What causes under-fragmented DNA during sequencing library construction?
High EDTA concentrations
Fragmentation is sensitive to EDTA concentration. Therefore, it is important for the sample dsDNA to be in low EDTA (0.1 mM) TE buffer. Under-fragmentation can occur if the sample dsDNA is in TE buffer containing 1 mM EDTA .
If the sample dsDNA is in TE buffer containing 1 mM EDTA , a buffer exchange will need to be performed using either a column- or bead-based purification method. Alternatively, a larger volume of Enzymatic Prep Reagent (up to a maximum of 3X the standard volume) can be used in the Enzymatic Preparation step.
Insufficient reagent mixing
Make sure to thoroughly mix the fragmentation master mix before and after adding it to the sample DNA. For the ligation, aliquot the Lotus Ligation Buffer carefully and mix the Ligation Master Mix thoroughly.