Hi Ying,

One thing I am concerned is that my files
> is kind of large, e.g., for each end I got a file with 20.7 gb, so totally
> 41.4
> gb for both ends of this sample. So do you think it is because the files
> are too
> big?


You'll want to use the FTP upload process to upload data this large to
Galaxy. See the upload page for details; we recommend FileZilla as a nice
GUI for performing FTP:

http://filezilla-project.org/


> Also when the genome center sent me the data, they already separate the two
> ends into two files, so I uploaded two files for one sample, but when do
> tophat
> analysis, do I need to merge those two files into one file?


You'll want to perform a single Tophat analysis on both files since they're
paired. However, no merging is required: Tophat will accept both files in a
single run; see the Tophat tool for details.


> do you know what is
> the usual parameter set up for paried-end RNA-seq analysis(with a 300 bp
> fragment)?


Tophat has a set of default parameters, but you'll likely want to experiment
with different values to see what works best for your data. Note that the
mean inner distance between pairs is (Insert_size-N*2 ) where N is the size
of each read fragment.

Best,
J.
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