Excessive Adaptor Dimer Formation (NGS Library Preparation (NEBNext Ultra II))

Question

Accepted Answer

Sharp 127 bp peak visible on Bioanalyzer representing adaptor dimers, reducing the proportion of desired library fragments and overall usable yield. Common causes: 1) Adaptor concentration too high relative to sample input amount, quality or type 2) Adaptor added directly to ligation master mix instead of to sample first, causing increased self-ligation 3) Adaptor dilution or titration not optimized when sample source changes (extraction method, tissue type, etc.) Solutions: 1) To recover samples with adaptor dimers, repeat bead cleanup using 0.9X bead ratio 2) Optimize adaptor dilution based on sample input, quality and type using adaptor titration experiment 3) Do NOT add adaptor to ligation master mix; add adaptor to sample first, mix, then add ligase master mix and ligation enhancer 4) Repeat adaptor titration if sample source changes (e.g., extraction method, tissue type)