problems of heterogeneity and reproducibility

[vivic21]

Hello,
We sequencing samples of 454 FLX 4/4 runs. We have problems of heterogeneity and reproducibility. Indeed according to the location on the picotiter plat the results of the same samples do not give the same. In addition, we are multiplexing (equimolar mixture), and the sizes are very variable (as we sequençons products of the same size).
I would like to know if the other person were confronted with this problem and how you solved this worries, knowing that we are already in contact with ROCHE that can not provide other lot numbers.
thank you for your help

[martin2]

Which library preparation kit did you use? General Library, Rapid Library, Amplicon I or II?

You mean the 4 samples were fragmeted/purified/bound to beads in large enough volumes so that you have enough beads to do 1 full FLX runs with each? But you actually sequenced always just in a 1/4 region? The sequencing kit is FLX or FLX+?

[vivic21]

In fact, we attack directly to the emPCR, the banks are composed of different samples each comprising a mid to identify them.
On a runFLX, so we spend 4 banks, each with a result to emPCR between 10 and 12%.