Hi Ying,<div><br></div><div><div class="gmail_quote"><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex;">One thing I am concerned is that my files<br>
is kind of large, e.g., for each end I got a file with 20.7 gb, so totally 41.4<br>
gb for both ends of this sample. So do you think it is because the files are too<br>
big? </blockquote><div><br></div><div>You&#39;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:</div><div>

<br></div><div><a href="http://filezilla-project.org/">http://filezilla-project.org/</a> </div><div> </div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex;">Also when the genome center sent me the data, they already separate the two<br>


ends into two files, so I uploaded two files for one sample, but when do tophat<br>
analysis, do I need to merge those two files into one file? </blockquote><div><br></div><div>You&#39;ll want to perform a single Tophat analysis on both files since they&#39;re paired. However, no merging is required: Tophat will accept both files in a single run; see the Tophat tool for details.</div>

<div> </div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex;">do you know what is<br>
the usual parameter set up for paried-end RNA-seq analysis(with a 300 bp<br>
fragment)?</blockquote><div><br></div><div>Tophat has a set of default parameters, but you&#39;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.</div>

<div><br></div><div>Best,</div><div>J.</div></div></div>