Tophat usage for paired-end reads

[vkartha]

Hey guys,
I have RNASeq data with paired end read fastq files for case and control samples. I am a beginner and have never ran programs like tophat, cufflinks etc before.

I wanted to know how I can use Tophat to accurately map these read files to the reference genome hg 19 build(assume I have the two fastq files for each end post quality filtering). What else do I have to keep in mind if I want to perform a DE analysis in the following steps?

[pbluescript]

That's a big question. I would recommend just trying it out and coming back with more specific questions if you need help.

Besides reading the manual for Tophat, be sure you set the -r and --mate-std dev options correctly for your library size. Doing this step incorrectly can have large effects on your mapping results.

[anurag.gautam]

Hi ,
I tried to map illumina ~2 million reads to Oryza sativa indica reference genome with its reference gtf file using different versions of Tophat 1.1.4, 1.3.0, 1.3.1, 1.3.2, 1.3.3 and the current one 1.4.1 .
I used the defalut options just to check if the mapping statistics really gets affected. As a result, I got the following stats:
Reads Used Reads Mapped
Tophat1.1.4 2,000,000 2,27,554
Tophat1.3.0 2,000,000 2,30,817
Tophat1.3.1 2,000,000 2,31,935
Tophat1.3.2 2,000,000 4,517
Tophat1.3.3 2,000,000 2,31,935
Tophat1.4.1 2,000,000 1,37,724

I wanted to know why the number of reads mapped is varying in each version even though using the same data. Secondly, why there is a drastic change in the mapping stats with version 1.3.2 and 1.4.1 as compared with other versions? Can please anybody throw some light on this matter