Library degrading after prep with Ovation Ultralow V2
Hello,
I am new to this forum and NGS but the have found endless answers here to every issue I have run into. Hopefully you can help me again with my own samples.
I am working with several low biomass samples amplified using the Nugen Ovation Ultralow Library System and keep running into issues with rapid degradation of my samples after pooling libraries and size selecting.
After ligation-amplification and clean-up, I pool samples (3 per project) and size select using the Blue Pippin 1.5% agarose gels and internal markers. Samples are visualized using Aligent Bioanalyzer High Sense labchip before quantifying for Illumina primers with qPCR.
For some reason, between my HS DNA visualization and qPCR the samples are rapidly degrading. After the first few rounds of qPCR showed such repeatedly lower low concentrations, I ran the pools on the HS DNA chip again and my previous sharp 520bp band is now a smear.
I follow a working protocol my lab has been using since before I started and has worked almost without fail for me over the past 5 months. Samples are eluted into MilliQ H2O that is UV sterilized between uses and maintained in 4C overnight or -20C for longer. Visualizing the individual libraries (3 per pool) shows no signs of degradation.
Could I be carrying over degrading molecules from my previous steps? Is the library kit not properly binding to fragments? All libraries with this issue were prepared with the same newly acquired library system kit. I use AmPure XP beads for clean up and my PI suggested they may be contaminated but I am not sure how that could have happened or what it could be.
Please and thank you for any advice or similar issues.
I am new to this forum and NGS but the have found endless answers here to every issue I have run into. Hopefully you can help me again with my own samples.
I am working with several low biomass samples amplified using the Nugen Ovation Ultralow Library System and keep running into issues with rapid degradation of my samples after pooling libraries and size selecting.
After ligation-amplification and clean-up, I pool samples (3 per project) and size select using the Blue Pippin 1.5% agarose gels and internal markers. Samples are visualized using Aligent Bioanalyzer High Sense labchip before quantifying for Illumina primers with qPCR.
For some reason, between my HS DNA visualization and qPCR the samples are rapidly degrading. After the first few rounds of qPCR showed such repeatedly lower low concentrations, I ran the pools on the HS DNA chip again and my previous sharp 520bp band is now a smear.
I follow a working protocol my lab has been using since before I started and has worked almost without fail for me over the past 5 months. Samples are eluted into MilliQ H2O that is UV sterilized between uses and maintained in 4C overnight or -20C for longer. Visualizing the individual libraries (3 per pool) shows no signs of degradation.
Could I be carrying over degrading molecules from my previous steps? Is the library kit not properly binding to fragments? All libraries with this issue were prepared with the same newly acquired library system kit. I use AmPure XP beads for clean up and my PI suggested they may be contaminated but I am not sure how that could have happened or what it could be.
Please and thank you for any advice or similar issues.