Library degrading after prep with Ovation Ultralow V2

[robicha7]

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.

[nucacidhunter]

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).

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?

Do you mean library degrades after size selection and clean up if you leave the library in fridge or RT?

If the library reagents are contaminated you would not have the library in first place.

[robicha7]

Yes, the library pool degrades after size selection and clean up while in the 4C fridge.

[robicha7]

Additional images

I will try to attach images of the qPCR concentrations, as well as before and after images visualized by the Bioanalyzer.

image 1 - qPCR values
image 2 - post qPCR HighSense visualizations (lane 3,4,5)
image 3 - post pooling and size selection HighSense visualizations (note that lane 1 size is off-set by misidentified upper marker)

[nucacidhunter]

I am not sure how you have come up with degradation conclusion. qPCR amplicons goes through excessive cycling (normally 40) and end product can have artifacts and also running them on BA without clean up will not give good indication of size.

The best test would be to use Qubit or other dsDNA specific quantification method to quantify the libraries after clean up (post size selection) and storage and also run them on BA. If there is degradation it more likely would be due to contamination of buffer used to elute libraries from bead clean up. Also note that DNA can stick to tubes and if concentration is low then some will be lost.

[robicha7]

Hello,

I have found half a solution - the library preparation kit is not contaminated nor unstable. You were correct that degradation was not the issue. Unexpected as the declining qPCR readings were, the actual issue seems to be size selection with Blue Pippin where we lose the majority (50-70% loss) of our expected product during elution. Thank you very much for your suggestions.