Hi everyone. Please let me know if this has been answered in another thread. I have a lab meeting coming up, so I'm a bit desperate to figure out this problem.
So I'm doing RNA-Seq on some yeast strains. I have two conditions, one wild time and one with a gene KO, and I have two replications of each for four total. I also have a sequenced and annotated genome, so I have reliable .GTF and .GFF files to work with.
Here's my issue: When I compare one WT data set to one KO data set, I get a gene expression change considered significant for ~200-300 genes out of 6100. When I try to merge my replicates, I wind up with something like 3000 significant changes. This gene KO shouldn't be affecting everything in the cell, so I"m assuming I'm doing something wrong. What is the appropriate way to use replication with Tuxedo? Should I be mapping my replicated together with Tophat? Should I map separately and then use cuffdiff to combine the mapped data sets?
Any help would be much appreciated.
-DrAlexander
So I'm doing RNA-Seq on some yeast strains. I have two conditions, one wild time and one with a gene KO, and I have two replications of each for four total. I also have a sequenced and annotated genome, so I have reliable .GTF and .GFF files to work with.
Here's my issue: When I compare one WT data set to one KO data set, I get a gene expression change considered significant for ~200-300 genes out of 6100. When I try to merge my replicates, I wind up with something like 3000 significant changes. This gene KO shouldn't be affecting everything in the cell, so I"m assuming I'm doing something wrong. What is the appropriate way to use replication with Tuxedo? Should I be mapping my replicated together with Tophat? Should I map separately and then use cuffdiff to combine the mapped data sets?
Any help would be much appreciated.
-DrAlexander
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