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A second peak between 1,000 – 9,000 bp, or an elevated baseline on a bioanalyzer trace (or similar microcapillary electrophoresis trace), that extends underneath the upper marker is an indication of overcycling (see Figure 4)). Similar features can be seen with other types of microcapillary electrophoresis traces.
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.
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Figure | Correct versus overcycled QuantSeq Library Results: Correctly cycled (10ng UHRR, 19 cycles, blue), overcycled (10ng UHRR, 24 cycles, red). Low input protocol modifications were used (skipping step 2, 1 hour incubation at 42 degrees C at step 4, reduced volumes of PB and PS at steps 16 and 29).
What causes overcycling?
Overcycling occurs when too many PCR cycles are used for the final library amplification. In this case, when the PCR reaction 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
How can I quantify overcycled libraries?
A qPCR assay for exact library quantification should be used additionally in addition to Bioanalyzer or similar resultsanalyses, if such a high molecular weight peaks occurs and/or overcycling is suspected.
What is the effect of overcycling on my data?
Overcycled libraries can still be sequenced. However, these may have more PCR duplicates, which can affect quantification accuracy and sample clustering (i.e., by PCA). Overcycling may lead to a distortion in gene expression quantification and hence should be avoided where possible.
For future QuantSeq library preps:
Should I sequence overcycled libraries or re-prep them?
In order to avoid possible biases in expression data, the best option would be to re-prep the libraries and use a lower number of PCR cycles for the library amplification. If the libraries cannot be re-prepped (i.e., due to precious or irreplaceable samples) it is best to proceed with sequencing.
How can I prevent overcycling?
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 avoidedover-cycling.
You can estimate the number of cycles to reduce according to the yield of overcycled libraries. To achieve half the yield, subtract 1 PCR cycle. To achieve 4x less yield, use 2 fewer cycles. To achieve 10x less yield, use 3 fewer PCR cycles.