# MetaIntegrator Real Example on GEO Data

#### Winston Haynes

#### 2018-10-05

# 1. Get Public Gene Expression Data

## Download Data

For this example, we will work through a small gene expression meta-analysis of systemic lupus erythematosus (SLE). We have identified public datasets that we will download from GEO for this analysis.

    #Download the data from GEO, this can take a little while
    sleData <- getGEOData(c("GSE11909","GSE50635", "GSE39088"))

## Label samples

All samples need to be assigned labels in the $class vector, 1 for ‘disease’ or 0 for ‘control’.

    sleData$originalData$GSE50635 <- classFunction(sleData$originalData$GSE50635, column = "subject type:ch1",
                                                  diseaseTerms = c("Subject RBP +", "Subject RBP -"))

    sleData$originalData$GSE11909_GPL96 <- classFunction(sleData$originalData$GSE11909_GPL96, column = "Illness:ch1",
                                                  diseaseTerms = c("SLE"))

    sleData$originalData$GSE39088 <- classFunction(sleData$originalData$GSE39088, column= "disease state:ch1",
                                                   diseaseTerms=c("SLE"))

    #Remove the GPL97 platform that was downloaded
    sleData$originalData$GSE11909_GPL97 <- NULL

# 2. Run Meta-Analysis

    sleMetaAnalysis <- runMetaAnalysis(sleData, runLeaveOneOutAnalysis = F, maxCores = 1)

# 3. Identify Gene Signature

## Filter Genes

Set up criteria to filter genes for whether or not they will be included in the disease signature.

    sleMetaAnalysis <- filterGenes(sleMetaAnalysis, isLeaveOneOut = F, effectSizeThresh = 1, FDRThresh = 0.05)

## Calculate Meta Score

Once you have identified a gene signature, you can calculate a score for each sample based on the geometric mean of the up-regulated genes minus the geometric mean of the down-regulated genes. This score will be elevated in SLE patients compared to healthy controls.

    sleScores <- calculateScore(filterObject = sleMetaAnalysis$filterResults[[1]], datasetObject = sleMetaAnalysis$originalData[[1]])

This score can now be used to examine the results. Most functions call this score in the background.

# 4. Examine Results

## Visualize gene effect sizes

We can visualize the effect sizes for all genes in the signature.

    heatmapPlot(metaObject = sleMetaAnalysis, filterObject = sleMetaAnalysis$filterResults[[1]])

## MetaScore Classification Performance

Receiver operating characteristic (ROC) curves and precision-recall (PRC) curves can be used to demonstrate the classification performance of the MetaScore.

### Single ROC Curve

    rocPlot(datasetObject = sleMetaAnalysis$originalData$GSE5063, filterObject = sleMetaAnalysis$filterResults[[1]])

### Multiple ROC Curves

Draw multiple ROC curves.

    multipleROCPlot(metaObject = sleMetaAnalysis, filterObject = sleMetaAnalysis$filterResults[[1]])

Draw multiple ROC curves with a summary ROC curve that represents an overall ROC estimate.

    summaryROCPlot(metaObject = sleMetaAnalysis, filterObject = sleMetaAnalysis$filterResults[[1]], bootstrapReps = 100)

Draw multiple ROC curves with a pooled ROC curve that represents a moving average ROC.

    pooledROCPlot(metaObject = sleMetaAnalysis, filterObject = sleMetaAnalysis$filterResults[[1]])

### Single PRC Plot

Draw a single PRC plot.

    prcPlot(datasetObject = sleMetaAnalysis$originalData$GSE5063, filterObject = sleMetaAnalysis$filterResults[[1]])

### Multiple PRC Curves

Draw multiple PRC curves.

    multiplePRCPlot(metaObject = sleMetaAnalysis, filterObject = sleMetaAnalysis$filterResults[[1]])

## Understand Sample Phenotypes

### Violin plot

With a violin plot, you can drill into subgroups within datasets to observe differences between populations, with the individual samples called out.

    violinPlot(filterObject = sleMetaAnalysis$filterResults[[1]], datasetObject = sleMetaAnalysis$originalData$GSE50635, labelColumn = "subject type:ch1")

### Regression plot

For continuous variables, generate regression plot to analyze relationships.

    sleMetaAnalysis$originalData$GSE50635$pheno$`age:ch1` <- as.numeric(sleMetaAnalysis$originalData$GSE50635$pheno$`age:ch1`)

    regressionPlot(filterObject = sleMetaAnalysis$filterResults[[1]], datasetObject = sleMetaAnalysis$originalData$GSE50635, continuousVariableColumn = "age:ch1", formattedVariableName = "Age")

## Forest plot

Forest plots allow us to examine individual genes across studies.

    forestPlot(geneName="IFI44L", metaObject = sleMetaAnalysis)

## Advanced Analyses

### Forward/backward search

Use forward and backward search to reduce the number of genes in the signature and improve classification performance.

    forwardRes <- forwardSearch( metaObject = sleMetaAnalysis, filterObject = sleMetaAnalysis$filterResults[[1]])

### immunoStates Deconvolution

immunoStates is a tool for estimating immune cell proportions based on gene expression profiles. immunoStateMeta() in MetaIntegrator allow you to estimate cell proportions, then use these cell proportions as input for a downstream meta-analysis (in place of genes).

    immunoMeta <- immunoStatesMeta( metaObject = sleMetaAnalysis)

immunoStates can also correct the underlying gene expression data for differences in cell proportions

    sleMetaImmuneCorrected <- immunoStatesDecov( metaObject = sleMetaAnalysis)

### LINCS tools

LINCS tools allows users to compare disease gene expression signatures to perturbation expression signatures identified by the LINCS consortium. lincsTools() will generate a broadly useful report of many different classes of molecules. The call to lincsCorrelate(), below, is one particular example of looking for a drug with a gene expression profile that reverses the SLE profile. Note that this requires downloading a significant amount of data, so the first execution will be slow.

    lincsHits <- lincsCorrelate( metaObject = sleMetaAnalysis, filterObject = sleMetaAnalysis$filterResults[[1]], dataset = "CP", direction = "reverse")

### Impute sex

Based on known marker genes, impute sex of samples. This can be useful for identifying sample labeling errors.

    imputedSex <- imputeSex( myDataset = sleMetaAnalysis$originalData$GSE11909_GPL96)
    head(imputedSex)

### COCONUT

COCONUT is a separate R package for correcting batch effects to merge multiple datasets into a single dataset. This is a wrapper function to call COCONUT on a MetaIntegrator object.

    coconutRes <- coconutMetaIntegrator( metaObject = sleMetaAnalysis)

### Pathway Analysis

Pathway analysis is commonly performed to provide biological interpretation for experiments. This is a wrapper function for deapathways, one R package for performing pathway analysis.
NOTE: This functionality will be added in future updates to MetaIntegrator

    #pathwayRes <- pathwayAnalysis( metaObject = sleMetaAnalysis)