Go Back   SEQanswers > Bioinformatics > Bioinformatics

Similar Threads
Thread Thread Starter Forum Replies Last Post
Forward-reverse read counts don't match Arvind Bhagwat Core Facilities 1 04-18-2016 10:11 AM
Solution found: For Cuffdiff/links 2.0.2 Make cufflinks FPKM match Cuffdiff FPKM NGSfan RNA Sequencing 4 04-16-2013 08:10 AM
Varscan header doesn't match the number of columns in the data rdeborja Bioinformatics 3 01-30-2013 09:52 AM
Library QC- cntrl loci enrichment doesn't match pre and post library generation yogioner Sample Prep / Library Generation 0 12-03-2012 04:40 PM
Cufflinks convert FPKM to Read Counts zee Bioinformatics 0 03-08-2010 07:35 PM

Thread Tools
Old 02-09-2017, 11:02 AM   #1
Junior Member
Location: Boston

Join Date: Feb 2017
Posts: 1
Default Very large FPKM from cuffdiff that doesn't match read counts

Hi Everyone,

I am relatively new to RNAseq, and hope can get some help from more experienced people here.

So I used cuffdiff to look at differential gene expression. I have 2 conditions and 3 replicates for each condition. When I look at the genes_read_group_tracking file, I found that for some genes, one of the replicates has very large FPKM values that doesn't match raw frags count.

For example this is what I see:

tracking_id condition replicate raw_frags internal_scaled_frags external_scaled_frags FPKM effective_length status
XLOC_009487 WT 0 4 4.21548 4.21548 1150.43 - OK
XLOC_009487 WT 1 5 5.39083 5.39083 1.14629 - OK
XLOC_009487 WT 2 8 7.56804 7.56804 1.60234 - OK
XLOC_009487 OE 1 2 2.29124 2.29124 0.476229 - OK
XLOC_009487 OE 0 5 4.84785 4.84785 0.995213 - OK
XLOC_009487 OE 2 5 4.07315 4.07315 0.77989 - OK

You can see that the FPKM for WT 0 is 1150 where as the raw frags is only 4.2. The other samples are fine. I observed this in multiple genes, and they don't always happen to the same sample. I also check the FPKMs from cufflinks, and they look normal. So seems that it's cuffdiff's problem.

Does anyone know why this happen and how to solve it? Appreciate the help!
fcchau is offline   Reply With Quote
Old 02-16-2017, 09:37 AM   #2
Senior Member
Location: California

Join Date: Jul 2014
Posts: 198

I think there is a decent amount of evidence out there that cuffdiff is less than optimal for gene-level DE analysis.


If you are only doing DGE, I'd recommend edgeR or DESeq2
fanli is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 07:04 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO