NOVEL MULTIPLEX BARCODED PAIRED-END DITAG (mbPED) SEQUENCING APPROACH AND ITS APPLICATION IN FUSION GENE IDENTIFICATION

A method of generating a barcoded Paired-End Ditag (bPED) nucleic acid fragment, comprising:

• (a) performing a first ligation by ligating a half-adaptor having one or two 3′-overhanging ends to a target nucleic acid to obtain a nucleic acid fragment with two ends each attached to one of the half-adaptor, the half adaptor comprising a half-barcode and a restriction enzyme (RE) recognition site;
• (b) performing a second ligation by ligating two of the half-adaptor 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 a virtual 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 and the second tags are the 5′- and 3′-ends of the target nucleic acid from which the two tags originate, and wherein the length of the half-adaptor is adapted to allow the length of bPED nucleic acid fragment fits one-step sequencing.