Using Solexa/Illumina methods to investigate plant pathogen variation and transcriptome

使用Illumina基因组分析仪（www.illumina.com），将进行除Hp 2之外的另外5个Hp种族（Noco 2、Maks 9、Cala 2、Waco 9、Hind 2）的重新测序，目的是鉴定显示多样化选择的基因。为了鉴定这些基因组区域内的表达模式，将使用Solexa测序建立基于基因表达的系列分析（SAGE）的mRNA谱分析方法（Velculescu等，1995 Science 270：484-487）。该新方法将基于SMART cDNA方案（www.clontech.com）以获得来自5'端的读段。这将揭示转录本的起始位置，也可用于表达水平的半定量分析，并提供有关不同基因在感染的不同阶段何时表达的信息。为了鉴定新的和验证预测的开放阅读框，将建立一种方法来对植物中生长的病原体的转录组进行测序。专性生物营养型病原体的转录组分析利用了Jones实验室新开发的用于富集植物中病原体表达的基因的方法（Rougon和Jones，未发表），该方法将与cDNA标准化技术相结合。Solexa测序方法依赖于将随机片段化（雾化）DNA连接到流动池。由于短cDNA不会随机片段化，因此将建立一种允许在随机片段化之前进行cDNA多联体化的方法。我们希望将这种cDNA方法应用于基因组尚未测序的病原体。目前可用于组装已证明对细菌DNA有用的短读段的不同方法将被测试并适用于cDNA从头组装。将开发一种计算方法，将所有数据联合收割机合并到一个数据库中，该数据库可以方便地获取有关种族之间基因组序列变异、表达水平、基因结构和可能功能的信息。所有数据都将公开提供。

Using the Illumina Genome Analyzer (www.illumina.com) re-sequencing 5 more races of Hp apart from Emoy2 (Noco2, Maks9, Cala2, Waco9, Hind2) will be carried out with the objective of identifying genes that show diversifying selection. To identify expression patterns within these genomic regions, a serial analysis of gene expression (SAGE)-based mRNA profiling method (Velculescu et al. 1995 Science 270: 484-487) will be established using Solexa sequencing. This novel method will be based on the SMART cDNA protocol (www.clontech.com) to obtain reads from 5' end. This will reveal where transcripts start and can also be used for semi-quantitative analysis of expression levels and to give information about when different genes are expressed during different stages of infection. To identify new and verify predicted open reading frames, a method will be established to sequence the transcriptome of the pathogen growing in planta. Transcriptome analysis of an obligate biotroph pathogen takes advantage of a newly developed method in the Jones lab for enriching genes expressed by pathogens in plants (Rougon and Jones, unpublished), which will be combined with a cDNA normalization technique. The Solexa sequencing approach relies on attachment of randomly fragmented (nebulised) DNA to a flow cell. Since short cDNAs do not fragment randomly, a method will be established which allows cDNA concatamerisation prior to random fragmentation. We would like to apply this cDNA method to pathogens whose genomes are not yet sequenced. Different methods currently available for assembling short reads that have proved useful with bacterial DNA, will be tested and adapted for cDNA de novo assembly. A computational method will be developed to combine all data into one database which allows easy access to information about variation of genome sequences between races, expression levels, gene structure and possible functions. All data will be made publicly available.