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

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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of a newly developed method in the Jones lab for enriching genes expressed by pathogens in plants (Rougon and Jones, unpublished), which will be combined with a cDNA normalization technique.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.