IC4R005-Genome-2013-24280374

Project Title

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

The Background of This Project

• 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).

Plant Culture & Treatment

• Sequence data sources:The IRGSP clone and PCR sequences of the O. sativa (japonica group, cultivar Nipponbare) genome deposited in the International Nucleotide Sequence Databases as of 25 February 2010 were used in construction of the MTP.
• Construction of the genome assembly based on a minimum tiling path and validation with the optical map
• Error corrections with Illumina and 454 reads
• Re-validation and annotation of final assembly

Research Findings

• 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.

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• 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, the researchers 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.
• To distinguish allelic differences between the genome sequence and the NIAS and CSHL re-sequenced individuals,the researchers examined three datasets, NIAS, CSHL, and NIAS + CSHL, that had average read depths of 7.9, 35.7, and 43.6, respectively. With respect to coverage, at least one read uniquely aligned to the pseudomolecules thereby covering 79.4% (NIAS), 90.5% (CSHL), or 90.6% (NIAS + CSHL) of the reference genome (Figure 1)

Figure 1 Depth of coverage of Illumina reads on the assembled genome. The depth of coverage of the Illumina reads at ≥1 read, ≥5 reads,and ≥10 reads are shown for the (A) NIAS, (B) CSHL, and (C) NIAS + CSHL datasets.

Labs working on this Project

• Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
• Cold Spring Harbor Laboratory (CSHL), Cold Spring Harbor, NY 11723, USA.
• Department of Plant Biology, Michigan State University, 612 Wilson Rd, Plant Biology Laboratories, East Lansing, MI 48824, USA.
• Perkin Elmer, Room 4096, 8490 Progress Drive, Frederick, MD 21701, USA.
• Laboratory for Molecular and Computational Genomics, University of Wisconsin-Madison, UWBiotechnology Center, 425 Henry Mall, Madison, WI 53706, USA.
• Integrated Center for Genes, Environment and Health, National Jewish Health, Denver,CO, USA.
• Dupont Pioneer, 7200 NW 62nd Ave, Johnston, IA 50131, USA.