We recently had a failed MiSeq run from an mRNA library (TruSeq stranded mRNA). Initially we though it was just a matter of overclustering, but on closer inspection we noticed the clusters looked strange. We did not get any clear, starlike clusters, instead we got small "specks" and much lower FWHM than normal. Upon rechecking the qPCR results we saw that we had two peaks in the melting curve. Thinking we might have primer-adapter dimers, we did two extra Ampure cleanups of the libraries and ran the products on our Bioanalyser and did another qPCR. The size distribution is now narrower than before, and the secondary peak in the melting curves is smaller, but still present. Is it normal to have two peaks in the melting curve? The libraries are all from a single species and tissue. We don't understand what went wrong with these libraries, and now we are uncertain if we should try another run with the extra cleanup steps, or repeat the library preps from the beginning.
Jon
Jon
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