Importance

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Abstract

The present disclosure provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles typically comprise steps of extension, ligation, and cleavage. In certain embodiments, the methods make use of extension probes containing phosphorothiolate linkages and agents capable of cleaving such linkages. Methods of determining information about a sequence using at least two distinguishably labeled probe families are provided, as are methods of performing multiple sequencing reactions on a single template. Automated sequencing systems, flow cells, image processing methods, and computer-readable media that store computer-executable instructions and/or sequence information that can be used in accordance with such methods are also provided. In certain embodiments, blocking oligonucleotides are provided to facilitate sequencing using disclosed methods.

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First Claim