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Nucleic Acids Research, 2000, Vol. 28, No. 7 E22-e22
© 2000 Oxford University Press

Octamer-primed sequencing technology: development of primer identification software

Gangwu Mei and Susan H. Hardin*

Department of Biology and Biochemistry, Institute of Molecular Biology, University of Houston, Houston, TX 77204-5513, USA

Octamer sequencing technology (OST) is a primer-directed sequencing strategy in which an individual octamer primer is selected from a pre-synthesized octamer primer library and used to sequence a DNA fragment. However, selecting candidate primers from such a library is time consuming and can be a bottleneck in the sequencing process. To accelerate the sequencing process and to obtain high quality sequencing data, a computer program, electronic OST or eOST, was developed to automatically identify candidate primers from an octamer primer library. eOST integrates the base calling software PHRED to provide a quality assessment for target sequences and identifies potential primer binding sites located within a high quality target region. To increase the sequencing success rate, eOST includes a simple dynamic folding algorithm to automatically calculate the free energy and predict the secondary structure within the template in the vicinity of the octamer-binding site. Several parameters were found to be important, including base quality threshold, the window size of the template sequence segment, and the threshold {Delta}G value. OST, coupled with the eOST software, can be used to sequence short DNA fragments or in the finishing assembly stage of large-scale sequencing of genomic DNA.

* To whom correspondence should be addressed. Tel: +1 713 743 2686; Fax: +1 713 743 2636; Email: shardin@uh.edu Present address: Gangwu Mei, Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Room N1521, Houston, TX 77030, USA


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