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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 clean-up (AmPure) then visualized again on Bioanalyzer High Sense labchip (strong peak and high concentration) before doing qPCR targeting Illumina primers.
After multiple runs of qPCR with increasingly lower and lower concentrations (full magnitude decreases), I visualized my samples again on the HS labchip to see what was happening and what was a sharp band before qPCR is now a smear. Between visualization post-size selection and qPCR I am seeing a rapid degradation of my sample pools!
The protocol I am following for each of the steps has a long history in this facility and been working well for me for the past couple months. All samples are maintained at 4C overnight or -20C if longer than that. I have since visualized the individual libraries and found no issue there but I am now up to 15 samples (in 5 separate pools) that all show this degradation.
Could the kit I am using for library prep (a newly acquired batch from Nugen) be causing this issue? Or the Blue Pippin reagents degrading adaptors slowly so it doesn’t appear on first visualization? My PI suggested contaminated AmPure beads but I am unsure of how the degradation didn’t happen until the very last round of AmPure when it is used frequently throughout the prep without issue.
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 clean-up (AmPure) then visualized again on Bioanalyzer High Sense labchip (strong peak and high concentration) before doing qPCR targeting Illumina primers.
After multiple runs of qPCR with increasingly lower and lower concentrations (full magnitude decreases), I visualized my samples again on the HS labchip to see what was happening and what was a sharp band before qPCR is now a smear. Between visualization post-size selection and qPCR I am seeing a rapid degradation of my sample pools!
The protocol I am following for each of the steps has a long history in this facility and been working well for me for the past couple months. All samples are maintained at 4C overnight or -20C if longer than that. I have since visualized the individual libraries and found no issue there but I am now up to 15 samples (in 5 separate pools) that all show this degradation.
Could the kit I am using for library prep (a newly acquired batch from Nugen) be causing this issue? Or the Blue Pippin reagents degrading adaptors slowly so it doesn’t appear on first visualization? My PI suggested contaminated AmPure beads but I am unsure of how the degradation didn’t happen until the very last round of AmPure when it is used frequently throughout the prep without issue.
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.