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Does anyone had problems with low fold enrichment after hybridization with Roche probes following their SeqCap protocol?
We use KAPA Hyper Plus / KAPA Hyper Prep for sample prep and follow the SeqCap protocol. Sample preps are quality checked and have concentration between 20-40ng/ul (median length of 320bp) and look very nice. They work well with 2 other designs.
With one design, after hybridization we get low fold enrichment by qPCR - only 50x, instead of >100x. First experiments with this probe set were nice, but after several runs the enrichment failed.
Fresh ethanol, good vortexing during washing (according to the new protocol), fresh beads, fresh reagents (the oldest one are UBO, but they work perfectly with other designs).
The other problem we observed was that after KAPA Hyper Prep in several samples some exons were missing in the repetitive manner (the same exons in different samples). It did not depend from the type of DNA extraction and this samples have low uniformity.
I asked tech support and we check the the possibilities but maybe there is someone that had the same problem and found the solution.
We use KAPA Hyper Plus / KAPA Hyper Prep for sample prep and follow the SeqCap protocol. Sample preps are quality checked and have concentration between 20-40ng/ul (median length of 320bp) and look very nice. They work well with 2 other designs.
With one design, after hybridization we get low fold enrichment by qPCR - only 50x, instead of >100x. First experiments with this probe set were nice, but after several runs the enrichment failed.
Fresh ethanol, good vortexing during washing (according to the new protocol), fresh beads, fresh reagents (the oldest one are UBO, but they work perfectly with other designs).
The other problem we observed was that after KAPA Hyper Prep in several samples some exons were missing in the repetitive manner (the same exons in different samples). It did not depend from the type of DNA extraction and this samples have low uniformity.
I asked tech support and we check the the possibilities but maybe there is someone that had the same problem and found the solution.