Library Not the Correct Insert Size (NGS Library Preparation (NEBNext Ultra II))

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Library fragment size distribution is shifted from expected range, with either smaller or larger inserts than intended, or discrepancy between fragment analyzer measurement and sequencer-determined insert size. Common causes: 1) PCR size bias: short fragments amplify more efficiently than long ones during PCR 2) FFPE DNA crosslinking inhibits amplification of longer PCR products (crosslinks cannot be reversed) 3) Incorrect size selection ratios or bead volumes used, or sample volume inaccurate due to evaporation 4) Sample not in correct reagent/buffer specified in previous protocol step before size selection 5) Clustering bias during sequencing: short fragments cluster more efficiently than long ones Solutions: 1) If sufficient input material available, perform size selection after ligation to narrow input size range; reduce PCR cycles 2) For FFPE samples: use less fragmentation before library prep to shift library towards longer inserts (crosslinks cannot be reversed) 3) Ensure sample is in correct reagent/buffer from previous step; use accurate volumes; top off evaporated samples with water to expected volume 4) For discrepancy between analyzer and sequencer: perform more stringent size selection (gel cut or Pippin Prep) to remove short fragments 5) Note: Additional size selection may result in lower library yields but increased average insert size