Hi all,
I'm running a library of synthetic amplicons (~175-250bp), and reading it out with custom primers on the MiSeq. I'm having a lot of problems getting enough cluster density. My last MiSeq run, I combined the custom sequencing primer with the Illumina Read 1 mix, and ran the mixed 20% 8pM phiX with with 80% 20pM library. I got ~1.5 million phiX reads and ~500K of the custom reads, with at total cluster density on ~225K/mm2. The custom reads looked fine and were what I expected of my library. I reran the Kapa QPCR quantitation again on my dilutions against phiX and it looked pretty accurate.
I was wondering what the concentration range of getting good cluster density is. Would it make sense to run 10X (200pM) of the library to get 10X the number of clusters? This seems ludicrously high. Does it something to do with the length of my library? Has anyone seen such a drastic difference between Kapa Library quantification and cluster density? I'm a little lost and want to figure stuff out without burning too many flowcells.
Thanks,
Sri
I'm running a library of synthetic amplicons (~175-250bp), and reading it out with custom primers on the MiSeq. I'm having a lot of problems getting enough cluster density. My last MiSeq run, I combined the custom sequencing primer with the Illumina Read 1 mix, and ran the mixed 20% 8pM phiX with with 80% 20pM library. I got ~1.5 million phiX reads and ~500K of the custom reads, with at total cluster density on ~225K/mm2. The custom reads looked fine and were what I expected of my library. I reran the Kapa QPCR quantitation again on my dilutions against phiX and it looked pretty accurate.
I was wondering what the concentration range of getting good cluster density is. Would it make sense to run 10X (200pM) of the library to get 10X the number of clusters? This seems ludicrously high. Does it something to do with the length of my library? Has anyone seen such a drastic difference between Kapa Library quantification and cluster density? I'm a little lost and want to figure stuff out without burning too many flowcells.
Thanks,
Sri
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