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Reduced coverage evenness of SureSelect QXT libraries with automation
Dear all,
We are trying to automate SureSelect QXT library prep with the Hamilton Star liquid handler, and encountered the issue of reduced coverage evenness. With automation, we had more coverage gaps even when the overall average coverage across the region of interest is slightly higher than the that of a manually prepped library. We are a clinical lab and coverage gaps have to be filled in by Sanger, which means that we would have to do more Sanger with automation of QXT library prep
.
Does anyone have similar experience?
I suspect the following possible reasons:
1. Incomplete MyOne beads transfer and loss of captured DNA because of this. I heard that lower input DNA may actually lead to poor coverage evenness. So maybe MyOne beads loss has similar effect?
2. On-deck hybridization: evaporation and volume loss during hybridization set up. The liquid handler takes 3-5 minutes to set up the hybridization on the deck, so I am sure there was some volume loss due to evaporation.
Any thoughts or comments are much appreciated. thanks!
We are trying to automate SureSelect QXT library prep with the Hamilton Star liquid handler, and encountered the issue of reduced coverage evenness. With automation, we had more coverage gaps even when the overall average coverage across the region of interest is slightly higher than the that of a manually prepped library. We are a clinical lab and coverage gaps have to be filled in by Sanger, which means that we would have to do more Sanger with automation of QXT library prep
. Does anyone have similar experience?
I suspect the following possible reasons:
1. Incomplete MyOne beads transfer and loss of captured DNA because of this. I heard that lower input DNA may actually lead to poor coverage evenness. So maybe MyOne beads loss has similar effect?
2. On-deck hybridization: evaporation and volume loss during hybridization set up. The liquid handler takes 3-5 minutes to set up the hybridization on the deck, so I am sure there was some volume loss due to evaporation.
Any thoughts or comments are much appreciated. thanks!