Found 4 recipes
Which genes lie in my copy number variation regions? Are these genes enriched for any biological functions or pathways?
This recipe provides a method for identifying biological functions for genes lying in copy number variation (CNV) regions. An example use of this recipe is a case where an investigator may want to examine their list of CNVs to see which genetic regions are amplified or deleted, and determine the biological functions or pathways associated with these amplified or deleted regions.
Copy number variations (CNVs) are large alterations to genomes, such as amplification or deletion of large segments of a chromosome. They can range in size from a focal aberration in a single gene to aberrations covering entire chromosome arms. These variations in the genome have been associated with different conditions, such as cancer. Many genomic analyses produce a set of genes which are assumed to be relevant to an underlying biological mechanism or phenotype. Thus, an investigator often has additional questions about the function or relatedness of these genes: Are they part of the same pathway? Do the gene products interact physically? Do the gene products localize to a specific part of the cell? Are the genes associated with certain stages of development? These questions, and others like them, can be answered by performing functional annotation of gene lists to better understand the underlying connections between genes.
In this particular example, we imagine a scenario in which an investigator identifies CNV regions that are amplified or deleted in glioblastoma multiforme (GBM) tumor samples, using a method called Genomic Identification of Significant Targets in Cancer (GISTIC, Mermel et al. (2011) Genome Biol.). Given a set of CNV regions, the goal is to infer which biological functions (e.g., metabolic and regulatory pathways, chemical perturbation signatures, etc.) are overrepresented in the set of reference genes that overlap with these regions. In particular, this recipe uses several Galaxy tools to find the overlap between CNV regions and reference genes obtained from the UCSC Table Browser. Then it uses the Molecular Signatures Database (MSigDB) to identify biological functions of the overlapped genes.
How can I use this recipe? This recipe may be modified to analyze CNV regions derived from any organism for which an annotated reference genome exists. Nor does the recipe depend on the algorithm used to identify these regions (GISTIC); any source of CNV data can be used. Once an investigator has pinpointed the CNV regions believed to be influencing their phenotype (disease state, cell type, etc.) of study he/she can use this recipe to identify functional pathways that may be affected by these copy number changes and draw closer to an understanding of the mechanisms behind these CNV effects.
How are SNP-related genes regulated in an expression dataset? Are these genes enriched for particular biological functions?
This recipe provides one method for identifying enriched biological functions in single-nucleotide polymorphisms (SNPs). An example use of this recipe is a case where an investigator may complete a genome-wide association study (GWAS) and wants to know the SNPs that are associated with certain genomic coordinates, in order to determine which genes have particular biological functions.
In this particular example, we imagine a scenario in which an investigator completes a GWAS study, obtaining a list of genomic coordinates that are associated with SNPs. However, simply knowing these genomic coordinates is not always informative; the investigator is also interested in knowing which genes lie in these regions, and what kinds of biological functions these genes may have. In this particular example, we are interested in answering two questions:
To answer the first question, we will find Gene Ontology functional annotations using Genomica. To answer the second question, we will use the Gene Set Enrichment Analysis (GSEA) module in GenePattern, comparing the SNP-associated genes to a gene expression dataset from a study of epithelial cancer stem cells. This study evaluated the ability of oncogenes to activate an embryonic stem cell program in differentiated adult tumor cells, by transforming human keratinocytes into squamous cell carcinomas using oncogenic Ras and IκBα, plus one of three genes: c-Myc, E2F3, and GFP (Wong et al. 2009. Cell Stem Cell). Comparisons between these three genes showed that c-Myc could re-activate the embroynic stem cell program. Comparing the SNPs to this gene expression dataset can determine whether this set of SNP-associated genes are differentially regulated in c-Myc samples, when compared to other genes such as GFP and E2F3.
How do I create a custom-generated gene set? Are there any commonalities between custom-generated gene sets, and MSigDB hallmark gene sets?
This recipe provides a method for identifying and visualizing similarities between diverse gene sets relevant to a study. An example use of this recipe is a case where an investigator may want to compare two phenotypes, such as two types of cancer, to determine which gene sets may be similar between these phenotypes.
Background information: What is Gene Set Enrichment Analysis, and why should I use it?
Gene sets are lists of genes that share similar functions, transcriptional regulation, chromosomal positions, pathways, or other biological processes. It is possible to identify gene sets that are enriched or over-represented in a particular phenotype, such as a specific disease. Gene Set Enrichment Analysis (GSEA) is a computational method which determines whether an a priori defined set of genes shows statistically significant, concordant differences between two phenotypes. GSEA can be used with a custom gene set generated by the user, or with the annotated, standardized gene sets which are available in the Molecular Signatures Database (MSigDB) collection. Completing GSEA on a gene expression dataset will identify those gene sets which are significantly enriched in a particular phenotype. Comparing similarities between the top gene sets following GSEA can yield unique insights into the mechanisms associated with a specific phenotype, which cannot be observed using a single-gene analysis.
Use case: Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition (Puissant et al. , Cancer Discov. 2013).
This study analyzed gene expression data generated from primary neuroblastoma tumors of two genetic classes: tumors harboring MYCN amplification (“MYCN amplified”) and tumors without MYCN amplification (“MYCN non-amplified”). MYCN amplified neuroblastoma is exquisitely dependent on the bromodomain and extra-terminal (BET) family of proteins. As such, treatment of MYCN amplified cell lines or tumors with JQ1, a small-molecule inhibitor of BET proteins, leads to dramatic transcriptional changes and induces cell death.
A training set of gene expression data was analyzed using GenePattern, and custom gene sets were generated representing the MYCN amplified and MYCN non-amplified datasets. The custom-generated gene sets were then concatenated with the Hallmark gene set from MSigDB using tools in Galaxy. Subsequently, a test gene expression dataset of neuroblastoma cell lines treated with JQ1 (treatment) or DMSO (control) was used to rank this collection of gene sets using single-sample Gene Set Enrichment Analysis (ssGSEA).
This analysis reveals that MYCN-associated gene sets are enriched in JQ1-associated datasets, and suggests that JQ1 functions to suppress transcriptional programs mediated by MYCN amplification. The resulting similarities of the top-ranked gene sets are visualized using ConstellationMap, a module available in GenePattern. This helps to highlight similarities and overlaps between gene sets.
This recipe recapitulates research by Pfefferle et al., in Genome Biology(2013), "Transcriptomic classification of genetically engineered mouse models of breast cancer identifies human subtype counterparts", conducted by Charles M Perou's group. This study encompasses the largest comprehensive genomic dataset to date to identify human-to-mouse disease subtype counterparts, consisting of three independent human breast cancer datasets and 385 DNA gene expression microarrays from 27 GEMMs of mammary carcinoma(Gene Expression Omnibus accession numbers GSE3165, GSE8516, GSE9343, GSE14457, GSE15263, GSE17916, GSE27101, and GSE42640). In the original study, the similarity between specific human and mouse subtypes was measured using gene set analysis (GSA)(Table 2 in the publication). To recapitulate this research, we will use Gene Set Enrichment Analysis module(v17) in GenePattern, as an independent method to further validate the research findings. This effort is supported by NCI Oncology Models Forum(OMF), a collaborative effort to credential cancer mouse models for translational research.