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Visualization of results Mary Piper Bioinformatics Consultant and - - PowerPoint PPT Presentation

Mar 24, 2024 919 likes •1.41k views

DataCamp RNA-Seq Differential Expression Analysis RNA - SEQ DIFFERENTIAL EXPRESSION ANALYSIS Visualization of results Mary Piper Bioinformatics Consultant and Trainer DataCamp RNA-Seq Differential Expression Analysis Visualizing results -

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DataCamp RNA-Seq Differential Expression Analysis

Visualization of results

RNA-SEQ DIFFERENTIAL EXPRESSION ANALYSIS

Mary Piper

Bioinformatics Consultant and Trainer

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DataCamp RNA-Seq Differential Expression Analysis

Visualizing results - Expression heatmap

# Subset normalized counts to significant genes sig_norm_counts_wt <- normalized_counts_wt[wt_res_sig$ensgene, ] # Choose a color palette from RColorBrewer library(RColorBrewer) heat_colors <- brewer.pal(6, "YlOrRd") display.brewer.all()

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DataCamp RNA-Seq Differential Expression Analysis

Visualizing results - Expression heatmap

# Run pheatmap pheatmap(sig_norm_counts_wt, color = heat_colors, cluster_rows = T, show_rownames = F, annotation = select(wt_metadata, condition), scale = "row")

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DataCamp RNA-Seq Differential Expression Analysis

Visualizing results - Volcano plot

# Obtain logical vector regarding whether padj values are less than 0.05 wt_res_all <- wt_res_all %>% rownames_to_column(var = "ensgene") %>% mutate(threshold = padj < 0.05) # Volcano plot ggplot(wt_res_all) + geom_point(aes(x = log2FoldChange, y = -log10(padj), color = threshold)) + xlab("log2 fold change") + ylab("-log10 adjusted p-value") + theme(legend.position = "none", plot.title = element_text(size = rel(1.5), hjust = 0.5), axis.title = element_text(size = rel(1.25)))

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DataCamp RNA-Seq Differential Expression Analysis

Visualizing results - Volcano plot

ggplot(wt_res_all) + geom_point(aes(x = log2FoldChange, y = -log10(padj), color = threshold)) xlab("log2 fold change") + ylab("-log10 adjusted p-value") + ylim=c(0, 15) + theme(legend.position = "none", plot.title = element_text(size = rel(1.5), hjust = 0.5), axis.title = element_text(size = rel(1.25)))

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DataCamp RNA-Seq Differential Expression Analysis

Visualizing results - Expression plot

Significant results: Normalized counts for significant genes:

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DataCamp RNA-Seq Differential Expression Analysis

Visualizing results - Expression plot

top_20 <- data.frame(sig_norm_counts_wt)[1:20, ] %>% rownames_to_column(var = "ensgene") top_20 <- gather(top_20, key = "samplename", value = "normalized_counts", 2:8)

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DataCamp RNA-Seq Differential Expression Analysis

Visualizing results - Expression plot

top_20 <- inner_join(top_20, rownames_to_column(wt_metadata, var = "samplename"), by = "samplename") ggplot(top_20) + geom_point(aes(x = ensgene, y = normalized_counts, color = condition)) + scale_y_log10() + xlab("Genes") + ylab("Normalized Counts") + ggtitle("Top 20 Significant DE Genes") + theme_bw() + theme(axis.text.x = element_text(angle = 45, hjust = 1)) + theme(plot.title = element_text(hjust = 0.5))

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DataCamp RNA-Seq Differential Expression Analysis

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DataCamp RNA-Seq Differential Expression Analysis

Let's practice!

RNA-SEQ DIFFERENTIAL EXPRESSION ANALYSIS

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DataCamp RNA-Seq Differential Expression Analysis

RNA-Seq DE analysis summary

RNA-SEQ DIFFERENTIAL EXPRESSION ANALYSIS

Mary Piper

Bioinformatics Consultant and Trainer

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DataCamp RNA-Seq Differential Expression Analysis

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DataCamp RNA-Seq Differential Expression Analysis

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DataCamp RNA-Seq Differential Expression Analysis

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DataCamp RNA-Seq Differential Expression Analysis

RNA-Seq Workflow: Raw data quality control

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DataCamp RNA-Seq Differential Expression Analysis

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DataCamp RNA-Seq Differential Expression Analysis

RNA-Seq Workflow: Alignment

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DataCamp RNA-Seq Differential Expression Analysis

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DataCamp RNA-Seq Differential Expression Analysis

RNA-Seq Workflow: Quantitation

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DataCamp RNA-Seq Differential Expression Analysis

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DataCamp RNA-Seq Differential Expression Analysis

Preparation for differential expression analysis: DESeq2 object

dds <- DESeqDataSetFromMatrix(countData = rawcounts, colData = metadata, design = ~ condition)

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DataCamp RNA-Seq Differential Expression Analysis

Let's practice!

RNA-SEQ DIFFERENTIAL EXPRESSION ANALYSIS

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DataCamp RNA-Seq Differential Expression Analysis

RNA-Seq DE analysis summary 2

RNA-SEQ DIFFERENTIAL EXPRESSION ANALYSIS

Mary Piper

Bioinformatics Consultant and Trainer

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DataCamp RNA-Seq Differential Expression Analysis

DESeq workflow - normalization

dds <- estimateSizeFactors(dds) normalized_counts <- counts(dds, normalized=TRUE)

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DataCamp RNA-Seq Differential Expression Analysis

Unsupervised clustering analyses: log transformation

# Log transformation of normalized counts vsd <- vst(dds, blind=TRUE)

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DataCamp RNA-Seq Differential Expression Analysis

Unsupervised clustering analyses

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DataCamp RNA-Seq Differential Expression Analysis

Unsupervised clustering analyses - heatmap

vsd %>% assay() %>% # Extract the vst matrix from the object cor() %>% # Compute pairwise correlation values pheatmap(annotation = metadata[ , c("column_name1", "column_name2])

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DataCamp RNA-Seq Differential Expression Analysis

Unsupervised clustering analyses - pca

# PCA plotPCA(vsd, intgroup="condition")

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DataCamp RNA-Seq Differential Expression Analysis

Running the DE analysis

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DataCamp RNA-Seq Differential Expression Analysis

Running the DE analysis

# Create DESeq object dds <- DESeqDataSetFromMatrix(countData = rawcounts, colData = metadata, design = ~ source_of_variation + condition) # Run analysis dds <- DESeq(dds)

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DataCamp RNA-Seq Differential Expression Analysis

DESeq2 workflow - model

# Plot dispersion estimates plotDispEsts(dds)

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DataCamp RNA-Seq Differential Expression Analysis

DESeq2 workflow - contrasts and LFC shrinkage

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DataCamp RNA-Seq Differential Expression Analysis

DESeq2 workflow - contrasts and LFC shrinkage

# Extract results for comparison of interest res <- results(dds, contrast = c("condition_factor", "level_to_compare", "base_level"), alpha = 0.05) # Shrink the log2 foldchanges res <- lfcShrink(dds, contrast = c("condition_factor", "level_to_compare", "base_level"), res = res)

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DataCamp RNA-Seq Differential Expression Analysis

DESeq2 workflow - LFC shrinkage

# Extract all results as a data frame res_all <- data.frame(res) %>% rownames_to_column(var = "ensgene") # Add gene annotations res_all <- left_join(x = res_all, y = grcm38[, c("ensgene", "symbol", "description")], by = "ensgene") res_all <- arrange(res_all, padj)

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DataCamp RNA-Seq Differential Expression Analysis

DESeq2 workflow - results exploration

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DataCamp RNA-Seq Differential Expression Analysis

DESeq2 workflow - results exploration

# Identify significant genes res_sig <- subset(res_all, padj < 0.05)

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DataCamp RNA-Seq Differential Expression Analysis

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DataCamp RNA-Seq Differential Expression Analysis

Let's practice!

RNA-SEQ DIFFERENTIAL EXPRESSION ANALYSIS

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DataCamp RNA-Seq Differential Expression Analysis

RNA-Seq next steps

RNA-SEQ DIFFERENTIAL EXPRESSION ANALYSIS

Mary Piper

Bioinformatics Consultant and Trainer

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DataCamp RNA-Seq Differential Expression Analysis

RNA-Seq next steps

Vignette: Bioconductor support site: (tag 'DESeq2') http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html https://support.bioconductor.org

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DataCamp RNA-Seq Differential Expression Analysis

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DataCamp RNA-Seq Differential Expression Analysis

Overview of goals

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DataCamp RNA-Seq Differential Expression Analysis

Significant genes interpretation

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DataCamp RNA-Seq Differential Expression Analysis

Significant genes interpretation

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DataCamp RNA-Seq Differential Expression Analysis

Significant genes interpretation

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DataCamp RNA-Seq Differential Expression Analysis

Conclusion

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DataCamp RNA-Seq Differential Expression Analysis

Congratulations!

RNA-SEQ DIFFERENTIAL EXPRESSION ANALYSIS