Of mice, men, and the genome.

In this issue Carninci et al. (2000) and the RIKEN Genome Exploration Research Group introduce one of the “marvels” of nature that many have predicted but that we are just beginning to grasp in full scope. This is the first largescale publication of a mammalian “transcriptosome,” or the genome as it is expressed, in which the sequenced clones are specifically enriched for full-length sequences. This massive effort represents the accumulated data from the RIKEN-MEI 4.02 release May 15, 2000 (http://genome.rtc.riken.go.jp/), including 914,452 3 end murine sequences representing 126,693 nonredundant clones found in >80 cDNA libraries generated from histologically and developmentally diverse tissues. Perhaps even more important than the public identification of so many new mouse cDNAs is the clear explanation of the methods used to construct the CapTrapped, full-length, normalized, and subtracted libraries. These methods were responsible for a sequence redundancy level <2% and a rate of novel sequences discovery that ran as high as 20%–39% for the most heavily subtracted sublibraries. It is these two factors that made it economically feasible to consider sequencing such a large part of the transcriptosome. As pointed out by the authors, these methods are likely to be quite useful to the other ongoing or proposed cDNA discovery and sequencing efforts, including the recently announced Mammalian Gene Collection (MGC, http://www.ncbi.nlm.nih.gov/ MGC/index.html). Of the transcripts obtained by these methods, 88% appear to contain at least one probable ATG start site and evidence of full-coding length, and many demonstrate large amounts of both 5 and 3 untranslated sequence. These data will allow other researchers to move quickly from the small fragments of genes derived from more traditional methods or from homology criteria to obtaining a fulllength actual cDNA clone. A number of features distinguish this effort from both prior methods and other large-scale cDNA sequencing projects, which also have used normalization and subtraction techniques. (Bonaldo et al. 1996; Marra 1999) The first difference is the Cap-Trapper method itself (outlined in Fig. 1 of Carninci et al. 2000, in this issue), on which they have made and published serial improvements since 1996 (Carninci et al. 1996, 1998; Carninci and Hayashizaki 1999). This is not a method for the timid or for those inexperienced with the most technically demanding RNA work. Although the authors mention that they have been able to perform the method with RNA generated from microdissected tissues (usually no more than microgram quantities at best), generally speaking, it has required milligram quantities of high-quality RNA as starting material. The subtracted and normalized material used in the final cloning steps is frequently present in only nanogram or even subnanogram level (J. Margolin, unpubl.). A second difference from prior efforts is the complete avoidance of the two methods that are the most likely causes of short and incomplete transcript insertion: There is no use of PCR and the cDNA is never amplified or otherwise manipulated in the plasmid state. Despite the improvements in PCR enzyme or enzyme mixture fidelity and processivity, the kinetics of the PCR reaction will inevitably favor short and easier to replicate templates. The PCR kinetic factor can be ameliorated but never eliminated with size fractionation schemas or by limiting cycle numbers. Inevitably, small amounts of contaminating shorter sequences or even sequences of the selected length that just happen to be easier to replicate or that were incompletely normalized will dominate the final product in geometric proportion to the amount of amplification (cycles) employed. The present methods avoid these pitfalls. Also, the Cap-Trapped, normalized, subtracted cDNA is cloned into a cre-lox containing a replacement vector where the ultimate circularization process does not favor shorter inserts. The importance of the cre-lox vector can not be understated, and it is hoped that Carninci et al. (2000, in this issue) will expeditiously publish both the vector and informatic details related to this project. A third and final difference is how these authors set up the normalization, using the original mRNA as the driver, and their use of RoT calculations for the subtraction against pools of RNA expressed from previously sequenced clones. Other authors cited above have used similar normalization techniques using in vitro–expressed RNA or singlestranded DNA. Some of the most efficient prior subtraction techniques have had to rely on fragmentation of the cDNA (often employing a cutting reE-MAIL jmargolin@txccc.orgl FAX (713) 8254038 Article and publication are at www.genome.org/cgi/ doi/10.1101/gr.162800. Insight/Outlook