3001 AP Note 04-06-070124.QXP

for SAGE library sequencing consists of constructing concatemers by ligation of SAGE tags. Concate-mers are cloned into vectors, then transformed in bacterial clones. With this method of SAGE library construction, clones composed of large inserts need to be sequenced using Sanger technology. Since lig-ation of ditags yields concatemers of various sizes, the efficiency of the SAGE protocol is limited by a small average size of cloned concatemers. When using the original SAGE protocol, both purification and concatenation of SAGE ditags are critical for optimal performance. A high-throughput sequencing method-the massively parallel picoliter-scale process provided by the Genome Sequencer System-offers an efficient approach to rapidly sequence DNA. 2 To date, the Genome Sequencer System has been successfully utilized in an increasing number of de novo sequencing projects, including sequencing the genomes of several bacteria and the mitochondrial genome of an extinct species of mammoth, as well as exploring the sequence diversity present in environmental samples. 2-6 Here, we report a further significant reduction in time and cost for SAGE library construction through the successful use of this newly available pyrosequencing method. This method reads short lengths of DNA, averaging 80-120 bases. In this application note, we describe how this technology is well adapted for directly sequencing SAGE ditags and is therefore ideally suited for rapid SAGE library construction. A high-throughput sequencing method has been developed and offers an efficient approach to rapidly sequence DNA. This pyrosequencing method is suitable for directly sequencing ditags for Serial Analysis of Gene Expression (SAGE), and is well adapted to reduce both time and cost of SAGE library construction. This strategy avoids the traditional concatemer construction step, which often requires extensive technical knowledge and experience for high-quality fragment construction. Directly sequencing SAGE ditags may therefore allow more laboratories to become involved in SAGE library construction in the future. Serial Analysis of Gene Expression (SAGE) remains a relevant technique that allows an accurate quantitative and qualitative analysis of cell transcription in a variety of physiological and pathological conditions. 1 Through a series of enzymatic manipulations , the SAGE method reduces cDNA molecules to tags of 14 bp (short SAGE) or 21 bp (long SAGE). Each tag represents one mRNA molecule. Tags are ligated to form concatemers that are cloned and sequenced. Comparing the sequence information from the tags with the GenBank database provides qualitative information about transcribed genes. The frequency of a specific tag within the SAGE …