Robust analysis of 50-transcript ends (50-RATE): a novel technique for transcriptome analysis and genome annotation

Complicated cloning procedures and the high cost of sequencing have inhibited the wide application of serial analysis of gene expression and massively parallel signature sequencing for genome-wide transcriptome profiling of complex genomes. Here we describe a new method called robust analysis of 50-transcript ends (50-RATE) for rapid and costeffective isolation of long 50 transcript ends ( 80 bp). It consists of three major steps including 50-oligocapping of mRNA, NlaIII tag and ditag generation, and pyrosequencing of NlaIII tags. Complicated steps, such as purification and cloning of concatemers, colony picking and plasmid DNA purification, are eliminated and the conventional Sanger sequencing method is replaced with the newly developed pyrosequencing method. Sequence analysis of a maize 50-RATE library revealed complex alternative transcription start sites and a 50 poly(A) tail in maize transcripts. Our results demonstrate that 50-RATE is a simple, fast and cost-effective method for transcriptome analysis and genome annotation of complex genomes.