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Old 08-18-2011, 02:54 PM   #1
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Location: S.F.-Bay area

Join Date: May 2011
Posts: 4
Default 454Jr run metrics - possible PCR primer contamination

I have a question regarding a couple of the run metrics obtained after performing amplicon (~500bp) sequencing on a 454 Jr. titanium platform.

If one were to not have sufficiently removed PCR primers during the AMPure purification of amplicon prior to library preparation, would one expect to see higher than usual Mixed reads, or both higher Dot AND Mixed reads in the Run Metrics at the completion of the run?

My "guess" is that I still have PCR primers in my amplicon library that were not removed by AMPure. Still, I'd be interested in reading what other users have to say regarding the Mixed vs. Dot reads in the event of primer contamination. - THANKS IN ADVANCE!

For instance:
Raw reads = ~210-230K
Passed Key reads =~97-99% of raw
Passed filters = 80 - 120K
Dot = ~2000 (same as typical run)
Mixed = >10000 (~ 5X higher compared to typical run)
% Dot+Mixed = >6%
Primer = 350

Run metrics typically obtained are:
Raw reads = ~210-230K
Passed Key reads =~97-99% of raw
Dot = ~2000 to 3500
Mixed = ~1000 to 2500
% Dot+Mixed = 1-3%
Primer = <40

Other possibly useful info regarding the runs:
Amplicon size = 500 bp
emPCR (Lib-A) kit used with 0.3 cpb
% DNA capture-bead recovery at enrichment = 6-9%
Loading only ~200-250K bead per PTP region (GS Jr.)

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