Octamer-primed sequencing technology: development of primer identification software.
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.
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Department of Biology and Biochemistry, Institute of Molecular Biology, University of Houston, Houston, TX 77204-5513, USA.
Hardin
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D000465:Algorithms; D000818:Animals; D001331:Automation; D017931:DNA Primers; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005069:Evaluation Studies as Topic; D019476:Insect Proteins / Q000235:genetics; D009690:Nucleic Acid Conformation; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013698:Templates, Genetic; D013816:Thermodynamics
D017931:DNA Primers; D029721:Drosophila Proteins; D019476:Insect Proteins; C096445:tim protein, Drosophila