Robust analysis of 5′-transcript ends (5′-RATE): a novel technique for transcriptome analysis and genome annotation

Abstract

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 5′-transcript ends (5′-RATE) for rapid and cost-effective isolation of long 5′ transcript ends (∼80 bp). It consists of three major steps including 5′-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 5′-RATE library revealed complex alternative transcription start sites and a 5′ poly(A) tail in maize transcripts. Our results demonstrate that 5′-RATE is a simple, fast and cost-effective method for transcriptome analysis and genome annotation of complex genomes.

Robust analysis of 5′-transcript ends: a high-throughput protocol for characterization of sequence diversity of transcription start sites

“…A substantial variation at the TSS of the transcripts of the same gene is expected, as shown in Figure 2, for the photosystem I complex PsaH subunit gene and also for other genes in maize 20 . This method can also possibly reveal an unusual 5¢-polyA tail structure at the beginning of the transcripts, as previously reported in maize 21 ( Fig. 2) and soybean (data not shown) 5¢-RATE libraries.…”

“…The 5¢-polyA tail structure has not been reported previously in any organism except for a late gene in poxvirus 23 . Similar 5¢-polyA tail structures can be found in FL cDNAs derived from plants, animals and viruses in the NCBI GenBank databases 21 . Further investigation of the formation and function of 5¢-polyA tails may reveal a novel mechanism of post-transcriptional processing and gene regulation in eukaryotic cells 24 .…”

“…We have successfully incorporated pyrosequencing into our newly developed method called 5¢ RATE 21 . This method is being used for investigating the transcript diversity at the 5¢ region and identifying putative TSSs and promoter regions of expressed genes in maize, soybean and rice in our laboratory.…”

Transcriptome Sequencing

“…Gowda et al (2006) used alkaline phosphatase and acid pyrophosphatase to convert 5' cap structures to monophosphates suitable for RNA linker ligation, while removing fragments that naturally harbor a 5' monophosphate or hydroxyl group. The technology was named Robust Analysis of 5'-Transcript Ends (5'-RATE).…”

“…The technology was named Robust Analysis of 5'-Transcript Ends (5'-RATE). Using this approach, they investigated the 5' ends of transcripts in maize and detected variance in 5' transcript start sites and 5' polyadenylation (Gowda et al, 2006). Ng et al (2006) used paired-end ditags (PETs) to investigate the 5' and 3' ends of transcripts simultaneously.…”