Library Overamplification with Heteroduplex Formation

Symptom

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.

1. 1 Too many PCR cycles performed relative to library input amount
2. 2 Insufficient PCR primer concentration or volume added to reaction
3. 3 PCR primer degradation due to improper storage temperature
4. 4 Too much input DNA template causing rapid primer depletion even with minimum 3 PCR cycles

1. 1 Start with number of PCR cycles provided in product manual; reduce cycles if overamplification observed
2. 2 Check PCR primer concentration and ensure correct volume added per manual; store primers at correct temperature
3. 3 For high DNA input: consider size selection after ligation, or use only a fraction of ligated library as PCR input
4. 4 Do not sequence overamplified libraries as data quality will be compromised; re-prepare with optimized cycle number

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