Agriculture and Agri-Food Canada, Lethbridge, Canada.
FEMS Microbiol Lett. 2011 Jul;320(2):152-9. doi: 10.1111/j.1574-6968.2011.02302.x. Epub 2011 May 25.
Molecular microbial ecology studies are heavily reliant on 'Universal' 16S rRNA gene primers for elucidating microbial community structure and composition, and yet primer design and optimization is often overlooked. Primers that exhibit minor biases due to primer-template mismatches can substantially alter the pool of amplicons from a community DNA sample, resulting in inaccurate conclusions. As a result, it is important that primers are critically evaluated against the most comprehensive data sets available before commencing molecular microbial community studies. We present a user-friendly, multi-platform (e.g. Windows, Linux, Mac) method named spyder for the in silico design and assessment of 16S rRNA gene primers. The method utilizes the Ribosomal Database Project's Probe Match feature coupled with a compact program (available at http://people.uleth.ca/~selibl/Spyder/Spyder.html) that aligns and identifies mismatches between primers and templates. To demonstrate the value of spyder, we assessed commonly used 'Universal' and phyla-specific primers and identified primer modifications that improved the coverage of target organisms by 5-42% as well as removed excessive degeneracies.
分子微生物生态学研究严重依赖“通用”16S rRNA 基因引物来阐明微生物群落结构和组成，然而引物设计和优化往往被忽视。由于引物-模板错配而表现出微小偏差的引物可以从群落 DNA 样本中大量改变扩增子库，从而导致不准确的结论。因此，在开始分子微生物群落研究之前，重要的是要根据可用的最全面的数据集对引物进行严格评估。我们提出了一种名为 spyder 的用户友好型、多平台（例如 Windows、Linux、Mac）方法，用于 16S rRNA 基因引物的计算机设计和评估。该方法利用核糖体数据库项目的 Probe Match 功能，结合一个紧凑的程序（可在 http://people.uleth.ca/~selibl/Spyder/Spyder.html 获得），该程序对齐并识别引物和模板之间的错配。为了展示 spyder 的价值，我们评估了常用的“通用”和类群特异性引物，并确定了改进目标生物覆盖范围 5-42%的引物修饰方法，同时去除了过多的简并性。
