FPKM for Normal vs Tumor

Question

[Ron]

Hi all,

I want to do calculate FPKM values of genes in Normal vs tumor samples.I have a doubt if this is the correct procedure to follow.

Run cufflinks on each sample to produce fpkm.gene.gtf.Merge the gtf's for all normals using cuff merge,merge the gtf for tumors similarly to produce two separate assemblies.

Thanks,
Ron

[dario.garvan]

If you intend to test for differences, you can simply use Cuffdiff.

Answer 1

[GouthamAtla]

Tophat --> Cuffdiff. Cuffdiff will output isoforms.read_group_tracking and genes.read_group_tracking which contains the FPKM values for each replicate.

A simple AWK command would convert it to a tabular form.

Your approach "Run cufflinks on each sample to produce fpkm.gene.gtf. Merge the gtf's for all normals using cuff merge,merge the gtf for tumors similarly to produce two separate assemblies." Merging all the GTFs for all normals is not a good idea as you need FPKM values for each replicate for inputting it to any software like clustering or heatmap. Hence, do not merge all the GTFs from all the normal samples, instead make a tabular format of FPKMs for each replicate.

[Ron]

I have to use cuffdiff separately on normals and tumors ,or together?Also I want to do a heatmap as u mentioned from this output.So the input to that data would be a table with FPKM values and having normals as well as tumors?

[GouthamAtla]

Spend some time on the cufflinks-cuffdiff manual.

http://cufflinks.cbcb.umd.edu/manual.html#cuffdiff

[Ron]

Cuffdiff required a merged transcript gtf file from all the samples for e.g.

cuffdiff merged_asm/merged.gtf top_SRR027863-65/accepted_hits.bam top_SRR027866-67/accepted_hits.bam

It can not just take the bam files as an input.

Kindly See this post: Meaning Of Fpkm Value Used By Cufflinks