Hi

I would like to plot RNASeq data that I have downloaded from TCGA in a PCA plot. I have found some great guides on how to plot the actual data in PCA using r in ggplot2 and such but my main question is what format data should I plot?

I currently have raw counts and RSEM data. Should I input raw counts into something like edgeR or deseq2 and filter for expression by cpm first? Should I normalise it? Should I stabilise variance using rlog2? Or convert to TPM and plot that? Argh I'm so confused. Grateful for any advice you can give me :)
Dear Elizabeth,

In the simplest scenario (4 samples; 4 genes; 1 experimental condition), your metadata object, which you may have to read in from a file, could look like:

    ID	Condition
    MA	M
    MB	M
    PA	P
    PB	P

...whilst your counts file could look like:

    	MA	MB	PA	PB
    gene1	45	46	25	22
    gene2	45	45	45	44
    gene3	10	10	9	4
    gene4	88	67	34	44

This could then be read into DESeq2 as:

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

