Wheat Chromosome 4D Survey Sequencing Analysis: Present Status and Perspectives

Sequencing wheat genome has always been viewed as a complex task because of its large size,ahigh repetitive DNA content and polyploidy. Several of previously mentioned constrains (largesize, polyploidy) can be overcome by flow sorting of individual chromosomes followed by shotgun-sequencing using next generation sequencing (NGS) technologies, and thus the IWGSC hasembraced this strategy. In this study we used 4D flow-sorted chromosome arms (obtained from cv Chinese Spring) toperform shotgun sequencing with a Roche 454 NGS platform producing sequence data equivalentto a 3.6x-chromosome coverage on single reads, plus 3kb paired end reads to overcome theproblem of repetitive regions during assembly. In order to reach a reliable and preliminary de novoassembly, short reads are being analyzed using different algorithms, strategies and parameters.The use of rapid alignment tools allow the identification of common (ie. emerging on variousassemblies) and trustable (emerging as more trustworthy variants when comparing differentassemblies) segments which have been in turn put through various validation steps. Multiple sources of evidence like EST collections, genetically mapped markers and gene contentsfor 4D chromosome where used to classify and assess both quality and completion of theassembly, pursuing the final goal of having additional metrics based on content,beyond contiglength and assembly coverage. The methodology of wheat chromosome assembly validationimplemented here, allows the integration of additional genomic information to help on gettingboth a measure of goodness and a strategy to improve short read assembly for plant genomes.