library concentrations variations

[dogcatcher]

Hi, everyone,

I made four libraries this week. But today when I ran a DNA gel, I found that the concentrations (DNA band intensity) of the four libraries have great difference. The concentration of the highest one is about 3 times of the lowest one. I am supposed to compare gene expression level changes among these four libraries. I wonder whether the library concentrations difference will affect RNA-sequencing results. For example, some reads may overpresent the library. Does anyone have similar situation?

Thanks!

[Brian Bushnell]

Standard RNA-seq analysis using FPKM is robust to (or, one might say, 'ignorant of') the absolute difference between sample concentrations, and only sensitive to relative differences between gene expressions. In other words, if you sequence the same sample twice - once at full concentration and once diluted by 50% - it shouldn't show any gene expression changes if you go by FPKM, other than in genes with only a very few reads mapped.

[dogcatcher]

Thank you very much!