A second peak between 1,000 – 9,000 bp, or an elevated baseline on a bioanalyzer trace (or similar microcapillary electrophoresis trace), is an indication of overcycling (Figure 4). 
The library prep has been very efficient and a lot of cDNA was generated. Hence, the PCR ran out of primers and template started to denature and reanneal improperly. Overcycling occurs when too many PCR cycles are used for the final library amplification, when the PCR runs out of primers and template and generated ds-cDNA starts to denature and reanneal improperly. This results in longer, bulky molecules that migrate at a lower speed on the Bioanalyzer chip or gels. This can interfere with exact library quantification for pooling and loading for sequencing if relying solely on the Bioanalyzer results.
Therefore, a qPCR assay for exact library quantification should be used additionally in addition to Bioanalyzer or similar results, if such a high molecular weight peaks occurs and overcycling is suspected.
Overcycled libraries may have more PCR duplicates which can affect quantification accuracy and sample clustering (i.e., by PCA).
For future QuantSeq library preps:
Perform the qPCR assay to determine the optimal number of PCR cycles required for library generation.
For similar samples, reduce the number of PCR cycles for library amplification (Endpoint PCR) to prevent overcycling.
Overcycling may lead to a distortion in gene expression quantification and hence should be avoided.
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