Compositions and methods for sequencing multiple regions of a template molecule using enzyme-based reagents

A method for sequencing a nucleic acid template molecule, comprising:

• a) providing at least one single-stranded nucleic acid template molecule immobilized to a support, wherein the at least one single-stranded nucleic acid template molecule is covalently attached or hybridized to a capture primer immobilized to the support;
• b) hybridizing at least one first sequencing primer to a first region of the nucleic acid template molecule and conducting one or more cycles of sequencing reactions, thereby generating at least one first extension duplex comprising a first sequencing read product hybridized to the template molecule;
• and
• c) contacting the first extension duplex with a double-stranded DNA specific enzyme having 5′ to 3′ exonuclease activity under a condition suitable to degrade the first sequencing read product and retain the template molecule, thereby removing at least a portion of the first sequencing read product from the first region of the template molecule;
• and
• d) washing the retained template molecule at a temperature between 22-27° C. to remove the double-stranded DNA specific enzyme having 5′ to 3′ exonuclease activity.