Discovering single nucleotide polymorphisms (SNPs) in an uncharacterized fungal genome using the software EagleView to evaluate 454 sequencing data

• Published in: Phytopathology Vol. 100; no. 6; p. S17
• Main Authors: Broders, K D, SanMiguel, P J, Westerman, R P, Woeste, KE, Boland, G J
• Format: Journal Article
• Language: English
• Published: 01-06-2010
• ISSN: 0031-949X

Benefits from high-throughput sequencing technology, such as 454 pyrosequencing, is most apparent for species with high societal or economic value but few genomic resources. However, it is questionable how well the sequencing of large numbers of short reads, for species with essentially no prior genome sequence information, will support SNP discovery. The focus of this research was to develop a method for identifying SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. To discover SNPs, genomic DNA from eight isolates of Sirococcus clavigignenti-juglandacearum were bulked in one four-region sequencing run on a Roche 454 GS_FLX. This yielded 71 million total bases comprising 217 thousand reads, 80% of which collapsed into 16,125,754 bases in 30,339 contigs upon assembly. By aligning the reads from multiple isolates we detected 298 SNPs using Roche's gsMapper. However, with no reference sequence available, it was difficult to detect true polymorphisms. EagleView software was used to manually examine each contig that contained one or more putative SNPs. The program confirmed 45 of the original 298 putative SNPs. Of those 45 SNPs, 16 were validated using standard sequencing. This research provides the framework for the rapid and cost-effective discovery of SNP markers for non-model organisms and proves to be especially useful in the case of asexual or clonal fungi with limited genetic variability.