Is PCR-free library preparation still advantageous?
In general, the original concerns about library PCR amplification (presented in papers from 2008) are no longer very relevant. This is due to the use of modern polymerases that are designed for complex samples like Kapa HiFi, NEB Q5, or QIAseq HiFi polymerase. The previous “standard”, the high-fidelity Phusion enzyme had tremendous disadvantages for complex samples (Quail et al. 2012 Optimal enzymes for amplifying sequencing libraries. Nature Methods volume 9, pages10–11(2012) https://www.nature.com/articles/nmeth.1814 ).
PCR-free libraries also have disadvantages, since they require significantly higher library QC efforts. Thus, we are charging a PCR-free Add-On fee for the preparation of PCR-free libraries.
What are your recommendations?
A great alternative to preparing the libraries completely PCR-free is the use of a single PCR cycle instead. This combines the advantages of both: It creates fully double-stranded library molecules that do not cause any problems in the library QC. In addition there will be no or only an extremely low PCR-bias introduced. Our recommendation is to submit the same amount of DNA sample as for PCR-free library preps (e.g. 1 ug) and then request the single PCR cycle library amplification.
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