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Quantitative evaluation of bias in PCR amplification and next-generation sequencing derived from metabarcoding
Marta Pawluczyk1, Julia Weiss, Matthew G Links
1Genetics, Instituto de Biotecnología Vegetal, Universidad Politécnica de Cartagena, 30202, Cartagena, Spain.
Analytical and Bioanalytical Chemistry
|January 12, 2015
Summary
PCR amplification and DNA sequencing can be biased. This study quantifies bias in universal plant DNA identification methods, finding significant variations across species and loci, even with next-generation sequencing.
Area of Science:
- Molecular Biology
- Genetics
- Bioinformatics
Background:
- Accurate organism identification via PCR and DNA sequencing relies on unbiased amplification.
- Universal primers are commonly used for broad-range species identification.
- Potential biases in PCR amplification and sequencing can affect identification accuracy.
Purpose of the Study:
- To quantify amplification bias at each step of DNA-based organism identification.
- To assess bias in endpoint PCR, quantitative PCR (qPCR), and next-generation sequencing (NGS).
- To evaluate bias across six universal loci and 48 plant species.
Main Methods:
- Endpoint PCR amplification using six universal loci across 48 plant species.
- Quantitative PCR (qPCR) to measure DNA quantity differences (Cq values) post-amplification.
- Next-generation sequencing (NGS) experiments on nine species using adaptor-specific primers.
Main Results:
- Significant amplification bias was observed in endpoint PCR, varying between loci and species.
- qPCR revealed substantial DNA quantity differences (up to 2,000-fold) across loci within single extractions.
- NGS experiments demonstrated significant biases towards specific species and loci, influenced by adaptor primers.
Conclusions:
- Universal primer-based PCR and sequencing methods exhibit significant bias in plant species identification.
- qPCR Cq values can help predict NGS sequencing bias.
- Awareness and mitigation of these biases are crucial for accurate molecular identification.
