Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores

A method comprising:

• (i) preparing a nucleic acid construct by ligating a polynucleotide bridging moiety to a 3′ terminus of a first strand and a 5′ terminus of a second strand of a double stranded nucleic acid, wherein the polynucleotide bridging moiety comprises a marker, wherein the first strand of the double stranded nucleic acid comprises a first nucleic acid sequence and the second strand of the double stranded nucleic acid comprises a second nucleic acid sequence that is complementary to the first nucleic acid sequence;
• (ii) using a single molecule sequencing apparatus to perform a sequencing reaction that enzymatically reads through the first strand, the bridging moiety, and the second strand to produce orthogonal proof-reading sequence information for a methylated nucleobase in the first strand of the construct;
• (iii) obtaining a signal from the marker in sequencing the bridging moiety that is indicative of the first strand having been sequenced;
• (iv) comparing the sequence information between the first nucleic acid sequence and the second nucleic acid sequence;
• and
• (v) determining the presence of a methylated nucleobase in the first sequence.