I am doing differential expression analysis on two samples, control and over-expression vector. I am using cuffdiff directly with the gtf file from the human genome as we are only interested in gene expression changes and not transcript level changes. Previously, some analysis was done on the same raw data using the same method and produced output files which match our latest runs near perfectly in terms of log2 fold change. However, in the initial analysis not done by us the p-values produced are very low (e.g. 10-5) however the new analysis we are doing has higher p values (e.g. 0.01 for the same gene). I am unsure what could be causing this difference, or if there is a setting in cuffdiff I am missing. There were no replicates for the sequencing runs.
If this time you had no replicates and the previous time you did have, then this could explain the difference (data are less robust). However, a p-value of 0.01 is totally good imho! I usually select < 0.05 so it would be in the range!