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What does it mean if I see other significant peaks in the melt analysis of my samples?
If the peak overlaps with the shoulder seen in the standards’ melting peaks, this is likely amplification of the Illumina® primers. This can occur when the primer/template ratio is too high, or when there are large amounts of unligated adapters. If the peak is significant, the quantification results are not reliable and the experiment should be repeated. Ensure that the correct amount of primer is added to the master mix and use a higher concentration of template if possible. If the problem persists, the library may need to go through size selection again to remove free adapters.
If multiple peaks are observed that do not correspond to the adapter peak, the most likely causes are either contamination or unsuccessful size selection during library preparation. Repeat the measurement with fresh dilutions of the library. If the problem persists, that library may need to undergo further size selection.