Patent ID: 8829172

Claim:
A method of generating a barcoded Paired-End Ditag (bPED) nucleic acid fragment, comprising: (a) performing a first ligation by ligating a half-adaptor to a target nucleic acid to obtain a nucleic acid fragment with the half-adaptor attached to the two ends thereof, the half adaptor comprising a half-barcode and a restriction enzyme (RE) recognition site, wherein the half-adaptor is: (1) a single sticky-end half-adaptor with one 3′-overhanging end; or (2) a double sticky-end half-adaptor with two 3′-overhanging ends; (b) performing a second ligation by ligating the half-adaptors at the two ends of the nucleic acid fragment to form a circularized nucleic acid construct, wherein the circularized nucleic acid construct comprises a full-size barcoded adaptor; and (c) digesting the circularized nucleic acid construct with a RE that cleaves at a defined distance from the RE recognition site, and thereby generating the bPED nucleic acid fragment, wherein the bPED nucleic acid fragment comprises: (i) the full-size barcoded adaptor, comprising: 1) a barcode, comprising no nucleotide or a user-defined known nucleotide sequence; and 2) 5′ and 3′ flanking nucleotide sequences, each of the flanking nucleotide sequences comprising the RE recognition site; (ii) a first tag; and (iii) a second tag, covalently linked to the first tag via the full-size barcoded adaptor; wherein the first tag corresponds to the 5′-end of the target nucleic acid, and the second tag corresponds to the 3′-end of the target nucleic acid.