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 (very rarely) encountered another significant problem: considerable amounts adapter-dimers were not visible on the Bioanalyzer traces of PCR-free libraries.
To accurately QC PCR-free Illumina libraries we recommend the following approach:
– 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.
– 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 represent the true molecule sizes and the true adapter-dimer content the closest.