IC4R011-RNA-Seq-2010-21122150

Project Title

• Massive parallel sequencing of mRNA in identification of unannotated salinity stress-inducible transcripts in rice (Oryza sativa L.)

The Background of This Project

• Microarray technology is limited to monitoring the expression of previously annotated genes that have corresponding probes on the array. Computationally annotated genes have not fully been validated, because ESTs and full-length cDNAs cannot cover entire transcribed regions. Here, mRNA-Seq (an Illumina cDNA sequencing application) was used to monitor whole mRNAs of salinity stress-treated rice tissues.

Plant Culture & Treatment

• Massive parallel sequencing of mRNA

Research Findings

Figure 1 Accumulation of 36-bp reads to cover whole transcripts. (a) Cumulative coverage of rice genome and annotated region. Data from nine technical replicates of reads from roots after salinity stress were accumulated. Cumulative coverage was calculated by using reads uniquely mapped on the rice genome (black) or the RAP2 annotated region (white). As the number of reads increased, the cumulative coverage approached a plateau. (b) Robustness of the measurement of transcripts in four different expression classes. Saturation of sequencing was estimated on the basis of the fraction of RAP2 genes supported by FL-cDNA sequences that had reached their final RPKM (reads per kilobase of exon model per million mapped reads) [16]. Vertical axis indicates the fraction of genes for which the RPKM was within 5% of the final value, and horizontal axis indicates the cumulative number of uniquely mapped reads. The fraction of highly expressed genes was almost unchanged, whereas those of genes with relatively low expression converged slowly. N indicates the number of transcripts in each of the four classes.

Labs working on this Project

• National Institute of Agrobiological Sciences (NIAS), Division of Genome and Biodiversity Research, 1-2, Kannondai 2-chome, Tsukuba, Ibaraki 305-8602, Japan.
• Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, 446-1, Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan.
• Hitachi Government & Public Corporation System Engineering Ltd., Solution Division 4, Research & Development Center Bioinformatics Group, 2-4-18 Toyo, Koto-ku, Tokyo 135-8633, Japan.

Corresponding Author

• mat@nias.affrc.go.jp