RNA-seq Method and pipeline
Oryza sativa Japonica Group Transcriptome or Gene expression
The emergence of NextGen sequencing technologies has generated much interest in the exploration of plant transcriptomes. Currently, Illumina Inc. (San Diego, CA) provides one of the most widely utilized sequencing platforms for gene expression analysis. While Illumina reagents and protocols perform adequately in RNA-sequencing (RNA-seq), alternative reagents and protocols promise a higher throughput at lower cost. We have developed a robust protocol to produce Illumina-compatible (GAIIx and HiSeq2000 platforms) RNA-seq libraries with several crucial improvements. First, we developed balanced adapter sequences for multiplexing of samples; second, the protocol captures strand-specific expression data using proven methodologies; third, we simplified the RNA purification, fragmentation and library size-selection steps thus drastically reducing the time and increasing throughput of library construction; fourth, we have included an RNA spike-in control for validation and normalization procedures and finally, we have made improvements on an informatics pipeline to incorporate significance tests for determining gene expression values and putative alternative splicing events. To demonstrate the potential of this pipeline, we characterized the transcriptome of the rice leaf. Our data supports novel genes models and can be used to improve current rice gene annotation. Additionally, using the rice transcriptome data, we compared different methods of calculating gene expression and discuss the advantages of a strand-specific approach to detect bona-fide anti-sense transcripts and to differentiate intronic transcripts that originate from pre-mRNA from intron retention events. Taking together, our results demonstrate the potential of this low cost and robust method for RNA-seq analysis.