Library Overamplification with Heteroduplex Formation (NGS Library Preparation (NEBNext Ultra II))

Question

Accepted Answer

High molecular weight fragments appear on Bioanalyzer after PCR due to single-stranded library fragments and heteroduplex formation when PCR primers are depleted. Data quality may be compromised upon sequencing. Common causes: 1) Too many PCR cycles performed relative to library input amount 2) Insufficient PCR primer concentration or volume added to reaction 3) PCR primer degradation due to improper storage temperature 4) Too much input DNA template causing rapid primer depletion even with minimum 3 PCR cycles Solutions: 1) Start with number of PCR cycles provided in product manual; reduce cycles if overamplification observed 2) Check PCR primer concentration and ensure correct volume added per manual; store primers at correct temperature 3) For high DNA input: consider size selection after ligation, or use only a fraction of ligated library as PCR input 4) Do not sequence overamplified libraries as data quality will be compromised; re-prepare with optimized cycle number