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  • medical oncology
    replied
    I have also run a library that was supplied too dilute, (10E8 not 10E9) and experienced high mixed reads. I would have expected better quality sequence. Can anyone explain this?

    Leave a comment:


  • WSL
    started a topic Questions on High% Dot+Mixed and library cpb

    Questions on High% Dot+Mixed and library cpb

    Hi all, I need some advices on my run result from the experts!

    Here is the data I got from GS Processer (ran with full amplicon processing pipeline):
    Raw Wells 209258
    KeyPass Wells 201705
    Passed Filter Wells 40695
    Dots 9053
    Mixed 56744
    Short Quality 94829
    Short Primer 3
    Passed filter wells 40695
    % Dot + Mixed 32.62
    %Short 47.02
    %Passed Filter 20.18

    Then I reprocess the data using the shot-gun pipeline and got the following data:
    Raw Wells 209491
    KeyPass Wells 200961
    Passed Filter Wells 80071
    Dots 10409
    Mixed 35704
    Short Quality 74172
    Short Primer 0
    Passed filter wells 80071
    % Dot + Mixed 22.95
    %Short 36.91
    %Passed Filter 39.84

    The first one is relating to a high number on %Dot + Mixed. Compared to previous runs, the percentages of these two filters have significantly increased (6% to 32%), especially in the "mixed" filter, increased from 4000 to 56000 (full processing) and 35700(shot-gun processing), does it mean that there are too many short DNA fragments per bead as the short % was also high?

    Then, I re-looked at the quantification and noticed I did not put my amplicon length into account when calculating the concentration of the library and have overestimated by 3-fold. So my input library has in fact reduced by 3-fold! Will this be the reason of a low number of passed filter wells (40695(full); 80071 (shot-gun))? Usually how much of %enrichment and number of reads will be affected when adjusting the cpd + 3 fold?

    However, if my input library cpb has been reduced by 3-fold, theoretically, I would have expected my result generated from an input of 0.3 molecules of library cpb (was using 1cpb to set up my emPCR), however my results seem suggesting I have too much DNA fragments per bead? Can anyone see which part of the procedure may have gone wrong therefore contributing to this result? Many Thanks!!!!!

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