Dear team,<br><br>I have a few questions regarding RNA-seq data analysis. I want to compare transcriptome profile of a bacterium between 5 time points, and for each time point I have three biological replicates. <br>Right now I have each of the replicates&#39; sequences aligned with Bowtie and assembled transcripts generates using Cufflinks. I understand that if I am just comparing two time points (if each of time point has only one sample, no replicate) I just need to cuffmerge the two time points and cuffdiff the merged file.<br>
<br>My question is, if I need to compare between 5 time points, should I do comparison pairwise?<br><br>And also, how should I include three biological replicates for each time point? I can only find &#39;include replicates&#39; in Cuffdiff. Could you check is the following methodology correct?<br>
<br><br>let&#39;s say I have samples in time 0 , 1hour, 2hours, 3 hours and 4 hours. I will name each of the replicates as 0hour-1, 0hour-2, 0hour-3,etc.<br><br>I will first compare expression between 0hour and 1hour.<br>
<br>I will use cuffmerge to merge 0hour-1, 0hour-2, 0hour-3, 1hour-1,1hour-2.1hour-3 to generate one cuffmerge file.<br>Then I will run cuffdiff using the merged file, include two groups, group 1 is 0 hour (add 0hour 1-3 in group 1) and group 2 is 1hour (add 1hour1-3 in group 2).<br>
<br>Does this sound reasonable?<br><br>Thank you very much,<br><br>Qian<br><br><br clear="all"><br>-- <br>Qian Dong<br>Bauer Lab, MCBD<br>Simon Hall: 313-317<br>212 S. Hawthorne Dr.<br>Bloomington, IN 47405<br><a href="mailto:Email%3Adong3@indiana.edu">Email:dong3@indiana.edu</a><br>
Lab Phone:812-855-8443<br>