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Old 04-23-2019, 08:36 AM   #13
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Location: Mountain View

Join Date: Feb 2019
Posts: 7

Originally Posted by a.boese View Post
I recently started using the iSeq and I had the same problem. I felt that my library prep concentration was too high and I got these results by changing the concentrations (I was using PCR products fragmented with the NEBNext Ultra II FS library prep kit):

200pM % PF 39.26% AVG %Q30 82.64%
100pM % PF 67.54%; AVG %Q30 89.15%
50pM %PF 73.92%; AVG %Q30 91.81%
We had some more issues with PF and they were corrected with the loading concentration being changed (raised or lowered depending on the % occupancy). We readily have 70% to 80% PF and the %Q30 have been in the 90th percentile.

The problem really ended up being miscalculation of our starting concentrations via qPCR due to an equation error.

50pM is the perfect loading concentration! Glad you (and I) were able to figure it out
ShyN is offline   Reply With Quote