Marco A. Marra

To accommodate the increasingly rapid rates of DNA sequencing we have developed and implemented an inexpensive, expeditious method for the purification of double-stranded plasmid DNA clones. The robust nature, high throughput, low degree of technical difficulty and extremely low cost have made it the plasmid DNA preparation method of choice in both our expressed sequence tag (EST) and genome sequencing projects. Here we report the details of the method and describe its application in the generation of more than 700 000 ESTs at a rate exceeding 16 000 per week.

Arabidopsis thaliana has emerged as a model system for studies of plant genetics and development, and its genome has been targeted for sequencing¹ by an international consortium (the Arabidopsis Genome Initiative; http://genome-www.stanford.edu/Arabidopsis/agi.html). To support the genome-sequencing effort, we fingerprinted more than 20,000 BACs (ref. 2) from two high-quality publicly available libraries^3,4,5, generating an estimated 17-fold redundant coverage of the genome, and used the fingerprints to nucleate assembly of the data by computer. Subsequent manual revision of the assemblies resulted in the incorporation of 19,661 fingerprinted BACs into 169 ordered sets of overlapping clones ('contigs'), each containing at least 3 clones. These contigs are ideal for parallel selection of BACs for large-scale sequencing and have supported the generation of more than 5.8 Mb of finished genome sequence submitted to GenBank; analysis of the sequence has confirmed the integrity of contigs constructed using this fingerprint data. Placement of contigs onto chromosomes can now be performed, and is being pursued by groups involved in both sequencing and positional cloning studies. To our knowledge, these data provide the first example of whole-genome random BAC fingerprint analysis of a eucaryote, and have provided a model essential to efforts aimed at generating similar databases of fingerprint contigs to support sequencing of other complex genomes, including that of human.

The laboratory mouse is the premier model system for studies of mammalian development due to the powerful classical genetic analysis¹ possible (see also the Jackson Laboratory web site, http://www.jax.org/) and the ever–expanding collection of molecular tools^2,3. To enhance the utility of the mouse system, we initiated a program to generate a large database of expressed sequence tags (ESTs) that can provide rapid access to genes^{4,5,6,7,8,9,10,11,12,13,14,15,16}. Of particular significance was the possibility that cDNA libraries could be prepared from very early stages of development, a situation unrealized in human EST projects^7,12. We report here the development of a comprehensive database of ESTs for the mouse. The project, initiated in March 1996, has focused on 5´ end sequences from directionally cloned, oligo–dT primed cDNA libraries. As of 23 October 1998, 352,040 sequences had been generated, annotated and deposited in dbEST, where they comprised 93% of the total ESTs available for mouse. EST data are versatile and have been applied to gene identification¹⁷, comparative sequence analysis^18,19, comparative gene mapping and candidate disease gene identification²⁰, genome sequence annotation^21,22, microarray development²³ and the development of gene–based map resources²⁴.