Illumina sequencing libraries are usually generated with Y-adapters. These are partly single-stranded and partly double stranded. A PCR-free library will thus still contain partly single-stranded regions. These single-stranded regions can lead to several types of Bioanalyzer artifacts:
Most commonly the libraries will appear about 70 to 100 nucleotides longer than expected. However, we have also encountered PCR-free libraries that ran as shorter molecules, as well as dramatically longer molecules. We have (rarely) encountered another significant problem: Considerable amounts adapter-dimers were not visible on the Bioanalyzer traces of PCR-free libraries.
Due to these potetnial problems, we recommend the following approach to accurately QC PCR-free Illumina libraries:
- Take a 1 ul aliquot of your library and run a short PCR (e.g. 6 cycles) with this aliquot.
- Clean up the PCR reaction with a spin column ( e.g. Qiagen Qiaquick, Zymo DNA -clean, ...); do NOT use Ampure beads (since these will reduce adapter dimers and defeat their diagnosis in this QC).
- Run the cleaned up PCR product on the Bioanalyzer again as well as the original PCR-free library.
The Bioanalyzer trace of the PCR product will then represent the true molecule sizes and the true adapter-dimer content the closest.
If we generate PCR-free libraries in our lab, the described additional QC steps for PCR-free libraries will necessitate significant additional costs for the library preparation. Please see the prices for the PCR-free Add-on.
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: fully double-stranded library molecules pose no problems in the library QC and there will only be an extremely low PCR-bias introduced and few duplicates generated.
Our recommendation is to submit the same amount of DNA sample as for PCR-free library preps (1 ug or more) and then only apply the single cycle of amplification.
Is PCR-free library preparation still advantageous?
In general, the original grave concerns about library PCR amplification (presented in papers from 2011) 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. In contrast, the previous "standard" 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 still have advantages, but they are now minimal.