Still getting the hang of the whole RNA-seq and gene annotation process. One factor I have been thinking about lately and after reading a publication I would like to ask a question in regards to:
1.In regards to filtering FPKM values in a spreadsheet, what value determines if a transcript is present/expressed / silenced and up-regulated. I generally assumed a value 'greater than' 0 would mean the transcript is expressed but I have read different. Can anyone shed light on this? Although different from FPKM, one paper was using the assumption that if RPKM > or equal to 3 then the transcript was over expressed. Can anyone explain?
2.I also have 3 different conditions: control, low and medium. When making comparisons I am looking at the top 1000 up-regulations and down-regulations and comparing these between Medium/Control Vs Low/Control ( Taking FPKM values,working out fold changes and Log2 values) using Blast2GO software. I have also thought about just comparing the top 1000 expressed transcripts of control Vs Medium Vs low based on FPKM values without working out top upreg/ downregs via fold-changes.Ultimately what would be the best way to go about comparisons, does my method make sense? What logical approaches have you guys taken to analysing an experiment with 3 treatments?
So far I have created some Venn diagrams to show transcripts present in each condition, shared between conditions, present in all conditions, heat maps using EdgeR and I am currently making use of Blast2GO for GO and annotation comparisons.I just need to be 100% confident in my methods.
Apologies for the very noobish questions but I guess I have to learn somewhere :D