I was thinking of mix up the of cells of population, but the sample that I sent is product of mega pre and the thing is I used exactly same sample for two different sequencing reaction which one of them is absolutely OK and the other one is rubbish. I though if it is mixed up the cells of population then I should get bad result of both reaction because I used the same sample, am I right?
The same thing as well for G & C percentage, also is not a reign full of C&G.
If I want break it down, basically I got a same lentiviral vector backbone for 3 samples and the difference is only in their insert gene, so I have same promoter and other elements in all of them and to make sure about the both junction side during cloning I sent them for sequencing.
The sequencing of 5’ in all of them OK but the 3’ sequencing in 2 of sample is bad and the other one ok.
I got three samples that they are same in promoter site and suppose to give exactly same sequence as well.
One of them has exactly the sequence that I expect but in the other two I can read the sequence only for 100 nucleotides, which isn’t enough to confirm the part that I am looking for. The strange thing is, the result become unreadable in exactly in same point in these two results.
What I thought might be some thing wrong by these two samples but on the hand I have sequencing of other part of these samples which is absolutely fine so the bad result couldn't be of poor sample.
The other possibility could be primer but the prime works and gave reading of 100 bp.
Any advise what could be the reason?
I thought something might be happened in company that I sent for sequencing.