Sequencing polynucleotides using nanopores

A sequencing system, comprising:

• a nanopore comprising a first side, a second side, and an aperture extending through the first and second sides;
• a polynucleotide disposed through the aperture of the nanopore such that a 3′ end of the polynucleotide is on the first side of the nanopore, and a 5′ end of the polynucleotide is on the second side of the nanopore;
• a primer hybridized to a primer binding sequence in the polynucleotide to form a duplex disposed on the first side of the nanopore, the duplex including a 3′ end corresponding to a 3′ end of the primer and at which a nucleotide is disposed;
• and
• circuitry configured to:
• (a) apply a first force disposing the 3′ end of the duplex within the aperture, wherein the nanopore inhibits translocation of the 3′ end of the duplex to the second side of the nanopore while the first force is applied;
• (b) measure a value of an electrical property of the 3′ end of the duplex and a single-stranded portion of the polynucleotide while applying the first force;
• (c) apply a second force to remove the 3′ end of the duplex from the aperture so that the duplex is extended on the first side of the nanopore by an additional nucleotide that is added to the 3′ end of the duplex;
• (d) repeat operations (a) through (c) for additional nucleotides;
• (e) after operation (d), apply a third force to dissociate the extended duplex from the polynucleotide;
• (f) after operation (e), repeating operations (a) through (d) to resequence the polynucleotide;
• and
• (g) identifying the nucleotide added in operation (c) and when operation (c) is repeated during operation (f) using the value measured in operation (b) and the value measured when operation (b) is repeated during operation (f).