Hello,
I am sequencing a library for targeted sequencing of CFTR composed by 68 amplicons. The library preparation cames always very well. However, when I sequence at 10pM I get a cluster of only 400. I have tried to sequence at 12pM but the situation seems to get worse. In fact, I get a cluster of only 300.
Do you have any clue why I am not able to get a higher cluster despite I am increasing the concentration of the library?
In our experience, it seems that the lenght of the DNA fragment at the end of the library preparation process has a great impact on the cluster production (smaller the fragment, lower the clustering). Maybe I would advice you to verify the size distribution of your library (using a labchip/bioanalyzer tool)?
Hello Maxime,
I completely agree with your hypothesis but our library is composed by two bands of 250 and 300 base pairs according to the bioanalyzer. I really doubt that the problem is the bridging of the library. However, maybe the fact that we have two size can influence negatively the clustering even though I do not see the reason.
I will keep you updated if I have any news. Thanks again for your availability.
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