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What should I do if my sample replicates show significant variation?
Check the melt curves to ensure there is a single product peak. If there are multiple peaks, this suggests the sample may have become contaminated or degraded, or that an error occurred during the size selection or adapter ligation steps or library preparation. If the melt analysis shows only one peak, the variation is probably caused by inaccurate pipetting. Create a new dilution of the sample and repeat the experiment, taking care to pipette the replicates accurately. If the problem persists, a new library may need to be created.