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Old 10-01-2012, 04:54 PM   #1
WSL
Junior Member
 
Location: NZ

Join Date: Sep 2012
Posts: 5
Question 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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Old 10-05-2012, 12:44 AM   #2
medical oncology
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Join Date: Mar 2010
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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?
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