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Over-amplification of a library reveals itself as an abnormal migration.
During over-amplification, when DNA replication can no longer take place due to reagent depletion, subsequent rounds of DNA denaturation and annealing result in the formation of improperly annealed, partially double-stranded DNA called heteroduplex structures. These run larger than expected on the Bioanalyzer™ or TapeStation™ (Agilent) native electrophoresis conditions.