A New Approach Using ‘Spectral Content Profiling’ for in-Depth Plant DNA QC during NGS Library Preparation

As NGS library construction is an expensive and laborious multiple step procedure, it is prudent to include quality control steps throughout the library construction process to ensure the quality of downstream data. Assessing the yield and purity of DNA extracts from plant tissue is important yet challenging due to the fragmented state of the isolated DNA and the presence of co-extracted metabolites like polysaccharides or phenolic derivatives. Furthermore, DNA quantification accuracy can be severely hampered by scattering effects by cell debris or magnetic bead residue while other components of the DNA isolation buffers, such as chaotropic salts and phenol can influence the success rate of downstream library preparation or amplification steps.

Here, we assess a novel approach for content QC during NGS shotgun library preparation of DNA samples extracted from perennial ryegrass (Lolium perenne) leaf tissue. This method is based on UV/Vis absorbance spectral data measured by the DropSense96 micro-volume plate reader by Trinean, and subsequent ‘spectral content profiling’ analysis to specifically quantify the isolated DNA as well as the amount of contaminating constituents in the sample contributing to the measured UV/Vis spectral shape. In this study we compare this novel approach with commonly used QC tools for NGS library preparation and assess the quality by Illumina HiSeq sequencing of 96 indexed, pooled and probe capture-enriched libraries.