High-throughput gene discovery in the rat

Abstract

The rat is an important animal model for human diseases and is widely used in physiology. In this article we present a new strategy for gene discovery based on the production of ESTs from serially subtracted and normalized cDNA libraries, and we describe its application for the development of a comprehensive nonredundant collection of rat ESTs. Our new strategy appears to yield substantially more EST clusters per ESTs sequenced than do previous approaches that did not use serial subtraction. However, multiple rounds of library subtraction resulted in high frequencies of otherwise rare internally primed cDNAs, defining the limits of this powerful approach. To date, we have generated >200,000 3' ESTs from >100 cDNA libraries representing a wide range of tissues and developmental stages of the laboratory rat. Most importantly, we have contributed to approximately 50,000 rat UniGene clusters. We have identified, arrayed, and derived 5' ESTs from >30,000 unique rat cDNA clones. Complete information, including radiation hybrid mapping data, is also maintained locally at http://genome.uiowa.edu/clcg.html. All of the sequences described in this article have been submitted to the dbEST division of the NCBI.

Figure 1

Summary of libraries created. This figure presents a graphical description of the cDNA libraries created throughout the gene discovery process. The operation of cDNA library normalization is represented by arrows labeled with an “N,” and serial subtraction operations are represented by arrows labeled with an “S.” Pooling of libraries is represented by multiple arrows converging on a single node.

Figure 2

Incremental discovery throughout gene discovery. This graph tracks the novelty per unit (1000 sequences) for the entire gene discovery project. Peaks in the incremental novelty rate correlate with sequencing of pooled subtracted cDNA libraries. The troughs in the graph correlate with sequencing single tissue non-normalized cDNA libraries.

Figure 3

Gene discovery in rat, mouse, and human. Discovery per 1000 sequences is presented to compare the rates of EST-based gene discovery among human, mouse and rat. The rat discovery rate is presented with and without the rat heart libraries to demonstrate the power of focused gene discovery with normalization and serial subtraction of cDNA libraries.

Figure 4

Sequence features of ESTs derived from oligo-dT-primed directionally cloned cDNA libraries. This figure presents the features commonly found in ESTs. The sequence is presented as viewed from the 3′ end, with all expected features shown. These include vector sequence before and after the insert sequence, as well as the flanking restriction sites, inserted library tag, and polyadenylation tail and signal sequence.