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What subnetworks of differentially expressed genes are enriched in my samples? What biological functions are they related to?

This recipe provides a method for identifying differentially expressed genes between two phenotypes, such as tumor and normal, to find subnetworks of interacting proteins and determine their functional annotations. An example use of this recipe is a case where an investigator may want to compare two phenotypes to determine which gene networks are similar between phenotypes, and to determine how functional annotation changes between phenotypes.

 

In particular, this recipe makes use of several GenePattern modules to identify differentially regulated genes, then uses several Cytoscape plugins to identify potential interactions between gene products, and to visualize the resulting network.

Why differential expression analysis? We assume that most genes are not expressed all the time, but rather are expressed in specific tissues, stages of development, or under certain conditions. Genes which are expressed in one condition, such as cancer tissue, are said to be differentially expressed when compared to normal conditions. To identify which genes change in response to specific conditions (e.g. cancer), we must filter or process the dataset to remove genes which are not informative.

Why protein interaction network analysis? Gene expression analysis results in a list of differentially expressed genes, but it does not explain whether these genes are connected biologically in a pathway or network. To better understanding the underlying biology that drives changes in gene expression analysis, we can perform network analysis to determine whether gene products (e.g. proteins) are reported to interact. To identify potential networks or pathways, we search for highly interconnected subnetworks within a large interaction network.

Does my gene expression dataset contain a module network of regulatory genes? Does the network have any special features?

This recipe provides one method for creating and visualizing a module network of regulatory genes. An example use of this recipe is a case where an investigator may want to evaulate an expression dataset to find regulatory genes such as transcription factors, and then determine if they are connected in a network.

 

In particular, the regulatory genes of interest are genes which regulate other genes associated with an embryonic stem cell (ESC) state. This 'stemness signature' is a feature common to ESCs, as well as induced pluripotent stem cells (iPSCs), and also in a compendium of human cancers, such as breast cancer. This recipe recapitulates research by Wong et al., in Cell Stem Cell (2008), "Module map of stem cell genes guides creation of epithelial cancer stem cells."

We use a gene expression dataset of primary human breast cancer tumor samples (described in Chin, K. et al, Cancer Cell, 2006), and create a module network by projecting a set of stemness regulators onto the gene expression dataset, using Genomica. A module network is a model which identifies regulatory modules from gene expression data, especially modules of co-regulated genes and their regulators. The module also identifies the conditions under which the regulation can occur.

After obtaining the module network, we visualize it using Cytoscape. Since the network is very large, we then filter it to just a subnetwork of stemness regulators and their connections, again using Cytoscape. This provides us with a visual representation of the stemness regulators as they appear projected onto a breast cancer tumor dataset.

 

What genes are essential to a cell’s survival in a specific environment?

This recipe provides a way to process the results of genome-wide CRISPR-Cas9 knockout screens. In these screens, single guide RNAs (sgRNAs) are designed to bind to and inhibit specific target DNA sequences in genes. Multiple sgRNAs may target the same gene to increase knockout efficiency. In positive screens, essential genes are identified through the sequencing of surviving cells post-selection. The loss of these ‘winning’ genes create cells that are resistant to the selective pressure. In negative screens, essential genes are identified by measuring which genes are lower in abundance post selection. These screens require a non-selected control, which is used to find which genes are essential to survival  under the given selective pressures (Miles et al., 2016). Since a large number of sgRNAs can be introduced in a single screen, many genes can be tested for a selection criteria. However, there are many factors to consider in processing of sequenced reads; often multiple sgRNAs in a library target the same gene but with different specificities and efficiencies, and read count distributions vary depending on library and study designs. Additionally, positive selection screens often result in relatively few sgRNAs that dominate the total sequenced reads. The MAGeCK (Li et al., 2014) method was specifically developed for CRISPR screen analyses with these conditions in mind.

How can we find the molecular mechanism responsible for resistance?

By looking at how the hits in the screen aggregate on an interaction network, we can get an idea of the mechanisms that are essential for the organism to survive an environmental challenge.  The network neighborhood that contains a high concentration of essential genes is strongly implicated as the molecular mechanism by which an organism handles the challenge.

We can find the network neighborhood that is enriched for the screen hits through an algorithm called network propagation (Carlin et al., in press) that is implemented as a feature of the popular network analysis program Cytoscape.  This algorithm will find the closely clustered hits and their network neighbors to build a network diagram of the resistance mechanism.  We can then use GeneMANIA plugin to find enriched terms that easily summarize the biological terms that are enriched in the diagram.

What is Model-based Analysis of Genome-wide CRIPSR/Cas9 Knockout (MAGeCK)?

Model-based Analysis of Genome-wide CRIPSR/Cas9 Knockout (MAGeCK) is an algorithm for identifying both positively and negatively selected sgRNAs and genes from genome-scale CRIPSR/Cas9 knockout screens. The MAGeCK method can be summarized by the following steps:

1. sgRNA read counts are median-ratio normalized.

2. Mean-variance modeling is then used to model each replicate. The statistical significance of each sgRNA is calculated using the learned mean-variance model.

3. Essential genes are determined by looking for genes with consistently highly significant sgRNAs using robust rank aggregation.

Use Case: MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens (Li et al. 2014).

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