Comparative Genomic Analyses among Coffee-Associated strains of Xylella fastidiosa. Xylella fastidiosa

With the aid of a biochip, carrying representative sequences from approximately 2200 sequences from the genome of isolate 9a5c from X. fastidiosa (Xf), microarray-based comparisons have been performed with 8 different Xf isolates obtained from coffee plants. Overall design: DNA from the 9a5c isolate was used as a reference in competitive hybridization experiments against DNA from 8 different Xf isolates obtained from coffee plants. Equimolecular amounts of each DNA have been labelled with either Cy3- or Cy5-dCTP and hybridized with a 9a5c Xf biochip. Statistical validation of fold change variations was performed with the aid of the Significance Analysis of Microarrays (SAM) method Tusher and coworkers, using the software developed by B. Narasimham and available at http://www-stat.stanford.edu/~tibs/SAM/index.html. Spots that showed a Reference/Test ratio 5:1 were considered to be missing in the test strain. The application of these criteria in a direct sequence comparison between strains 9a5c and Temecula-1, which have been completely sequenced, provided an estimated error rate below 0.3%

本研究使用携带苛养木杆菌（Xylella fastidiosa, Xf）菌株9a5c基因组中约2200条代表性序列的生物芯片，针对8株从咖啡植株中分离得到的苛养木杆菌开展了微阵列比较分析。
整体实验设计如下：以菌株9a5c的DNA作为参照，与8株咖啡来源的苛养木杆菌DNA进行竞争性杂交实验。将等摩尔量的各菌株DNA分别用Cy3-或Cy5-dCTP标记，随后与苛养木杆菌9a5c生物芯片进行杂交。
借助Tusher等人提出的微阵列显著性分析（Significance Analysis of Microarrays, SAM）方法，对杂交信号的倍数变化进行统计学显著性验证，所用分析软件由B. Narasimham开发，可从http://www-stat.stanford.edu/~tibs/SAM/index.html获取。当参考信号与检测信号的比值达到5:1时，该信号点对应的序列被判定为在检测菌株中缺失。将该判定标准应用于已完成全基因组测序的菌株9a5c与Temecula-1之间的直接序列比对后，估算得到的错误率低于0.3%