High EDTA concentrations and insufficient reagent mixing are the two main causes of under-fragmented DNA during sequencing library construction.

DNA fragmentation is highly sensitive to EDTA (Ethylenediaminetetraacetic acid) concentration. The sample double-stranded DNA must be in a low EDTA (0.1mM) TE buffer for best results. If the sample double-stranded DNA is in a TE buffer containing 1mM EDTA, it can result in under fragmentation.

For proper DNA fragmentation, the fragmentation master mix must be mixed thoroughly before and after it is added to the sample DNA. Under-fragmentation can occur if it is not mixed properly.