Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 407, Issue 7, Pages 1841-1848Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-014-8435-y
Keywords
Metabarcoding; Next-generation sequencing; Ion torrent; Cq value; PCR efficiency
Funding
- Comunidad Autonoma de la Region de Murcia Project
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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 2,000-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 qPCR amplification.
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