My cutoff for differentially expressed genes (DEGs) is **adjusted P < 0.05** and

**|log2FoldChange| > 1**. I used this code in DESeq2 to get those DEGs:

```
library(DESeq2)
cts <- as.matrix(read.csv("GE_counts_Sst.csv",row.names="gene"))
coldata <- read.csv('coldata_Sst.csv', row.names = 1)
dds <- DESeqDataSetFromMatrix(countData = cts,
colData = coldata,
design = ~ condition + gender)
keep <- rowSums(counts(dds)) >= 10
dds <- dds[keep,]
dds$condition <- relevel(dds$condition, ref = "Sst_Input")
dds <- DESeq(adds)
res <- results(dds, alpha = 0.05, lfcThreshold = 1, altHypothesis = "greaterAbs",
contrast = c("condition","Sst_IP","Sst_Input"), name = "condition_Sst_IP_vs_Sst_Input")
summary(res)
```

The summary of **res** is

```
out of 18461 with nonzero total read count
adjusted p-value < 0.05
LFC > 1.00 (up) : 131, 0.71%
LFC < -1.00 (down) : 907, 4.9%
outliers [1] : 7, 0.038%
low counts [2] : 1432, 7.8%
(mean count < 2)
```

Then I used the package EnhancedVolcano to plot a volcano plot from **res** using the cutoff **adjusted P < 0.05** and

**|log2FoldChange| > 1**:

```
library(EnhancedVolcano)
FC <- 1
q <- 0.05
keyvals <- rep('grey75', nrow(res))
names(keyvals) <- rep('NS', nrow(res))
keyvals[which(abs(res$log2FoldChange) > FC & res$padj > q)] <- 'grey50'
names(keyvals)[which(abs(res$log2FoldChange) > FC & res$padj > q)] <- 'log2FoldChange'
keyvals[which(abs(res$log2FoldChange) < FC & res$padj < q)] <- 'grey25'
names(keyvals)[which(abs(res$log2FoldChange) < FC & res$padj < q)] <- '-Log10P'
keyvals[which(res$log2FoldChange < -FC & res$padj < q)] <- 'blue'
names(keyvals)[which(res$log2FoldChange < -FC & res$padj < q)] <- 'Signif. down-regulated'
keyvals[which(res$log2FoldChange > FC & res$padj < q)] <- 'red'
names(keyvals)[which(res$log2FoldChange > FC & res$padj < q)] <- 'Signif. up-regulated'
EnhancedVolcano(res,
lab = rownames(res),
x = 'log2FoldChange',
y = 'padj',
xlab = bquote(~Log[2]~ 'fold change'),
ylab = bquote(~-Log[10] ~ adjusted~italic(P)),
title = 'res',
pCutoff = 0.05,
FCcutoff = 1,
colCustom = keyvals,
colAlpha = 0.5,
drawConnectors = FALSE,
widthConnectors = 0.5,
colConnectors = 'grey50',
gridlines.major = TRUE,
gridlines.minor = FALSE,
border = 'partial',
borderWidth = 1.5,
borderColour = 'black',
xlim = c(-15,15),
ylim = c(0,5))
```

However, I find the shape of volcano plot not very typical

if you can't see the image, click here

If I extracted the result using default in DESeq2 (which is adjusted p-value < 0.1 and |log2FoldChange| > 0)

```
res1 <- results(dds, contrast = c("condition","Sst_IP","Sst_Input"), name = "condition_Sst_IP_vs_Sst_Input")
```

the summary of **res1** would be

```
out of 18461 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up) : 4817, 26%
LFC < 0 (down) : 5632, 31%
outliers [1] : 7, 0.038%
low counts [2] : 358, 1.9%
(mean count < 1)
```

Then I again used the cutoff **adjusted P < 0.05** and

**|log2FoldChange| > 1**for volcano plot

```
FC <- 1
q <- 0.05
keyvals <- rep('grey75', nrow(res1))
names(keyvals) <- rep('NS', nrow(res1))
keyvals[which(abs(res1$log2FoldChange) > FC & res1$padj > q)] <- 'grey50'
names(keyvals)[which(abs(res1$log2FoldChange) > FC & res1$padj > q)] <- 'log2FoldChange'
keyvals[which(abs(res1$log2FoldChange) < FC & res1$padj < q)] <- 'grey25'
names(keyvals)[which(abs(res1$log2FoldChange) < FC & res1$padj < q)] <- '-Log10P'
keyvals[which(res1$log2FoldChange < -FC & res1$padj < q)] <- 'blue'
names(keyvals)[which(res1$log2FoldChange < -FC & res1$padj < q)] <- 'Signif. down-regulated'
keyvals[which(res1$log2FoldChange > FC & res1$padj < q)] <- 'red'
names(keyvals)[which(res1$log2FoldChange > FC & res1$padj < q)] <- 'Signif. up-regulated'
EnhancedVolcano(res1,
lab = rownames(res1),
...
xlim = c(-15,15),
ylim = c(0,10))
```

The volcano plot looks more typical

if you can't see the image, click here

However, it shows a lot more DEGs in **res1** than in **res**.
Is it because in **res**, I filtered the DEGs twice in DESeq2 and in the volcano plot?

I was wondering which method is correct. What result from DESeq2 should I use to generate a volcano plot that shows all data points and labels the correct number of DEGs in colors? Any comments would be appreciated.