Use of ordered deletions in genome sequencing.
Previous attempts to use the non-random approach for sequencing long DNA fragments have met with little success. As a result, nearly all genomic sequencing is done by the random (shotgun) approach, and the economy promised by the non-random approach has so far not materialized. Here we describe a simple system based on the use of ordered deletions that can be incorporated in the common strategies for genome sequencing. Long genomic fragments are cloned in the pAL-F cosmid and fragmented by digestion with specific restriction endonucleases. The digests are religated to subclone individual restriction fragments. The subclones are then subdivided by overlapping deletions and used for sequencing. We present the nucleotide sequences of two cosmid inserts from chromosome IV of Drosophila (containing the ci gene and the 5' end of the zfh-2 gene) that were determined by this method. This is the first report of successful sequencing of long genomic fragments by the use of overlapping deletions. Our calculations show that, with the present approach, sequence data can be acquired at a rate comparable to the shotgun approach but with significantly reduced numbers (approximately 30%) of sequencing runs. Hence, the use of ordered deletions should allow significant savings in both the amount and cost of sequencing work.
Ahmed
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Department of Biological Sciences, University of Alberta, Edmonton, Canada. asad.ahmed@ualberta.ca
Podemski
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D000818:Animals; D003360:Cosmids / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D017344:Genes, Insect / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA / Q000379:methods*; D017384:Sequence Deletion; D014157:Transcription Factors
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D014157:Transcription Factors; C092937:ci protein, Drosophila; C077729:Zfh2 protein, Drosophila