Quantitative evaluation of bias in PCR amplification and Next Generation Sequencing derived from metabarcoding samples

Unbiased identification of organisms by PCR reactions using universal primers followed by DNA sequencing assumes positive amplification. We used six universal loci spanning 48 plant species and quantified the bias at each step of the identification process from end point PCR to Next-Generation Sequencing. End-point amplification was significantly different for single loci and between species. Quantitative PCR revealed that Cq threshold for various loci, even within a single DNA extraction, showed 2000-fold differences in DNA quantity after amplification. Next Generation Sequencing (NGS) experiments in nine species showed significant biases towards species and specific loci using adaptor-specific primers. NGS sequencing bias may be predicted to some extent by the Cq values of Q-PCR amplification.This work was funded by the Comunidad Autónoma de la Región de Murcia Project “Molecular markers in conservation and management of the flora of Murcia Region”. This work was published in Analytical and Bioanalytical Chemistry (407(7): 1841-8