Gowda Malali, Li Haumeng, Alessi Joe, Chen Feng, Pratt Richard, Wang Guo-Liang
Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2006;34(19):e126. doi: 10.1093/nar/gkl522. Epub 2006 Sep 29.
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 (approximately 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.
复杂的克隆程序和高昂的测序成本阻碍了基因表达序列分析（SAGE）和大规模平行签名测序（MPSS）在复杂基因组全基因组转录组图谱分析中的广泛应用。在此，我们描述了一种名为5'-转录本末端稳健分析（5'-RATE）的新方法，用于快速且经济高效地分离长5'转录本末端（约80 bp）。它包括三个主要步骤，即mRNA的5'-寡聚帽化、NlaIII标签和双标签的生成以及NlaIII标签的焦磷酸测序。省去了诸如连接体的纯化和克隆、菌落挑选及质粒DNA纯化等复杂步骤，并且用新开发的焦磷酸测序方法取代了传统的桑格测序方法。对玉米5'-RATE文库的序列分析揭示了玉米转录本中复杂的可变转录起始位点和5'多聚（A）尾。我们的结果表明，5'-RATE是一种用于复杂基因组转录组分析和基因组注释的简单、快速且经济高效的方法。
