Why do I see larger peaks in my cfDNA library amplification?
Overamplification can result if too many PCR cycles are used. Once PCR primers are depleted, library fragments may be single stranded or form heteroduplexes that appear as high molecular weight fragments in capillary gel electrophoresis instruments (Figure 3). These peaks are different from the di-nucleosomal peaks that are expected from cfDNA as seen in early PCR cycles. We recommend starting with the number of PCR cycles recommended in the protocol (https://www.twistbioscience.com/products/ngs/library-preparation/universal-adapter-system) and reducing PCR cycles if overamplification is observed.
| 6 Cycles | 7 Cycles |
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| 8 Cycles | 9 Cycles |
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Figure 3. Capillary gel electropherogram from Bioanalyzer High Sensitivity Assay (Agilent) comparing amplified cfDNA libraries with increasing PCR Cycles. Cycles 8 and 9 show overamplification phenotypes.
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