Hello,
I am having an issue with library pools rapidly degrading after pooling and size selection.
I am prepping low biomass samples with Nugen’s Ovation Ultralow Library System V2 kit. Samples are visualized after amplification and clean-up (AmPure) on Aligent Bioanalyzer DNA 1000 labchip before being pooled and size selected on Blue Pippin 1.5% agarose gel cassettes with internal markers. Pooled samples are cleaned-up (AmPure again) then visualized again on Bioanalyzer High Sense labchip before doing qPCR targeting Illumina primers.
After multiple runs of qPCR with increasingly lower concentrations, I visualized my samples again on the HS labchip again and what was once a defined band before qPCR is now a smear. Between visualization post-size selection and qPCR I am seeing a rapid degradation of my sample pools but can find no other evidence of contamination. I have repeated the procedure but end up with similar results even with all new reagents (excluding library prep kit).
The protocol I am following has been working well for me for the past couple months. All samples are maintained at 4C overnight or -20C if longer than day. When visualizing the original individual libraries maintained in freezer, there is no sign of degradation.
Could the kit I am using for library prep be causing this issue? Or the method of selection? How could I parse out the contaminating agent in the future?
I am still new to NGS but SeqAnswers hasn’t let me down yet for finding answers. Please let me know what advice you have.
I am having an issue with library pools rapidly degrading after pooling and size selection.
I am prepping low biomass samples with Nugen’s Ovation Ultralow Library System V2 kit. Samples are visualized after amplification and clean-up (AmPure) on Aligent Bioanalyzer DNA 1000 labchip before being pooled and size selected on Blue Pippin 1.5% agarose gel cassettes with internal markers. Pooled samples are cleaned-up (AmPure again) then visualized again on Bioanalyzer High Sense labchip before doing qPCR targeting Illumina primers.
After multiple runs of qPCR with increasingly lower concentrations, I visualized my samples again on the HS labchip again and what was once a defined band before qPCR is now a smear. Between visualization post-size selection and qPCR I am seeing a rapid degradation of my sample pools but can find no other evidence of contamination. I have repeated the procedure but end up with similar results even with all new reagents (excluding library prep kit).
The protocol I am following has been working well for me for the past couple months. All samples are maintained at 4C overnight or -20C if longer than day. When visualizing the original individual libraries maintained in freezer, there is no sign of degradation.
Could the kit I am using for library prep be causing this issue? Or the method of selection? How could I parse out the contaminating agent in the future?
I am still new to NGS but SeqAnswers hasn’t let me down yet for finding answers. Please let me know what advice you have.
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