Genome annotation by high-throughput 5′ RNA end determination

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Abstract
Complete gene identification and annotation, including alternative transcripts, remains a challenge in understanding genome organization. Such annotation can be achieved by a combination of computational analysis and experimental confirmation. Here, we describe a high-throughput technique, trans-spliced exon coupled RNA end determination (TEC-RED), that identifies 5′ ends of expressed genes in nematodes. TEC-RED can distinguish coding regions from regulatory regions and identify genes as well as their alternative transcripts that have different 5′ ends. Application of TEC-RED to ≈10% of the Caenorhabditis elegans genome yielded tags 75% of which experimentally verified predicted 5′-RNA ends and 25% of which provided previously unknown information about 5′-RNA ends, including the identification of 99 previously unknown genes and 32 previously unknown operons. This technique will be applicable in any organisms that have a trans-splicing reaction from spliced leader RNA. We also describe an efficient sequential method for concatenating short sequence tags for any serial analysis of gene expression-like techniques.