Pawluczyk Marta, Weiss Julia, Links Matthew G, Egaña Aranguren Mikel, Wilkinson Mark D, Egea-Cortines Marcos
Genetics, Instituto de Biotecnología Vegetal, Universidad Politécnica de Cartagena, 30202, Cartagena, Spain.
Anal Bioanal Chem. 2015 Mar;407(7):1841-8. doi: 10.1007/s00216-014-8435-y. Epub 2015 Jan 11.
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.
通过使用通用引物进行PCR反应，随后进行DNA测序来无偏倚地鉴定生物体，这一过程假定为阳性扩增。我们使用了跨越48种植物物种的6个通用基因座，并对从终点PCR到下一代测序的鉴定过程的每个步骤中的偏差进行了量化。单基因座以及不同物种之间的终点扩增存在显著差异。定量PCR显示，即使在单次DNA提取中，不同基因座的Cq阈值在扩增后的DNA量上也显示出2000倍的差异。对9个物种进行的下一代测序（NGS）实验表明，使用接头特异性引物时，对物种和特定基因座存在显著偏差。NGS测序偏差在一定程度上可以通过qPCR扩增的Cq值来预测。
