SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
one gene one RPKM cufflinks or DEseq frankyue50 Bioinformatics 5 10-30-2014 01:37 AM
RPKM and multiple reads, tophat and cufflinks mgogol Bioinformatics 6 09-03-2012 03:46 PM
very high RPKM values from Cufflink honey Bioinformatics 21 03-08-2012 07:00 AM
some gene no RPKM in every library by Cufflinks stunlay RNA Sequencing 0 08-19-2011 10:06 AM
tophat/cufflinks bam vs. RPKM mgogol Bioinformatics 5 04-26-2010 10:58 AM

Reply
 
Thread Tools
Old 11-30-2011, 01:30 AM   #1
hlwright
Member
 
Location: Liverpool, UK

Join Date: Feb 2011
Posts: 30
Default Cufflinks v 1.2.0 high number of RPKM 0

I have three SOLiD human RNA-Seq libraries which I am analysing using cuffdiff. The libraries have a large number of reads which MAY be pcr duplicates (~90%), but as they are single-end reads I have no way of knowing for sure if they are pcr duplicates or if they are genuine reads which just happen to align to the same location.

I have two sets of BAM files: (1) all uniquely mapped reads, (2) uniquely mapped reads with "duplicates" removed. The library sizes for BAMs(1) are 23, 26 and 49 million, and for BAMs(2) are 2,3 and 4.5 million.

I am comparing the cuffdiff output on these two sets of BAM files. I am using a hg19mRNA gtf file which I have made compatible with cufflinks using this cuffcompare command as recommended in the cufflinks manual:

Code:
cuffcompare -s hg19.fa -CG -r hg19mRNA.gtf hg19mRNA.gtf
My cuffdiff command looks like this:

Code:
cuffdiff -p 8 -b hg19.fa cuffcmp.combined.gtf file1.bam file2.bam file3.bam
When I look at the RPKM values generated by cuffdiff I see that for the BAM(1) files containing all the reads, only 839 genes get RPKM values in at least 1 of my 3 samples. Cuffdiff on the BAM(2) files with "duplicates" removed generates RPKM values for 19,129 genes. I have positive controls in my samples which do not get RPKM values in the first scenario BAM(1) but do get RPKM values as expected in scenario 2 BAM(2).

Can anyone explain what is going on here?

Thanks
HELEN
hlwright is offline   Reply With Quote
Old 11-30-2011, 08:08 AM   #2
hlwright
Member
 
Location: Liverpool, UK

Join Date: Feb 2011
Posts: 30
Default

Just to let everyone know I have had a pm from Cole Trapnell and my problem is being caused by a bug in the current version of Cufflinks.

This option corrects the RPKM values:
Code:
--max-bundle-frags 100000000
Cole explains:
Quote:
That option is there so that Cufflinks and Cuffdiff automatically skip extremely deeply sequenced loci, which can cause the programs to hang or take a really long time. The skipped loci get marked as such, and receive zero FPKM. However, in 1.2.0, there's a bug that causes other genes downstream of the large loci to receive incorrect values, and I'm currently working on the fix. The option I told you about basically turns off this feature, so the bug is never triggered.
Thanks for your help, Cole.

Best wishes
HELEN
hlwright is offline   Reply With Quote
Reply

Tags
cufflinks cuffdiff, pcr duplicates, rpkm

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 09:20 PM.


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