Hello to you all,
I want to perform DNA seq and I am having issues in the step of ligating the adaptors to the sequences.
To give you more details: We want to find the different virus detected in an individual. For that purpose, we eliminated the host genome using DNAses and RNAses on the lysate, and then inactivated the nucleases and performed an additional RNA/DNA isolation step with GeneJet Viral DNA/RNA isolation kit. Then, using the Ovation system by Tekan, samples were RT to dsDNA. Then DNA was purified again using Qiagen columns.
At this point, samples were quantified using nanodrop (most of them were at around 100ng/ul with good ratios), and Qubit with equally good results. Size profiles were assessed with Bioanalyzer HS DNA chip, and profiles looked ok.
We performed then library prep with NEBNExtUltra II starting from 300ng and then a qPCR to measure number of sequences containing adaptors, and there was barely any detection. We checked using a Bioanalyzer the profiles again and there was indeed no shift of the samples, what should be observed after the ligation with the adapters.
We repeated the protocol with two samples in parallel, one from our cohort, another a commercial DNA used as a control. The control sample worked perfectly. There was no detection of adapters on our sample.
Has anyone been on a similar situation? Any idea on what may be the issue here?
Thanks,
J
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