomas exhibit hyper-methylation at CTCF binding sites, compromising binding of this methylation-sensitive insulator protein. Applicants also demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to aberrantly interact with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Thus, Applicants have uncovered that IDH mutations may promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

Abstract: Embodiments disclosed herein provide methods of using somatic mutations in mitochondrial genomes to retrospectively infer cell lineages in native contexts and to serve as genetic barcodes to measure clonal dynamics in complex cellular populations. Further, somatic mutations in mitochondrial DNA (mtDNA) are tracked by single cell genomic approaches for simultaneous analysis of single cell lineage and state. Applicants further show that mitochondrial mutations can be readily detected with contemporary single cell transcriptomic and epigenomic technologies to concomitantly capture gene expression profiles and chromatin accessibility, respectively.

Abstract: The subject matter disclosed herein is generally directed to compositions and methods for treating diffuse gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-gliomas). Disclosed herein are gene signatures specific for tumor cell types and compositions for treatment of H3K27M gliomas. In one embodiment, PRC1 is targeted in a treatment regimen for H3K27M-gliomas.

Abstract: The present invention relates to methods of deriving genetic information from RNA-seq libraries, that can enable an overlay of genetic information (such as cancer driver mutations) onto single-cell transcriptomes and permitting efficient identification, localization, and quantification of certain cells of interest within a population as well as provide low-cost selection and sequencing of any portion of a transcript, including at the 5? end.

Abstract: The present application generally to the diagnosis and treatment of diseases resulting from the alteration of chromatin boundaries between topologically-associated domains. In particular, the present application relates to detection of mutations causing DNA hypermethylation phenotypes, CpG methylation within CTCF binding motifs, and aberrant gene expression caused by altered chromatin topology. Applicants show that IDH mutant gliomas exhibit hyper-methylation at CTCF binding sites, compromising binding of this methylation-sensitive insulator protein. Applicants also demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to aberrantly interact with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Thus, Applicants have uncovered that IDH mutations may promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

Abstract: The present invention advantageously provides for novel gene signatures, tools and methods for the treatment and prognosis of epithelial tumors. Applicants have used single cell RNA-seq to reveal novel expression programs of malignant, stromal and immune cells in the HNSCC tumor ecosystem. Malignant cells varied in expression of programs related to stress, hypoxia and epithelial differentiation. A partial EMT-like program (p-EMT) was discovered that was expressed in cells residing at the leading edge of tumors. Applicants unexpectedly linked the p-EMT state to metastasis and adverse clinical features that may be used to direct treatment of epithelial cancers (e.g., HNSCC). Applicants also show that metastases are dynamically regulated by the tumor microenvironment (TME). Finally, a computational modeling approach was developed that allows analysis of malignant cells in bulk sequencing samples.

Abstract: The present invention provides for single-molecule profiling of combinatorial protein modifications and single-molecule profiling of combinatorial protein modifications combined with single-molecule sequencing of protein/nucleic acids complexes. High-throughput single-molecule imaging was applied to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. Applicants identified bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. Applying genetic and chemical perturbations of chromatin enzymes show a preferential affect on nucleosomes harboring specific modification states. The present invention also combines this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes.

Abstract: This invention relates generally to compositions and methods for identifying genes and gene networks that respond to, modulate, control or otherwise influence tumors and tissues, including cells and cell types of the tumors and tissues, and malignant, microenvironmental, or immunologic states of the tumor cells and tissues. The invention also relates to methods of diagnosing, prognosing and/or staging of tumors, tissues and cells, and provides compositions and methods of modulating expression of genes and gene networks of tumors, tissues and cells, as well as methods of identifying, designing and selecting appropriate treatment regimens. The invention also relates to the modulation of complement activity to shift cellular immunity and obtain an effective therapeutic response.

Abstract: Disclosed are methods for shearing and tagging chromatin DNA. The disclosed methods include contacting chromatin DNA with at least one transposome, that includes a transposase enzyme. The transposon is made up of a first DNA molecule that includes a first transposase recognition site and a second DNA molecule that includes a second transposase recognition site, wherein the transposase integrates the first and second DNA molecules into chromatin DNA. The first and second DNA molecules of the transposon can be disconnected, such that upon integration of the transposon the chromatin bound DNA is sheared and tagged with the first and second DNA molecules, for example to prepare a library of sheared and tagged chromatin DNA fragments.