Venu R C, Zhang Yuan, Weaver Brian, Carswell Peter, Mitchell Thomas K, Meyers Blake C, Boehm Michael J, Wang Guo-Liang
Department of Plant Pathology, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2011;722:167-78. doi: 10.1007/978-1-61779-040-9_12.
Deep transcriptome profiling of pathogen-infected tissues enhances the understanding of molecular mechanisms underlying host-pathogen interactions. Illumina's next generation sequencing technology sequencing-by-synthesis (SBS) is a powerful tool to rapidly sequence genomes and transcriptomes at an affordable rate. We modified the procedure for SBS library construction to significantly increase the efficiency of library construction. Using our improved method, two Sclerotinia homoeocarpa libraries were constructed from mycelia grown in potato dextrose broth (PDB) or potato dextrose agar (PDA) for 96 h, respectively, and two creeping bentgrass libraries were constructed from leaves 96 h after inoculation with S. homoeocarpa or water sprayed, respectively. About 4-7 million mRNA signatures were sequenced from each library. Sequence analysis using BLAST was performed against sequenced fungal genomes and rice genomic sequence to identify the expressed genes in both S. homoeocarpa mycelia and creeping bentgrass. Bioinformatic analysis identified many expressed genes in the pathogen and host. A public database to access the sequence data was developed at http://www.dstidb.org . Our results demonstrate how SBS technology can unravel transcriptome complexity during the creeping bentgrass-S. homoeocarpa interaction.
对病原体感染组织进行深度转录组分析，有助于加深对宿主 - 病原体相互作用潜在分子机制的理解。Illumina的新一代合成测序（SBS）技术是一种强大的工具，能够以可承受的成本快速对基因组和转录组进行测序。我们改进了SBS文库构建程序，以显著提高文库构建效率。使用我们改进的方法，分别从在马铃薯葡萄糖肉汤（PDB）或马铃薯葡萄糖琼脂（PDA）中培养96小时的菌丝体构建了两个果生核盘菌文库，以及分别从接种果生核盘菌或喷水96小时后的匍匐翦股颖叶片构建了两个匍匐翦股颖文库。每个文库测序约400 - 700万个mRNA特征序列。使用BLAST对已测序的真菌基因组和水稻基因组序列进行序列分析，以鉴定果生核盘菌菌丝体和匍匐翦股颖中表达的基因。生物信息学分析在病原体和宿主中鉴定出许多表达的基因。在http://www.dstidb.org上建立了一个用于访问序列数据的公共数据库。我们的结果证明了SBS技术如何揭示匍匐翦股颖与果生核盘菌相互作用过程中的转录组复杂性。
