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Old 05-09-2014, 12:00 AM   #1
PhD Student
Location: La Jolla

Join Date: Jun 2012
Posts: 33
Unhappy TopHat 2 & Tophat for Illumina - Different FPKM?

Hi all,

First let me just say I am new to RNA-seq analysis. I have a weird issue in Galaxy where I am experiencing different transcript expression FPKM values when compared to my reference based on whether I use TopHat v2 or TopHat for Illumina.

Here are my two workflows:
1. FastQ file --> convert to Sanger FastQ with FastQ Trimmer --> Align with TopHat2 using built in reference mm10 ---> Run Cufflinks Single Read.

2. FastQ file ..> convert to Sanger FastQ with FastQ Trimmer --> Align with Tophat for Illumina using built in reference mm10 ---> Run with Cufflinks Single Read.

Option 1 results in Cufflinks FPKM values of 0 for all transcripts. Option 2 gives actual FPKM results.

What is the difference between these two programs? The only difference I can notice (as I don't know how to ''view'' bam files as text) is in the insertions and deletions tables in TopHat on Galaxy. TopHat2 lists my insertions and deletions in first column based on chromosomes, followed by location. Tophat for Illumina gives me gene names in the format of ENSMUST00000022345, for example, followed by location.

Any help would be greatly appreciated!
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