Data from: Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers.

High-fidelity “proofreading” polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.

在下一代测序项目的文库构建流程中，研究人员常使用高保真校读型DNA聚合酶（high-fidelity proofreading polymerase），以尽可能降低最终产出的序列数据中的错误。这类聚合酶所提升的模板保真度，往往会以降低模板特异性为代价；而基于扩增片段长度多态性（Amplified Fragment Length Polymorphism, AFLP）技术的文库制备方法，或许尤其容易受到该问题的影响。本研究对比了采用标准Taq DNA聚合酶与两种高保真聚合酶所得到的AFLP图谱。结果显示，相较于高保真聚合酶，标准Taq DNA聚合酶所产生的峰数更少且峰强度更高，这表明Taq在选择性扩增与引物序列完全匹配的模板时表现更优。鉴于校读型聚合酶的高准确性在测序应用中仍具备重要价值，我们认为采用替代性文库制备方法或许会更为有效。