Zoological Museum, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
BMC Genomics. 2009 Nov 30;10:565. doi: 10.1186/1471-2164-10-565.
Complementary-DNA based amplified fragment length polymorphism (cDNA-AFLP) is a commonly used tool for assessing the genetic regulation of traits through the correlation of trait expression with cDNA expression profiles. In spite of the frequent application of this method, studies on the optimization of the cDNA-AFLP assay design are rare and have typically been taxonomically restricted. Here, we model cDNA-AFLPs on all 92 eukaryotic species for which cDNA pools are currently available, using all combinations of eight restriction enzymes standard in cDNA-AFLP screens.
In silco simulations reveal that cDNA pool coverage is largely determined by the choice of individual restriction enzymes and that, through the choice of optimal enzyme combinations, coverage can be increased from <40% to 75% without changing the underlying experimental design. We find evidence of phylogenetic signal in the coverage data, which is largely mediated by organismal GC content. There is nonetheless a high degree of consistency in cDNA pool coverage for particular enzyme combinations, indicating that our recommendations should be applicable to most eukaryotic systems. We also explore the relationship between the average observed fragment number per selective AFLP-PCR reaction and the size of the underlying cDNA pool, and show how AFLP experiments can be used to estimate the number of genes expressed in a target tissue.
The insights gained from in silico screening of cDNA-AFLPs from a broad sampling of eukaryotes provide a set of guidelines that should help to substantially increase the efficiency of future cDNA-AFLP experiments in eukaryotes. In silico simulations also suggest a novel use of cDNA-AFLP screens to determine the number of transcripts expressed in a target tissue, an application that should be invaluable as next-generation sequencing technologies are adapted for differential display.
基于互补 DNA 的扩增片段长度多态性（cDNA-AFLP）是一种常用于通过将性状表达与 cDNA 表达谱相关联来评估性状遗传调控的工具。尽管这种方法经常被应用，但关于 cDNA-AFLP 检测设计优化的研究很少，而且通常仅限于分类学。在这里，我们使用 cDNA-AFLP 筛选中常用的 8 种限制酶的所有组合，对目前可获得 cDNA 池的 92 种真核生物进行 cDNA-AFLP 的计算机模拟。
计算机模拟显示，cDNA 池的覆盖率主要取决于单个限制酶的选择，并且通过选择最佳的酶组合，可以在不改变基础实验设计的情况下，将覆盖率从<40%提高到 75%。我们发现覆盖数据中存在系统发育信号，这主要是由生物体的 GC 含量介导的。然而，对于特定的酶组合，cDNA 池覆盖率仍然具有高度的一致性，这表明我们的建议应该适用于大多数真核系统。我们还探讨了每个选择性 AFLP-PCR 反应中观察到的平均片段数与潜在 cDNA 池大小之间的关系，并展示了 AFLP 实验如何用于估计目标组织中表达的基因数量。
从广泛的真核生物采样中进行 cDNA-AFLP 的计算机筛选获得的见解提供了一组指南，应该有助于极大地提高未来真核生物 cDNA-AFLP 实验的效率。计算机模拟还表明，cDNA-AFLP 筛选可用于确定目标组织中表达的转录本数量，这一应用在将下一代测序技术应用于差异显示时应该非常有价值。
