Low Library Yield with Intact Input DNA (NGS Library Preparation (NEBNext Ultra II))

Question

Accepted Answer

Library yield is significantly lower than expected based on input amount, despite successful completion of all enzymatic steps. Common causes: 1) Input DNA is damaged or degraded (especially FFPE samples) 2) NEBNext adaptor denatured during dilution (diluted in water or inappropriate buffer) 3) Prolonged storage of sample after A-tailing step, especially with low input amounts 4) Ligation incubation temperature exceeded 20°C causing DNA end breathing and reduced ligation efficiency Solutions: 1) For FFPE samples: shear input DNA on Covaris instrument in 1X TE Buffer and use NEBNext FFPE DNA Repair Mix (NEB #M6630) after shearing 2) When diluting NEBNext adaptors, use 10 mM Tris-HCl (pH 7.5-8.0) with 10 mM NaCl; keep adaptor on ice until use 3) Avoid prolonged storage after A-tailing; for low inputs avoid overnight storage and proceed immediately to adaptor ligation 4) Ensure ligation incubation temperature does not exceed 20°C during the entire incubation period