Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data.

BACKGROUND: Rice research has been enabled by access to the high quality reference genome sequence generated in 2005 by the International Rice Genome Sequencing Project (IRGSP). To further facilitate genomic-enabled research, we have updated and validated the genome assembly and sequence for the Nipponbare cultivar of Oryza sativa (japonica group). RESULTS: The Nipponbare genome assembly was updated by revising and validating the minimal tiling path of clones with the optical map for rice. Sequencing errors in the revised genome assembly were identified by re-sequencing the genome of two different Nipponbare individuals using the Illumina Genome Analyzer II/IIx platform. A total of 4,886 sequencing errors were identified in 321 Mb of the assembled genome indicating an error rate in the original IRGSP assembly of only 0.15 per 10,000 nucleotides. A small number (five) of insertions/deletions were identified using longer reads generated using the Roche 454 pyrosequencing platform. As the re-sequencing data were generated from two different individuals, we were able to identify a number of allelic differences between the original individual used in the IRGSP effort and the two individuals used in the re-sequencing effort. The revised assembly, termed Os-Nipponbare-Reference-IRGSP-1.0, is now being used in updated releases of the Rice Annotation Project and the Michigan State University Rice Genome Annotation Project, thereby providing a unified set of pseudomolecules for the rice community. CONCLUSIONS: A revised, error-corrected, and validated assembly of the Nipponbare cultivar of rice was generated using optical map data, re-sequencing data, and manual curation that will facilitate on-going and future research in rice. Detection of polymorphisms between three different Nipponbare individuals highlights that allelic differences between individuals should be considered in diversity studies.

Assembling genomic DNA sequences with PHRAP.

The PHRAP assembly program provides rapid comparison, alignment, and assembly of large sets of DNA sequences. PHRAP compares sequences by searching for pairs of perfectly matching "words" or sequence regions that meet certain criteria. If a match is found, PHRAP then tries to extend the alignment into overlapping sections called contigs. PHRAP uses quality values produced by the PHRED basecaller to strike a balance between tolerance of discrepancies and prevention of stacking repeat sequences. The PHRAP assembly algorithm is generally used as part of the PHRED/PHRAP/Consed software suite for sequence analysis. This unit presents instructions for basic usage of the PHRAP assembler, including preparation of the input files (Support Protocols 1 and 2) and explanation of output files (Basic Protocols 1 and 2). Several command line options for changing the PHRAP assembly parameters are also discussed (Basic Protocol 3).

Maize genome sequencing by methylation filtration.

Gene enrichment strategies offer an alternative to sequencing large and repetitive genomes such as that of maize. We report the generation and analysis of nearly 100,000 undermethylated (or methylation filtration) maize sequences. Comparison with the rice genome reveals that methylation filtration results in a more comprehensive representation of maize genes than those that result from expressed sequence tags or transposon insertion sites sequences. About 7% of the repetitive DNA is unmethylated and thus selected in our libraries, but potentially active transposons and unmethylated organelle genomes can be identified. Reverse transcription polymerase chain reaction can be used to finish the maize transcriptome.