DescriptionNGS applications in plant pathogen diagnosticsDullemans Annette, Verstappen Els, Houwers Ilse, Verbeek Martin, Van der Lee Theo, Van der Vlugt René and Bonants Peter. Wageningen UR, BU Biointeractions & Plant Health, PO. Box 16, 6700 AA Wageningen, The Netherlands, email@example.comRoutine diagnostics of plant pathogens often involve methods such as ELISA and real-time (TaqMan) PCR. These methods only detect known pathogens. Next Generation Sequencing (NGS) is a valuable method to identify new pathogens. We developed a (semi) automated pipeline to use HiSeq Illumina Sequencing to detect pathogens in different hosts. Total DNA or RNA was isolated from symptomatic plant tissue to generate sequence libraries. Individual samples were labelled with tags to pool multiple samples in one run. The sequence reads generated from a single sample were analyzed in a CLC Genomics Work Bench pipeline. Host reads were removed and the remaining reads were used in a de novo assembly. The resulting contig sequences were compared with sequences in public and in-house databases to identify the origin of the non-host sequences. General knowledge on plant pathogens is essential to identify the pathogen contigs. The read coverage of the pathogen genomes expressed as the Reads per Kb per million reads, and therefore the analytical sensitivity, depends on number of reads, genome size, and the abundance of the pathogen in the host. To investigate whether NGS can be a useful tool in routine screening, RNA samples from an inspection service were sequenced. Samples infected with known pathogens, detected by real-time (Taqman) PCR could be confirmed by NGS. Several examples of NGS will be presented.
|2 Dec 2015
|Testa - EPPO Conference on diagnostics for plant pests
Project: LNV project