Workshop from Alignment to Differential Expression
Today we are going to:
- Explore GTF File
- Run featureCounts to get gene counts
- Use DESeq2 to normalize data and visualization
- Use DESeq2 to do differential expressed genes
- Differential expressed genes filtering and draw heatmap
Log into BioHPC
First, we will log into the log into a compute node,install a necessary package and then log into a compute node
- Set up a WebGui session on BioHPC
- Log into via the VNC
- Open a terminal window -- you should be in the directory /archive/nanocourse/June2018/trainXX
- Copy data from /archive/nanocourse/June2018/shared/session2 to your train directory
ln -s /archive/nanocourse/June2018/shared/session2 /archive/nanocourse/June2018/trainXX/session2 ls /archive/nanocourse/June2018/trainXX/session2
Exploring GTF File
- How many lines in the GTF file?
wc -l session2/gencode.gtf
- How would you view the first 5 lines of the GTF file?
head -n 5 session2/gencode.gtf
- What are the first 5 lines of the file?
description: evidence-based annotation of the mouse genome (GRCm38), version M10 (Ensembl 85)
- Why might this information be important?
Indicates the version of the annotation use and when it was generated.
- How many transcripts are there for Tnnt2? (hint: use grep, cut, uniq 9th field (attributes) transcript ids have ENSMUST in transcript_id field semicolon ’;’ can be used as a delimiter)
grep 'Tnnt2' session2/gencode.gtf | cut -f9 | grep ENSMUST | cut -f2,8 -d ';' | sort | uniq
- How many transcripts are protein coding (hint: transcript_type)?
grep 'Tnnt2' session2/gencode.gtf | cut -f9 | grep ENSMUST | grep 'transcript_type "protein_coding"' | cut -f2,8 -d ';' | sort | uniq | wc
Run FeatureCounts to get counts on a file
module load subread/1.6.1 featureCounts -p -g gene_name -a session2/gencode.gtf -o heart_e11_5_rep1.cts session2/alignments/heart_e11_5_rep1.dedup.bam
featureCounts will print statistics on the screen. There is one line in the second box: "Successfully assigned fragments : 376082 (72.3%)"
Depending on how your samples are prepared, this value varies: * If you are using poly-A extraction, we expect this value to be at least 70% * If you are extracting total RNA, this value will be around 50% * When you have a percent less than 40%, you should look into this matter to figure out why
Now Let's take a look at featureCounts results
head heart_e11_5_rep1.cts.summary head heart_e11_5_rep1.cts
- What are the counts for the following genes?
- Tnnt2: 7752
- Myh6: 6347
- Myh7: 8586
- Kcng2: 2989
- Gja5: 5746
Run feature counts on all of the files.
Then we need to combine all the counts into a matrix. We will be using an in-house Perl script "concat_cts.pl" to do this. It takes in the names of all genes (genenames.txt)
There are 2 parameter you need to provide: 1. The output folder -o (here we use the current directory ./ ) 2. the patter of all of the cts files (.cts)
perl session2/scripts/concat_cts.pl -o ./ session2/counts/*.cts head countTable.txt
Before we are ready for differential expression we need to create a design file. Thank full we have already done that for you.
Also lets copy 2 files to your home directory
cp design_se.txt /home2/trainXX cp countTable.txt /home2/trainXX
Use RStudio on demand
- Set up a RStudio session on BioHPC
Install edgeR for gene differential expression analysis
For pheatmap package, click "package"->"install" Type 'a' if program asks you if you want to update packages. Install from:CRAN Packages: pheatmap Check "Install dependencies" Click "Install"
Click "Yes" if prompt window asks you if you want to use a personal library.
After everything finished, load the libraries using
Set working directory
Session-> Set working directory -> choose directory
#Read data matrix and sample file cfile<-read.table("countTable.txt",header=T,row.names=1) coldata<-read.table("design_se.txt",header=T,sep="\t") head(counts) head(coldata) #Reorder the counts columns to match the order of sample file cfile = cfile[c("heart_e11_5_rep1", "heart_e11_5_rep2", "heart_p0_rep1", "heart_p0_rep2")] #It is good to set your control group label as the baseline. Especially you are going to use intercept group = relevel(factor(coldata$SampleGroup),ref="heart_e11_5") cds = DGEList(cfile,group=group)
Pre-filtering the low-expressed genes
Filter for keeping a gene if cpm (counts per million) exceeds 1 in at least 2 samples.
cds = cds[ rowSums(cpm(cds)>=1) >= 2, ,keep.lib.sizes=FALSE]
Exploratory analysis and some vizulization
Use cpm() function to get log2 transformed normalized counts
rld <- cpm(cds, log=TRUE)
Calculate the distance between sample pairs and do hierarchical clustering
sampleDists = dist(t(rld)) sampleDists plot(hclust(sampleDists))
Use heatmap to show sample correlation**
Use MDS plot to check the relationship of replicates.
points = c(15,16) colors = rep(c("red","blue"),4) plotMDS(cds, col=colors[group], pch=points[group]) legend("bottomleft", legend=levels(group), pch=points, col=colors, ncol=2)
Make a design matrix for samplegroups
samplegroup <- factor(coldata$SampleGroup) design<-model.matrix(~samplegroup) design
Normalize data and estimate dispersion. What is the norm.factors per sample?
cds = calcNormFactors(cds) cds
Use glmFit() and glmLRT() to test for differential expression. What are the top 10 differential genes sorted by logFC?
cds$samples cds <- estimateDisp(cds,design) fit <- glmFit(cds, design) lrt <- glmLRT(fit, coef=2) res <- topTags(lrt, n=dim(cfile),sort.by="logFC") res[1:10,]
Make a dataframe with column for genes
res_df = cbind(gene_name = rownames(res), data.frame(res)) write.table(res_df,"edgeR.res.tsv",quote=F,sep="\t",row.names=F) head(res_df)
Filter for genes that are logFC >= 1 & FDR <= 0.01
res_filt = res_df[(abs(res_df$logFC)>=1 & res_df$FDR<=0.01),]
Draw a heatmap of differential expressed genes meeting filter criteria.
res_filt_rld = rld[rownames(rld) %in% res_filt$gene_name,] pheatmap(res_filt_rld,scale="row",show_rownames = F)