Please HELP! TCGA RNAseq data and Limma

[Mous]

Hi,

Very much a beginner here. I downloaded TCGA RNA seq data, and unzipped the file, and now have a folder of about 80 .txt files with different names. I want to run differential expression on these files. I have downloaded R, and uploaded the limma package. I am not quite sure how to run the differential expression. I don't know how to load these files into limma. I have read several tutorials, but am essentially lost. I need beginner steps please. Any help would be greatly appreciated.

[Richard Finney]

Let's try and break this down by asking some questions ...

Have you read up on how the TCGA rnaseq txt files were prepared?
What are the inputs to LIMMA?
You say you want "differential expression"? Between what groups of samples?

[Mous]

Hi,

I have read about the TCGA files, and their output formats. Essentially, I am looking at level 3 data and have downloaded spljxn.quantification.txt, gene.quantification.txt, exon.quantification.txt. I am looking at head and neck squamous cancer with matched normal tissue. I am not sure which files are the normal and which ones are the cancer. I made sure to download all "TN", tumor matched, expression profiles.

Currently, I do not even know what to input into Limma. Ive loaded and installed it. Again, pretty new, so whatever help you can give would be greatly appreciated.

[Mous]

I am confused by the format in which these .txt files are in, vs the files that are needed for doing the differential expression.

These are the column headers in exon.quantification.txt
"exon raw_counts median_length_normalized RPKM"

I know I need to generate a targets file. Are these the files that go into the "FileName" column?

[GenePool]

Hi Mous,

We've put this data into GenePool and much of the RNA-Seq data is freely available to the community. We've linked all of the genomics data (such as RNA-Seq) to the relevant patient & sample metadata. GenePool makes it fairly simple to search for the samples you are interested in and conduct the differential expression analyses you wish to do.

Try it out!

Here are some related threads:

http://seqanswers.com/forums/showthread.php?t=42471

http://seqanswers.com/forums/showthread.php?t=48485

Good luck!

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