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Components of variance in transcriptomics based on electrophoretic separation of cDNA fragments (cDNA-AFLP)

机译：基于电泳分离cDNA片段（cDNA-AFLP）的转录组学方差成分

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摘要

The sources of variance and errors in transcriptomics based on the electrophoretic separation of amplified cDNA fragments were investigated using cDNA-amplified fragment length polymorphism (AFLP). Transcriptome profiles of the plant-pathogenic fungus Verticillium longisporum were generated by a standard cDNA-AFLP protocol followed by electrophoretic separation of amplified DNA fragments in flatbed polyacrylamide gels with fluorescence detection as well as by capillary electrophoresis (DNA sequencer). The total variance was partitioned into contributions of cDNA synthesis, adapter ligation, preamplification, amplification, and electrophoresis. Parameters of computer-aided peak recognition and matching were investigated and strategies improving matching success based on double passage with different signal intensity thresholds were developed. The overall quality of data was similar for cDNA-AFLP and microarray hybridization. Variance of cDNA-AFLP was independent of signal intensity, whereas microarray data showed higher variance for low-intensity signals. Capillary electrophoresis significantly reduced the number of wrongly matched and unmatched signals as compared with flatbed gels. These results are also likely to apply to related electrophoresis-based transcriptome analysis techniques such as mRNA differential display.
机译：使用cDNA扩增的片段长度多态性（AFLP）研究了基于电泳分离扩增的cDNA片段的转录组学方差和错误来源。通过标准的cDNA-AFLP方案生成植物病原性真菌黄萎病菌的转录组图谱，然后通过电泳检测在平板聚丙烯酰胺凝胶中电泳分离扩增的DNA片段，并通过毛细管电泳（DNA测序仪）进行分离。将总变异分为cDNA合成，衔接子连接，预扩增，扩增和电泳的贡献。研究了计算机辅助峰识别和匹配的参数，并提出了基于不同信号强度阈值的两次通过提高匹配成功率的策略。 cDNA-AFLP和微阵列杂交的总体数据质量相似。 cDNA-AFLP的方差与信号强度无关，而微阵列数据显示低强度信号的方差更高。与平板凝胶相比，毛细管电泳显着减少了错误匹配和未匹配信号的数量。这些结果也可能适用于相关的基于电泳的转录组分析技术，例如mRNA差异显示。

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